[Analyses on the quantity, structure and allocation equity of stomatologists in China].

Objective: To study the quantity, structure and allocation equity of stomatologists, in order to provide bases and advices for improving the allocation of stomatologists in China. Methods: On the basis of data from China Health Statistics Yearbooks and Brief Book of Administrative Divisions of the People's Republic of China, the general situation of stomatologists was analyzed by descriptive analysis. Gini coefficient was used to evaluate the allocation equity in 2010 and 2020. Results: The total number of stomatologists reached 2 780 hundred in 2020, which increased by 150.5% compared with that in 2010. The overall quality structure of stomatologists had improved. The gender proportion was balanced and the age distribution was reasonable. The team was mainly composed by the young people, in which the numbers under 44 years old accounted for 71.6% (1 991 hundred/2 780 hundred). The proportion of personnel with senior professional titles decreased to 7.9% (220 hundred/2 780 hundred) while the total number increased to 220 thousand. The distribution of stomatologists by population was fair. Gini coefficients of the whole country as well as the eastern, central and western regions were less than 0.3. Conclusions: The quantity, quality and allocation equity of stomatologists were still insufficient in China. It is necessary to optimize the human resources allocation for stomatologists. It is suggested to increase the talents supply through supply-side reform, medicine-education collaboration and multi-agent participation. And it is suggested to optimize criterions to improve the quality of stomatologists.

[Effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats].

Objective: To explore the effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats. Methods: The method of experimental study was adopted. The polyvinyl alcohol/sodium alginate microspheres (simple microspheres), P311 microspheres, and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) microspheres were prepared by water-in-oil emulsification, and then their morphology was observed under a light microscope/inverted fluorescence microscope. Chitosan solution was prepared, chitosan solution and ß-glycerol phosphate disodium hydrate were mixed to prepare simple thermosensitive hydrogels, and thermosensitive hydrogels loaded with simple microspheres or P311 microspheres were prepared by adding corresponding substances in simple thermosensitive hydrogels. The morphological changes of the prepared four liquids in the state of tilt was observed at 37 ℃. After being freeze-dried, the micromorphology of the prepared four liquids was observed under a scanning electron microscope. Eighteen 3-4-week-old male Sprague-Dawley rats were divided into normal group without any treatment, dressing group, chitosan group, hydrogel alone group, simple microspheres-loaded hydrogel group, and P311 microspheres-loaded hydrogel group, which were inflicted with one full-thickness skin defect wound on both sides of the back spine and were dealt correspondingly, with 3 rats in each group. Rats with full-thickness skin defects in the five groups were collected, the wound healing was observed on post injury day (PID) 0 (immediately), 5, 10, and 15, and the wound healing rates on PID 5, 10, and 15 were calculated. The wound and wound margin tissue of rats with full-thickness skin defects in the five groups on PID 15 and normal skin tissue in the same site of rats in normal group were collected, hematoxylin and eosin staining was conducted to observe the histological changes, immunohistochemical staining was performed to observe the expressions of CD31 and vascular endothelial growth factor (VEGF), and Western blotting was conducted to detect the protein expressions of CD31 and VEGF. The number of samples was all three. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, and Bonferroni correction. Results: Simple microspheres were spherical, with loose and porous surface. The surfaces of P311 microspheres and FITC-BSA microspheres were smooth without pores, and the FITC-BSA microspheres emitted uniform green fluorescence. The diameters of the three microspheres were basically consistent, being 33.1 to 37.7 µm. Compared with chitosan solution and simple thermosensitive hydrogel, the structures of the two microspheres-loaded hydrogels were more stable in the state of tilt at 37 ℃. The two microspheres-loaded hydrogels had denser network structures than those of chitosan solution and simple thermosensitive hydrogel, and in the cross section of which microspheres with a diameter of about 30 µm could be seen. Within PID 15, the wounds of rats in the five groups were healed to different degrees, and the wound healing of rats in P311 microspheres-loaded hydrogel group was the best. On PID 5, 10, and 15, the wound healing rates of rats in dressing group and chitosan group were (26.6±2.4)%, (38.5±3.1)%, (50.9±1.5)%, (47.6±2.0)%, (58.5±3.6)%, and (66.7±4.1)%, respectively, which were significantly lower than (59.3±4.8)%, (87.6±3.2)%, (97.2±1.0)% in P311 microspheres-loaded hydrogel group (P<0.05 or P<0.01). The wound healing rates of rats in hydrogel alone group on PID 10 and 15, and in simple microspheres-loaded hydrogel group on PID 15 were (76.0±3.3)%, (84.5±3.6)%, and (88.0±2.6)%, respectively, which were significantly lower than those in P311 microspheres-loaded hydrogel group (P<0.05). The epidermis, hair follicles, and sebaceous glands could be seen in the normal skin of rats in normal group, without positive expressions of CD31 or VEGF. The wounds of rats in P311 microspheres-loaded hydrogel group on PID 15 were almost completely epithelialized, with more blood vessels, hair follicles, sebaceous glands, and positive expressions of CD31 and VEGF in the wounds than those of rats with full-thickness skin defects in the other four groups, and more protein expressions of CD31 and VEGF than those of rats in the other five groups. Conclusions: The P311 microspheres-loaded thermosensitive chitosan hydrogel can release the encapsulated drug slowly, prolong the drug action time, and promote wound healing in rats with full-thickness skin defects by promoting wound angiogenesis and re-epithelialization.

[Effects of reactive oxygen species-responsive antibacterial microneedles on the full-thickness skin defect wounds with bacterial colonization in diabetic mice].

Objective: To study the effects of reactive oxygen species (ROS)-responsive antibacterial microneedles (MNs) on the full-thickness skin defect wounds with bacterial colonization in diabetic mice. Methods: Experimental research methods were adopted. The ROS-responsive crosslinker N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA) was first synthesized, and then the polyvinyl alcohol (PVA)-TSPBA MNs, PVA-ε-polylysine (ε-PL)-TSPBA MNs, PVA-TSPBA-sodium hyaluronate (SH) MNs, and PVA-ε-PL-TSPBA-SH MNs were prepared by mixing corresponding ingredients, respectively. The PVA-TSPBA MNs were placed in pure phosphate buffer solution (PBS) and PBS containing hydrogen peroxide, respectively. The degradation of MNs immersed for 0 (immediately), 3, 7, and 10 days was observed to indicate their ROS responsiveness. The standard strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) cultured in Luria-Bertani medium containing hydrogen peroxide were divided according to the random number table (the same grouping method below) into blank control group (without any treatment, the same below) and 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group with which PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL were co-cultured, respectively. Bacterial growth was observed after 24 h of culture, and the relative survival rate of bacteria was calculated (n=3). The mouse fibroblast cell line 3T3 cells at logarithmic growth stage (the same growth stage below) were divided into blank control group and 0 g/L ε-PL group, 1.0 g /L ε-PL group, 5.0 g /L ε-PL group, and 10.0 g /L ε-PL group in which cells were cultured in medium with the extract from PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, respectively. Cell growth was observed after 24 h of culture by optical microscopy, and the relative survival rate of cells was detected and calculated by cell counting kit 8 (CCK-8) assay to indicate the cytotoxicity (n=6). Both PVA-TSPBA MNs and PVA-TSPBA-SH MNs were taken, the morphology of the two kinds of MNs was observed by optical microscopy, and the mechanical properties of the two kinds of MNs were tested by microcomputer controlled electronic universal testing machine (denoted as critical force, n=6). Six male BALB/c mice aged 6-8 weeks (the same gender and age below) were divided into PVA-TSPBA group and PVA-TSPBA-SH group, with 3 mice in each group. After pressing the skin on the back of mice vertically with the corresponding MNs for 1 minute, the skin condition was observed at 0, 10, and 20 min after pressing. Another batch of 3T3 cells were divided into blank control group, 0 g/L ε-PL group and simple 5.0 g/L ε-PL group which were cultured with the extract of PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, and 5.0 g/L ε-PL+SH group which were cultured with the extract of PVA-ε-PL-TSPBA-SH MNs with 5.0 g/L ε-PL. The CCK-8 assay was performed to detect and calculate the relative survival rate of cells cultured for 24, 48, and 72 h to indicate the cell proliferation activity (n=6). Eighteen BALB/c mice were induced into diabetic mice model by high-sugar and high-fat diet combined with streptozotocin injection and then divided into sterile dressing group, 0 g/L ε-PL+SH group, and 5.0 g/L ε-PL+SH group, with 6 mice in each group. A full-thickness skin defect wound was made on the back of each mouse, and S. aureus solution was added to make a full-thickness skin defect wound with bacterial colonization model for diabetic mouse. The wounds of mice in 0 g/L ε-PL+SH group and 5.0 g/L ε-PL+SH group were covered with PVA-ε-PL-TSPBA-SH MNs with the corresponding concentration of ε-PL, and the wounds of mice in the 3 groups were all covered with sterile surgical dressings. The wound healing was observed on post injury day (PID) 0, 3, 7, and 12, and the wound healing rate on PID 3, 7, and 12 was calculated. On PID 12, the skin tissue of the wound and the wound margin were stained with hematoxylin and eosin to observe the growth of new epithelium and the infiltration of inflammatory cells. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Mann-Whitney U test, and Bonferroni test. Results: With the extension of the immersion time, the PVA-TSPBA MNs in PBS containing hydrogen peroxide gradually dissolved and completely degraded after 10 days of immersion. The PVA-TSPBA MNs in pure PBS only swelled but did not dissolve. After 24 h of culture, there was no growth of S. aureus in 5.0 g/L ε-PL group or 10.0 g/L ε-PL group, and there was no growth of E. coli in 10.0 g/L ε-PL group. The relative survival rate of S. aureus was significantly lower in 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group than in blank control group (P<0.05 or P<0.01). The relative survival rate of E. coli was significantly lower in 5.0 g/L ε-PL group and 10.0 g/L ε-PL group than in blank control group (P<0.01). After 24 h of culture, the cells in blank control group, 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group all grew well, and the relative survival rate of cells was similar among the groups (P>0.05). The needle bodies of PVA-TSPBA MNs and PVA-TSPBA-SH MNs were both quadrangular pyramid-shaped and neatly arranged, and the needle bodies of PVA-TSPBA-SH MNs was more three-dimensional and more angular. The critical force of PVA-TSPBA-SH MNs was significantly higher than that of PVA-TSPBA MNs (Z=3.317, P<0.01). The MNs in PVA-TSPBA+SH group penetrated the skin of mice at 0 min after pressing, and the pinholes partially disappeared after 10 min and completely disappeared after 20 min, while the MNs in PVA-TSPBA group failed to penetrate the skin of mice. After 24, 48, and 72 h of culture, the proliferation activity of the cells in 5.0 g/L ε-PL+SH group was significantly higher than that of blank control group (P<0.05 or P<0.01). In sterile dressing group, the wounds of mice healed slowly and exuded more. The wound healing speed of mice in 0 g/L ε-PL+SH group was similar to that of sterile dressing group in the early stage but was faster than that of sterile dressing group in the later stage, with moderate exudation. The wound healing of mice in 5.0 g/L ε-PL+SH group was faster than that in the other two groups, with less exudation. The wound healing rates of mice in 5.0 g/L ε-PL+SH group were (40.6±4.2)%, (64.3±4.1)%, and (95.8±2.4)% on PID 3, 7, and 12, which were significantly higher than (20.4±2.7)%, (38.9±2.2)%, and (59.1±6.2)% in sterile dressing group and (21.6±2.6)%, (44.0±1.7)%, and (82.2±5.3)% in 0 g/L ε-PL+SH group (P<0.01). The wound healing rates of mice in 0 g/L ε-PL+SH group on PID 7 and 12 were significantly higher than those in sterile dressing group (P<0.05 or P<0.01). On PID 12, the wounds of mice in 5.0 g/L ε-PL+SH group were almost completely epithelialized with less inflammatory cell infiltration, the wounds of mice in 0 g/L ε-PL+SH group were partially epithelialized with a large number of inflammatory cell infiltration, and no obvious epithelialization but a large number of inflammatory cell infiltration was found in the wounds of mice in sterile dressing group. Conclusions: The composite MNs prepared by TSPBA, PVA, ε-PL, and SH can successfully penetrate mouse skin and slowly respond to ROS in the wound to resolve and release antibacterial substances, inhibit bacterial colonization, and promote the repair of full-thickness skin defect wounds with bacterial colonization in diabetic mice.

[Effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 on the migration and motility of human dermal microvascular endothelial cells under hypoxia and the mechanism].

Objective: To explore the effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 (BNIP3) on the migration and motility of human dermal microvascular endothelial cells (HDMECs) under hypoxia and the mechanism. Methods: The experimental research method was applied. (1) HDMECs were divided into normoxia group received routine culture and hypoxia 6, 12, 24 h groups treated under hypoxia with oxygen volume fraction of 2% for corresponding time according to the random number table (the same grouping method below). Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the healing rate of scratch was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The number of sample was 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with those of normoxia group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxia 6, 12, 24 h groups were significantly increased (P<0.01). (2) After 6 hours of culture, compared with that of hypoxia+ unloaded group, the BNIP3 protein expressions of cells in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were significantly decreased (P<0.05 or P<0.01). The red fluorescence denoting BNIP3 protein expression of cells in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+ unloaded group was strong, and the red fluorescence of cells in hypoxia+ BNIP3 knockdown group was significantly decreased compared with that in hypoxia+ unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+ unloaded group basically healed, while the remaining scratch area in the other three groups were large. The healing rates of scratch of cells in normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)%, and (57±4)%, respectively. The healing rate of scratch of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group (P<0.01) and hypoxia+ BNIP3 knockdown group (P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+ unloaded group was significantly larger than that in normoxia+ unloaded group, the range of cell movement in hypoxia+ BNIP3 knockdown group was significantly smaller than that in hypoxia+ unloaded group, and the curve movement velocity of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group (P<0.01). (3) After 6 hours of culture, compared with hypoxia+ unloaded group, the LC3Ⅱ protein expressions of cells in hypoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were decreased significantly (P<0.05 or P<0.01). After 6 hours of culture, the red fluorescence denoting LC3 protein expressions of cells was weak in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group, the red fluorescence of cells was significantly enhanced in hypoxia+ unloaded group, and the red fluorescence of cells was significantly inhibited in hypoxia+ BNIP3 knockdown group. Conclusions: BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration of HDMECs by BNIP3.

[Effects and mechanism of mitochondrial transcription factor A and cytochrome c oxidase pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1].

Objective: To investigate the effects and mechanism of mitochondrial transcription factor A (TFAM) and cytochrome c oxidase (COX) pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1 (TRAP1). Methods: The cardiomyocytes were isolated from 135 neonatal Sprague-Dawley rats (aged 1-3 d) and cultured for the following experiments. (1) Cells were collected and divided into normoxia blank control (NBC) group, hypoxia blank control (HBC) group, hypoxia+ TRAP1 over-expression control (HTOC) group, and hypoxia+ TRAP1 over-expression (HTO) group according to the random number table (the same grouping method below), with 1 bottle in each group. Cells in NBC group were cultured routinely, cells in HBC group were cultured in hypoxic condition for 6 hours after routine culture, cells in HTOC and HTO groups were respectively added with TRAP1 over-expression empty virus vector and TRAP1 over-expression adenovirus vector virus suspension for transfection for 48 hours after routine culture and then cultured in hypoxic condition for 6 hours. The protein expression of TFAM of cells in each group was detected by Western blotting. (2) Cells were collected and divided into NBC, HBC, HTOC, HTO, HTO+ TFAM interference control (HTOTIC), and HTO+ TFAM interference (HTOTI) groups, with 1 well in each group. Cells in the former 4 groups were dealt with the same methods as the corresponding groups in experiment (1). Cells in HTOTIC and HTOTI groups were respectively added with TFAM interference empty virus vector and TFAM interference adenovirus vector virus suspension for transfection for 48 hours, and the other processing methods were the same as those in HTO group. The content of ATP of cells in each group was determined by ATP determination kit and microplate reader, and the COX activity of cells in each group was determined by COX activity assay kit and microplate reader. (3) Cells were collected and divided into NBC group, normoxia+ sodium azide (NSA) group, HBC group, and hypoxia+ sodium azide (HSA) group, with 1 well in each group. Cells in NBC and HBC groups were respectively dealt with the same methods as the corresponding groups in experiment (1). Cells in NSA and HSA groups were respectively added with 32 nmol sodium azide at 30 min before experiment or hypoxia, and then cells in HSA group were cultured in hypoxic condition for 6 hours. The content of ATP was determined by the same method as above. The above three experiments were repeated for three times. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with that in NBC group, the protein expression of TFAM of cells in HBC group was significantly decreased (P<0.01). Compared with that in HBC group or HTOC group, the protein expression of TFAM of cells in HTO group was significantly increased (P<0.01). (2) Compared with 0.552±0.041 and 1.99±0.15 in NBC group, the COX activity (0.270±0.044) and ATP content (1.09±0.11) of cells in HBC group were significantly decreased (P<0.01). Compared with 0.269±0.042 and 1.17±0.12 in HBC group and those in HTOC group, the COX activity (0.412±0.032 and 0.404±0.016) and ATP content (1.75±0.06 and 1.69±0.07) of cells in HTO and HTOTIC groups were significantly increased (P<0.01). Compared with those in HTO and HTOTIC groups, the COX activity (0.261±0.036) and ATP content (1.23±0.07) of cells in HTOTI group were significantly decreased (P<0.01). (3) Compared with that in NBC group, the ATP content of cells in NSA and NBC groups was significantly decreased (P<0.01). Compared with that in HBC group, the ATP content of cells in HSA group was significantly decreased (P<0.01). Conclusions: TRAP1 can increase the COX activity of cardiomyocytes by raising the expression of TFAM, and finally alleviate the impairment in energy production of cardiomyocytes caused by hypoxia.

[CD38 dictates cardiac damage through mitochondrial apoptotic pathway under hypoxic-ischemic conditions].

Objective: To explore the mechanism of CD38-mediated cardiac damage under hypoxic-ischemic (H/I) conditions. Methods: Twenty CD38(-/-) male mice (8-week-old) and 20 wild-type (WT) male C57BL/6J mice (8-week-old) were randomly selected to construct the model of approximately 25% of the total body surface area (TBSA) burn injury. The cardiomyocytes (CMs) were separated from neonatal mice (1day) to construct the H/I injury model. Ad-CD38 adenovirus was transfected into CD38(-)/- primary CMs to callback CD38 expression. Animal experiments were grouped into WT-control group, CD38(-/-)-control group, WT-burn group, and CD38(-/-)-burn group (10 mice in each group). Primary CMs were divided into 6 groups: WT-normoxia group, CD38(-/-)-normoxia group, CD38(-/-)+Ad-CD38-normoxia group, WT-H/I group, CD38(-/-)-H/I group, CD38(-/-)+Ad-CD38-H/I group. The release of lactic dehydrogenase (LDH) from CMs and the cell viability were measured to estimate the level of myocardial injury. Ultrastructure of cardiomyocytes was examined by electron microscope. CD38 protein level and mitochondrial apoptosis-related proteins were detected by Western blot. Flow cytometry was used to detect mitochondrial reactive oxygen species (MitoSOX) of CMs under H/I condition. Cardiac function of mice was detected by ultrasonic apparatus. Results: (1) Animal experiments: The expression level of cardiac CD38 in WT-burn group was significantly higher than that in sham group (P<0.001). The heart function of CD38(-/-)-burn group was obviously better than WT-burn group [ejection fraction (EF)%: (84.70±2.31)% vs (76.10±2.96)%, shortening fraction (FS)%: (48.90±5.00)% vs (38.10±2.80)%] (both P<0.001). (2) Cell experiments: The expression level of cardiac CD38 in WT CMs under H/I condition was significantly higher than that in WT CMs under normoxia condition (P<0.05). The level of LDH, apoptotic cell and MitoSOX in CD38(-/-)-H/I group were fewer than WT-H/I group and CD38(-/-)+Ad-CD38(-)H/I group [(11.2±3.0)% vs (18.2±3.4)% and (17.6±4.0)%, (13.0±2.8)% vs (23.1±4.9)% and (23.3±6.0)%, (162±11)% vs (228±18)% and (220±18)%] (all P<0.001). The levels of cleaved-caspase3, Cytochrome-C in CD38(-/-)-H/I group were significantly lower than those in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (P<0.001). The cell viability in CD38(-/-)-H/I group was higher than that in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (0.355±0.043 vs 0.280±0.051 and 0.291±0.024) (all P<0.05). Electron microscopy results showed that structure of mitochondria in CD38(-/-)-H/I group was better than in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group. Conclusion: Overexpression of CD38 contributes to cardiac damage by stimulating mitochondrial apoptotic pathway.

[Effect and mechanism of cardiac adipose triglyceride lipase overexpression on burn-induced cardiac injury].

Objective: To explore the effect and potential mechanism of cardiac adipose triglyceride lipase (ATGL) overexpression on burn-induced cardiac injury. Methods: Eight-week-old C57BL/6J mice with cardiac ATGL overexpression driven by the myosin heavy chain (MHC) promoter (MHC-ATGL burn group) and wild-type (wild-type burn group) mice were randomly chose to the following experiments with burn injury after 24 h (n=8/group), MHC-ATGL mice and wild-type mice with corresponded age and sex were included as control. Cardiac ATGL protein expression, serum levels of cardiac troponin T and cardiac kinase-MB (CK-MB), cardiac free fatty acid and reactive oxygen species were detected. The wild-type and MHC-ATGL burn groups were not only compared with their corresponded control groups, but also compared between each other. Results: The hair color and development were shown little difference between each group. ATGL protein expression is elevated in wild-type burn group (1.00±0.68 vs 3.09±0.93, P=0.023) and decreased in MHC-ATGL burn group (17.84±2.41 vs 10.36±2.22, P<0.001), while ATGL protein expression is still increased in MHC-ATGL burn group compared with wild-type burn group (P<0.001). Serum levels of cardiac troponin T and CK-MB were both elevated in wild-type burn group and MHC-ATGL burn group [(0.456±0.131) vs (0.076±0.019) µg/L and (0.219±0.089) vs (0.060±0.019) µg/L, (1 421±162) vs (221±67) U/L and (761±142) vs (221±41) U/L] (all P<0.001), while serum levels of cardiac troponin T and CK-MB was still decreased in MHC-ATGL burn group compared with wild-type burn group (P<0.001). In addition, cardiac free fatty acid was increased in wild-type burn group and little difference was found in MHC-ATGL burn group [(2.54±0.51) vs (0.46±0.27) mmol/L, P<0.001, and (0.81±0.38) vs (0.59±0.25) mmol/L, P=0.251], while cardiac free fatty acid was significant reduction in MHC-ATGL burn group compared with wild-type burn group (P<0.001). Levels of cardiac reactive oxygen species was both elevated in wild-type burn group and MHC-ATGL burn group [(1.89±0.23) vs (1.00±0.18) and (1.38±0.17) vs (0.95±0.13)] (both P<0.001), while levels of cardiac reactive oxygen was reduction in MHC-ATGL burn group compared with wild-type burn group (P<0.001). Conclusion: Cardiac ATGL overexpression may protect against burn-induced cardiac injury through reducing free fatty acid and reactive oxygen species production.

[In vitro study of the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes].

Objective: To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro. Methods: The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased (t=12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P<0.05 or P<0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased (t=6.88, 10.56, 5.76, 9.91, P<0.05 or P<0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased (t=6.01, P<0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups (F=1.09, P>0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups (t=43.05, 11.07, 5.39, P<0.05 or P<0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups (t=11.18, 12.71, P<0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased (t=4.82, 4.44, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased (t=4.39, 6.27, P<0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group (t=10.13, P<0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=3.28, P<0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group (t=4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P<0.05 or P<0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased (t=5.51, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=5.97, P<0.05). Conclusions: After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.

[In vitro study of effects of transient receptor potential vanilloid 1 on autophagy in early hypoxic mouse cardiomyocytes and the mechanism].

Objective: To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro. Methods: The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco' s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 µmol/L capsaicin group, hypoxia+ 1.0 µmol/L capsaicin group, and hypoxia+ 10.0 µmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 µmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 µmol/L chloroquine, 10.0 µmol/L capsaicin, and 50 µmol/L chloroquine+ 10.0 µmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 µmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 µmol/L capsaicin group were treated with 10.0 µmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t(3 h)=4.891, 5.890, 4.928; t(6 h)=9.790, 6.750, 10.590; t(9 h)=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 µmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 µmol/L capsaicin group and hypoxia+ 10.0 µmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 µmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 µmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01). Conclusions: TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.

[Role of hexokinase â ¡ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro].

Objective: To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro. Methods: The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments. (1) The cells were divided into 6 groups according to the random number table (the same grouping method below), i. e., normal control 3, 6, and 9 h groups and ischemia-hypoxia 3, 6, and 9 h groups, with 4 wells in each group. After being regularly cultured for 48 h with Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), the cells in normal control 3, 6, and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3, 6, and 9 h, respectively, and the cells in ischemia-hypoxia 3, 6, and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3, 6, and 9 h, respectively. Cell viability was measured by the cell counting kit 8 (CCK-8) method. (2) The cells were grouped and treated the same as those in experiment (1), with 1 well in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3Ⅱ, p62, and hexokinase Ⅱ. (3) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, and ischemia-hypoxia 9 h+ 2-deoxyglucose (2-DG) group, with 4 wells in each group. After a regular culture for 48 h, the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h; the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium, and the cells in ischemia-hypoxia 9 h+ 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 µmol), and then they were cultured with hypoxia for 9 h. Cell viability was measured by CCK-8 method. (4) The cells were grouped and treated the same as those in experiment (3), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, and p62. (5) The cells were grouped and treated the same as those in experiment (3), with 2 wells in each group. Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes. (6) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ hexosinase Ⅱ small interfering RNA1 (HK-ⅡsiRNA1) group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group, with 4 wells in each group. The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h, and the cells in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then also cultured for 48 h. The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h, and the cells in simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h. Cell viability was measured by CCK-8 method. (7) The cells were grouped and treated the same as those in experiment (6), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, p62, and hexokinase Ⅱ. Except for experiment (5), each experiment was repeated 3 times. Data were processed with one-way analysis of variance and lest significant difference t test, and Bonferroni correction. Results: (1) The viabilities of cardiomyocytes in ischemia-hypoxia 3, 6, and 9 h groups were 0.450±0.022, 0.385±0.010, and 0.335±0.015, respectively, which were significantly lower than 0.662±0.026, 0.656±0.028, and 0.661±0.021 of the corresponding normal control 3, 6, and 9 h groups, respectively (t=6.21, 9.12, 12.48, P<0.01). (2) Compared with those of corresponding normal control 3, 6, and 9 h groups, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3, 6, and 9 h groups were significantly increased (t(3 h)=16.15, 10.99, 5.30, t(6 h)=6.79, 10.42, 9.42, t(9 h)=15.76, 16.51, 7.20, P<0.05 or P<0.01). (3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353±0.022, which was significantly lower than 0.673±0.027 of normal control group (t=9.29, P<0.01). The viability of cardiomyocytes in ischemia-hypoxia 9 h+ 2-DG group was 0.472±0.025, which was significantly higher than that of simple ischemia-hypoxia 9 h group (t=3.60, P<0.05). (4) Compared with those of normal control group, the LC3Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=9.45, 8.40, P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group were significantly decreased (t=4.39, 4.74, P<0.05). (5) In cardiomyocytes of normal control group, only single autophagosome/autolysosome with bilayer membrane structure was observed. Compared with that of normal control group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly. Compared with that of simple ischemia-hypoxia 9 h group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group was significantly decreased. (6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358±0.023, which was significantly lower than 0.673±0.026 in normal control group (t=9.12, P<0.01). The viabilities of cardiomyocytes in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were 0.487±0.027 and 0.493±0.022, respectively, which were significantly higher than the viability in simple ischemia-hypoxia 9 h group (t=3.63, 4.28, P<0.05). (7) Compared with those of normal control group, the LC3Ⅱ/Ⅰratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=6.08, 6.31, 4.83, P<0.05 or P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were significantly decreased (t=5.10, 7.76, 15.33, 4.17, 8.42, 12.11, P<0.05 or P<0.01). Conclusions: Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro, which decreases the viability of cardiomyocytes by impairing autophagic flow. To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.

[Effects of change in the activity of vacuolar adenosine triphosphatase of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism].

Objective: To explore the effects of change of activity of vacuolar adenosine triphosphatase (V-ATPase) of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism. Methods: The myocardial lysosomes were extracted from the hearts of 12 SD rats with ultra-high speed gradient density centrifugation, then Western blotting and transmission electron microscope observation were conducted for identification. One hundred and twenty rats were divided into pure burn group, ATP group, normal control group, and bafilomycin group according to the random number table, with 30 rats in each group. Rats in pure burn group and ATP group were inflicted with 40% TBSA full-thickness scald on the back. Immediately after injury, rats in pure burn group were intraperitoneally injected with lactated Ringer's solution in 4 mL·%TBSA(-1)·kg(-1,) and rats in ATP group were intraperitoneally injected with ATP in 0.4 mg/kg at 12 h before burn, immediately after burn, and 12 h after burn. Rats in normal control group did not receive any treatment, and rats in bafilomycin group were intraperitoneally injected with bafilomycin A1 in 0.3 mg/kg at the same time points as those of ATP group. At 24 h after burn, 30 rats from each group were collected for determining activity of V-ATPase of myocardial lysosome with coupled-enzyme assay and the expression of myocardium autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and P62 by Western blotting. Left ventricular arterial blood was collected to detect the content of 5 items of myocardial enzyme spectrum and cardiac troponin T (cTnT). Data were processed with one-way analysis of variance and t test. Results: (1) After identification, both the expression level of lysosome-related membrane protein 1 and purity of lysosome in the sample were high, and the structure of lysosome was intact. (2) At 24 h after burn, the activity values of V-ATPase of myocardial lysosome in rats of pure burn group, ATP group, normal control group, and bafilomycin group were (2.03±0.67), (3.01±0.58), (4.29±0.26), and (1.83±0.52) µmol·mg(-1)·h(-1,) respectively. The activity value of V-ATPase of myocardial lysosome in rats of pure burn group was significantly lower than the values in ATP group and normal control group (with t values respectively 3.14 and 8.87, P values below 0.01). The activity values of V-ATPase of rats in normal control group were significantly higher than those in bafilomycin group (t=11.87, P<0.01). At 24 h after burn, the expressions of myocardial LC3 and P62 in pure burn group were significantly higher than those in ATP group and normal control group (with t values from 3.73 to 5.88, P values below 0.01). The expressions of myocardial LC3 and P62 in normal control group were significantly lower than those in bafilomycin group (with t values respectively 2.64 and 3.07, P<0.05 or P<0.01). At 24 h after burn, the content of 5 items of myocardial enzyme spectrum and cTnT in pure burn group was significantly higher than that in ATP group and normal control group (with t values from 3.24 to 16.72, P values below 0.01). The content of 5 items of myocardial enzyme spectrum and cTnT in normal control group was significantly lower than that in bafilomycin group (with t values from 2.39 to 10. 70, P values below 0.01). Conclusions: The activity of V-ATPase of myocardial lysosome decreased in rats after severe burn, which can result in myocardial damage by inhibiting myocardial autophagy flux.

[Effect of rapamycin on the migration of human epidermal cell line HaCaT and its possible molecular mechanism].

OBJECTIVE: To explore the effects of rapamycin on the migration of human epidermal cell line HaCaT, and to analyze its molecular mechanism. METHODS: HaCaT cells were conventionally cultured with RPMI 1640 culture medium containing 10% fetal calf serum (hereinafter referred to as culture medium). (1) According to the random number table, HaCaT cells in logarithmic phase were divided into control group and 1, 5, 50, 100, 200 nmol/L rapamycin groups, with 6 wells in each group. The cells in rapamycin groups were cultured with culture medium containing rapamycin in corresponding mass concentration, and the cells in control group were cultured with culture medium containing dimethyl sulfoxide (DMSO) instead. After being conventionally cultured for 4 hours, proliferative activity of cells was determined with microplate reader (denoted as absorbance value). (2) HaCaT cells in logarithmic phase were grouped and cultured as that in experiment (1), with 1 well in each group. After being conventionally cultured for 4 hours, range of movement of cells in 3 hours was observed under live cell imaging workstation, and their curvilinear movement speeds were calculated. Then the suitable concentration of rapamycin was selected for experiments (3) and (4). (3) HaCaT cells in logarithmic phase were divided into control group and rapamycin group according to the random number table, with 1 well in each group. The cells in rapamycin group were cultured with culture medium containing 50 nmol/L rapamycin, and the cells in control group were cultured with culture medium containing DMSO. After being conventionally cultured for 4 hours, cells were collected for scratch assay. Wound area was observed at post scratching hour (PSH) 0, 5, 10, and 15, and the migration rates of cells at PSH 5, 10, and 15 were calculated respectively. (4) HaCaT cells in logarithmic phase were grouped and cultured as that in experiment (3), with 1 well in each group. Activity of focal adhesion kinase (FAK) was determined with Western blotting (denoted as the ratio of gray value of phosphorylated FAK to that of FAK). Above-mentioned experiments were independently repeated for three or five times. Data were processed with one-way analysis of variance, LSD test, and t test. RESULTS: (1) Proliferative activity of cells in control group and 1, 5, 50, 100, 200 nmol/L rapamycin groups was respectively 1.22±0.28, 1.29±0.38, 1.12±0.27, 1.20±0.29, 1.15±0.30, 1.39±0.40, without statistically significant differences among these groups (F=2.112, P=0.068). (2) The ranges of movement of cells in 1, 5 nmol/L rapamycin groups were similar to the range of movement of cells in control group, while those of cells in 50, 100, 200 nmol/L rapamycin groups were obviously smaller than the range of movement of cells in control group. There were statistically significant differences in cell curvilinear movement speeds among the 6 groups (F=3.525, P=0.004). The curvilinear movement speeds of cells in 1, 5 nmol/L rapamycin groups were respectively (0.8±0.4) and (0.8±0.8) µm/min, and they were similar to the curvilinear movement speed of cells in control group [(0.9±0.5) µm/min, with P values above 0.05]. The curvilinear movement speeds of cells in 50, 100, 200 nmol/L rapamycin groups were respectively (0.7±0.5), (0.7±0.4), (0.7±0.4) µm/min, and they were significantly lower than the curvilinear movement speed of cells in control group (with P values below 0.01). Thus, 50 nmol/L rapamycin was selected for experiments (3) and (4). (3) Compared with those of control group, wound areas of rapamycin group showed no obvious change at PSH 0 and 5, while they were obviously increased at PSH 10 and 15. At PSH 5, migration rate of cells in control group [(17.5±2.6)%] was similar to that in rapamycin group [(15.8±3.5)%, t=1.951, P>0.05]. Migration rates of cells of rapamycin group at PSH 10 and 15 [(42.5±4.0)% and (71.3±9.2)%, respectively] were obviously decreased as compared with those of control group [(46.9±6.7)% and (88.0±7.7)%, with t values respectively 2.732 and 6.746, P values below 0.01]. (4) Compared with that in control group (0.46±0.14), FAK activity of cells in rapamycin group (0.16±0.08) was significantly down-regulated (t=4.967, P<0.01). CONCLUSIONS: FAK signal pathway is sensitive to rapamycin in HaCaT cells. Inhibition effects of rapamycin on migration of HaCaT cells may be mediated by down-regulated activity of FAK.

Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.).

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F1 plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) x UR206. Hybrid seeds were produced through backcrossing F1 with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F2 individuals derived from a cross UR206 x UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.

One hundred and one new microsatellite loci derived from ESTs (EST-SSRs) in bread wheat.

Four hundred and seventy-eight microsatellite markers derived from expressed sequence tags (EST-SSRs) were screened among three mapping populations (W-7984xOpata 85, WOpop; LumaixHanxuan, LHpop; WenmaixShanhongmai, WSpop). The number of polymorphic EST-SSR primer pairs found in WOpop, LHpop and WSpop was 92, 58 and 29 respectively. A total of 101 EST-SSR loci amplified from 88 primer sets were distributed over the 20 chromosomes of the reference maps (no markers were located on chromosome 4B). These 101 mapped EST-SSR markers add to the existing 450 microsatellite loci previously mapped in bread wheat. Seventy-four of the 101 loci showed significant similarities to known genes, including 24 genes involved in metabolism, 4 in cellular structures, 9 in stress resistance, 12 in transcription, 2 in development, 2 transporters and 21 storage proteins. Besides gliadin and glutenin, most of the 53 genes with putative functions were mapped for the first time by EST-SSR markers in bread wheat. Sequence alignment of the mapped wheat EST-SSR loci allowed tentative assignment of functionality to the other members of grasses family. Colinearity combined with homology information offers an attractive approach to comparative genomics.

Assessment of population genetic structure in common wild rice Oryza rufipogon Griff. using microsatellite and allozyme markers.

The genetic structure of five natural populations of common wild rice Oryza rufipogon Griff. from China, was investigated with 21 microsatellite loci and compared to estimates of genetic diversity and genetic differentiation detected by 22 allozyme loci. Microsatellite loci, as expected, have much higher levels of genetic diversity (mean values of A = 3.1, P = 73.3%, Ho = 0.358 and He = 0.345) than allozyme loci (mean values of A = 1.2, P = 12.7%, Ho = 0.020 and He = 0.030). Genetic differentiation detected by microsatellite loci ( FST = 0.468, mean I = 0.472) was higher than that for allozyme loci ( FST =0.388, mean I = 0.976). However, microsatellite markers showed less deviation from Hardy-Weinberg expectation (Wright's inbreeding coefficient FIS = -0.069) than do allozymes ( FIS = 0.337). These results suggest that microsatellite markers are powerful high-resolution tools for the accurate assessment of important parameters in population biology and conservation genetics of O. rufipogon, and offer advantages over allozyme markers.

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat.

Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.