[A prospective study on application of human umbilical cord mesenchymal stem cells combined with autologous Meek microskin transplantation in patients with extensive burns].

Objective: To investigate the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) combined with autologous Meek microskin transplantation on patients with extensive burns. Methods: The prospective self-controlled study was conducted. From May 2019 to June 2022, 16 patients with extensive burns admitted to the 990th Hospital of PLA Joint Logistics Support Force met the inclusion criteria, while 3 patients were excluded according to the exclusion criteria, and 13 patients were finally selected, including 10 males and 3 females, aged 24-61 (42±13) years. A total of 20 trial areas (40 wounds, with area of 10 cm×10 cm in each wound) were selected. Two adjacent wounds in each trial area were divided into hUCMSC+gel group applied with hyaluronic acid gel containing hUCMSCs and gel only group applied with hyaluronic acid gel only according to the random number table, with 20 wounds in each group. Afterwards the wounds in two groups were transplanted with autologous Meek microskin grafts with an extension ratio of 1∶6. In 2, 3, and 4 weeks post operation, the wound healing was observed, the wound healing rate was calculated, and the wound healing time was recorded. The specimen of wound secretion was collected for microorganism culture if there was purulent secretion on the wound post operation. In 3, 6, and 12 months post operation, the scar hyperplasia in wound was assessed using the Vancouver scar scale (VSS). In 3 months post operation, the wound tissue was collected for hematoxylin-eosin (HE) staining to observe the morphological changes and for immunohistochemical staining to observe the positive expressions of Ki67 and vimentin and to count the number of positive cells. Data were statistically analyzed with paired samples t test and Bonferronni correction. Results: In 2, 3, and 4 weeks post operation, the wound healing rates in hUCMSC+gel group were (80±11)%, (84±12)%, and (92±9)%, respectively, which were significantly higher than (67±18)%, (74±21)%, and (84±16)% in gel only group (with t values of 4.01, 3.52, and 3.66, respectively, P<0.05). The wound healing time in hUCMSC+gel group was (31±11) d, which was significantly shorter than (36±13) d in gel only group (t=-3.68, P<0.05). The microbiological culture of the postoperative wound secretion specimens from the adjacent wounds in 2 groups was identical, with negative results in 4 trial areas and positive results in 16 trial areas. In 3, 6, and 12 months post operation, the VSS scores of wounds in gel only group were 7.8±1.9, 6.7±2.1, and 5.4±1.6, which were significantly higher than 6.8±1.8, 5.6±1.6, and 4.0±1.4 in hUCMSC+gel group, respectively (with t values of -4.79, -4.37, and -5.47, respectively, P<0.05). In 3 months post operation, HE staining showed an increase in epidermal layer thickness and epidermal crest in wound in hUCMSC+gel group compared with those in gel only group, and immunohistochemical staining showed a significant increase in the number of Ki67 positive cells in wound in hUCMSC+gel group compared with those in gel only group (t=4.39, P<0.05), with no statistically significant difference in the number of vimentin positive cells in wound between the 2 groups (P>0.05). Conclusions: The application of hyaluronic acid gel containing hUCMSCs to the wound is simple to perform and is therefore a preferable route. Topical application of hUCMSCs can promote healing of the autologous Meek microskin grafted area in patients with extensive burns, shorten wound healing time, and alleviate scar hyperplasia. The above effects may be related to the increased epidermal thickness and epidermal crest, and active cell proliferation.

[Role of enolase 1 in hepatocellular carcinoma and possible mechanism].

Objective: To investigate the role of enolase 1 (ENO1) in hepatocellular carcinoma (HCC) and possible mechanism. Methods: Real-time PCR and Western blot were used to measure the expression of ENO1 in HCC tissue, adjacent tissue, hepatoma cells, and normal hepatocytes. The siRNA interference technique was used for ENO1 knockout in HepG2 cells, and then CCK-8, colony formation assay, and transwell assay were used to measure the proliferation, migration, and invasion abilities of HepG2 cells. Real-time PCR and Western blot were used to measure the expression of proteins and genes involved in the activation of the Notch signaling pathway. The two-independent-samples t test and a one-way analysis of variance were used for comparison. Results: HCC tissue and HepG2 cells had significantly higher expression of ENO1 than adjacent tissue and normal hepatocytes (P < 0.05). There were significant reductions in the proliferation, migration, and invasion abilities of HepG2 cells after siRNA interference (P < 0.05). There were also significant reductions in the expression of N1ICD, snail, slug, HEY1, HES1, and HES5 (P < 0.05). Conclusion: ENO1 may promote the development of HCC, possibly by participating in the regulation of the Notch signaling pathway.

[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.

[Effects and mechanism of allogeneic platelet rich plasma on collagen synthesis in wound healing].

Objective: To investigate the effects and mechanism of allogeneic platelet rich plasma (PRP) on collagen in wound surface at different time. Methods: A total of 50 clean 7-week rats were selected for this study, including 10 rats for platelet-rich blood plasma preparation, 20 rats for PRP group and 20 rats for control group, 0.1 ml allogenic PRP and 0.1 ml saline were smeared respectively on wound surfaces of PRP and control group, wound regeneration and healing were examined. Cellular and histological morphology alteration was observed via Masson staining, type Ⅰ and type Ⅲ collagen protein and mRNA expression level were detected by Western blot and real-time PCR. T test was applied for comparison between two samples and one-way ANOVA was utilized for comparison between two groups. Results: The wound healing rate of PRP group was higher than that of control group on 3(rd,) 6(th,) 10(th) and 15(th) day (30.33±3.35 vs.18.35±2.04, 55.51±2.74 vs.36.83±2.34, 79.64±1.40 vs.56.92±1.44, 86.88±2.12 vs.65.80±1.76) after wound surface formation, there were statistic differences (t=13.66-50.48, all P<0.05). The wound collagen of PRP group form faster and coarser, and the fibers arrayed more densely in Masson staining. The protein expression of type Ⅰ collagen(1.92±0.09 vs.1.18±0.11) and type Ⅲ collagen(1.16±0.05 vs.0.74±0.11) of PRP group were higher than that of control group (t=22.99, P<0.01; t=17.62, P<0.05); the mRNA expression of type Ⅰ collagen(5.17±0.11 vs.1.79±0.18, 6.97±0.09 vs.1.96±0.08, 6.00±0.26 vs.2.10±0.05, 4.95±0.11 vs.3.58±0.09)and type Ⅲ collagen(2.35±0.08 vs.1.44±0.05, 3.08±0.05 vs.1.84±0.06, 3.48±0.07 vs.2.36±0.09, 4.42±0.07 vs.2.77±0.10) were higher than that of control group on 3(rd,) 6(th,) 10(th) and 15(th) day after wound surface formation, there were significant differences (t=43.37-188.37, all P<0.05). Conclusion: The allogeneic platelet rich plasma may promote fibroblasts secreted collagen by activated and releasing all kinds of growth factors, especially type Ⅰ and type Ⅲ collagen to accelerate the wound healing.

[The role of autophagy in the curcumin induced proliferation in human laryngeal cancer Hep2 cell].

Objective:The aim of this study is to study the effect of 3-methyladenine (3-MA) on the autophagy and apoptosis of human laryngeal cancer Hep 2 cells induced by curcumin. Method:The proliferation of human laryngeal cancer Hep2 cells was observed by MTT assay. The autophagy level was detected by AO acridine orange staining. Annexin VFITC/PI double staining was used to detect the apoptosis of Hep2 cells. The expression of LC3, Beclin1, Bcl-2 and Bax protein were detected by Western blot. Result:MTT assay showed that curcumin inhibited the proliferation of Hep2 cells in a dose/time dependent manner. The apoptosis rate of curcumin combined with 3-MA increased (P<0.05). Acridine orange staining showed that 3-MA combined with curcumin could significantly reduce the autophagy level of laryngeal carcinoma Hep2 cells. The expression of Bcl-2, Bclin-1 and LC3 Ⅱ was decreased, while the expression of Bax protein was increased (P<0.05). Conclusion:Curcumin can induce apoptosis of Hep 2 cells and induce the development of protective autophagy. The inhibitory effect of curcumin on the apoptosis of laryngeal carcinoma Hep2 cell line was significantly enhanced by 3-MA.

Downregulation of miR-181a upregulates sirtuin-1 (SIRT1) and improves hepatic insulin sensitivity.

AIMS/HYPOTHESIS: Sirtuin-1 (SIRT1) is a potential therapeutic target to combat insulin resistance and type 2 diabetes. This study aims to identify a microRNA (miRNA) targeting SIRT1 to regulate hepatic insulin sensitivity. METHODS: Luciferase assay combined with mutation and immunoblotting was used to screen and verify the bioinformatically predicted miRNAs. miRNA and mRNA levels were measured by real-time PCR. Insulin signalling was detected by immunoblotting and glycogen synthesis. Involvement of SIRT1 was studied with adenovirus, inhibitor and SIRT1-deficient hepatocytes. The role of miR-181a in vivo was explored with adenovirus and locked nucleic acid antisense oligonucleotides. RESULTS: miR-181a targets the 3' untranslated region (3'UTR) of Sirt1 mRNA through a miR-181a binding site, and downregulates SIRT1 protein abundance at the translational level. miR-181a is increased in insulin-resistant cultured hepatocytes and liver, and in the serum of diabetic patients. Overexpression of miR-181a decreases SIRT1 protein levels and activity, and causes insulin resistance in hepatic cells. Inhibition of miR-181a by antisense oligonucleotides increases SIRT1 protein levels and activity, and improves insulin sensitivity in hepatocytes. Ectopic expression of SIRT1 abrogates the effect of miR-181a on insulin sensitivity, and inhibition of SIRT1 activity or SIRT1 deficiency markedly attenuated the improvement in insulin sensitivity induced by antisense miR-181a. In addition, overexpression of miR-181a by adenovirus impairs hepatic insulin signalling, and intraperitoneal injection of locked nucleic acid antisense oligonucleotides for miR-181a improves glucose homeostasis in diet-induced obesity mice. CONCLUSIONS/INTERPRETATION: miR-181a regulates SIRT1 and improves hepatic insulin sensitivity. Inhibition of miR-181a might be a potential new strategy for treating insulin resistance and type 2 diabetes.