Sema3A secreted by sensory nerve induces bone formation under mechanical loads / 国际口腔科学杂志·英文版

Bone formation and deposition are initiated by sensory nerve infiltration in adaptive bone remodeling. Here, we focused on the role of Semaphorin 3A (Sema3A), expressed by sensory nerves, in mechanical loads-induced bone formation and nerve withdrawal using orthodontic tooth movement (OTM) model. Firstly, bone formation was activated after the 3rd day of OTM, coinciding with a decrease in sensory nerves and an increase in pain threshold. Sema3A, rather than nerve growth factor (NGF), highly expressed in both trigeminal ganglion and the axons of periodontal ligament following the 3rd day of OTM. Moreover, in vitro mechanical loads upregulated Sema3A in neurons instead of in human periodontal ligament cells (hPDLCs) within 24 hours. Furthermore, exogenous Sema3A restored the suppressed alveolar bone formation and the osteogenic differentiation of hPDLCs induced by mechanical overload. Mechanistically, Sema3A prevented overstretching of F-actin induced by mechanical overload through ROCK2 pathway, maintaining mitochondrial dynamics as mitochondrial fusion. Therefore, Sema3A exhibits dual therapeutic effects in mechanical loads-induced bone formation, both as a pain-sensitive analgesic and a positive regulator for bone formation.

Influence of long non-coding RNA plasmacytoma variant translocation 1 in cell proliferation, invasion and glucose transporter 3 expression in glioma / 中华神经医学杂志

Objective:To study the influence of long non-coding RNA (LncRNA) plasmacytoma variant translocation 1 (PVT1) in glucose transporter 3 (GLUT3) expression, and cell proliferation and invasion in glioma.Methods:(1) The correlation between PVT1 and GLUT3 gene expressions and their influences in overall survival (OS) were analyzed using data from 222 cases of primary gliomas from Chinese Glioma Genome Atlas mRNAseq_325 data set. (2) Fifteen glioma specimens, including 8 from patients with low-grade glioma (LGG group) and 7 from patients with glioblastoma (GBM group), were collected in our hospital from January 2019 to December 2019; the PVT1 expression was detected by fluorescence in situ hybridization (FISH); the GLUT3 protein expression was detected by immunohistochemistry. (3) Normal human astrocyte (NHA) and glioblastoma cell lines U87, LN229 and U251 (NHA group, U87 group, ln229 group and U251 group) were cultured in vitro; real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the PVT1 and GLUT3 mRNA expressions; Western blotting was used to detect the GLUT3 protein expression; U87 and LN229 cells were divided into PVT1 overexpression plasmid group and blank plasmid group, PVT1 short hairpin RNA (shRNA) group and negative control shRNA group; the GLUT3 mRNA and protein expressions were detected by RT-qPCR and Western blotting. (4) In U87 and LN229 cells of negative control shRNA group and PVT1 shRNA group, CCK-8 assay and colony formation assay were used to detect the cell proliferation and Transwell assay was used to detect the cell invasion. (5) Ten female BALB/c-nu nude mice were randomly divided into experimental group and control group ( n=5); the U87 cells from PVT1 shRNA group and negative control shRNA group were transplanted into the mice to establish subcutaneously transplanted tumor models. The animals were sacrificed and the tumors were weighed and measured 4 weeks after transplantation; the Ki-67 and GLUT3 protein expressions were detected by immunohistochemistry. Results:(1) The gene expressions of PVT1 and GLUT3 were positively correlated in the 222 cases of primary glioma from mRNAseq_325 data set ( r=0.514, P=0.000); the OS of patients in the PVT1 high-expression group or GLUT3 high-expression group was significantly shorter as compared with that in the PVT1 low-expression group or GLUT3 low-expression group, respectively ( P<0.05). (2) As compared with the low-grade glioma group, the glioblastoma group had significantly increased PVT1 and GLUT3 protein expressions ( P<0.05). (3) As compared with NHA cells, the U87, LN229 and U251 cells had significantly increased PVT1 and GLUT3 mRNA and protein expressions ( P<0.05). As compared with those in the blank plasmid group, the GLUT3 mRNA and protein expressions were significantly increased in the U87 and LN229 cells of PVT1 overexpression plasmid group ( P<0.05); as compared with those in the negative control shRNA group, the GLUT3 mRNA and protein expressions were significantly decreased in the U87 and Ln229 cells of PVT1 shRNA group ( P<0.05). (4) As compared with negative control shRNA group, PVT1 shRNA group had significantly reduced optical density value, significantly smaller numbers of clone formation and invasive cells in U87 and LN229 cells ( P<0.05). (5) As compared with those in the control group, the subcutaneous transplanted tumor volume was significantly smaller, the subcutaneous transplanted tumor mass and Ki-67 and GLUT3 protein expressions were significantly lower in the experimental group ( P<0.05). Conclusion:Down-regulation of PVT1 can decrease the GLUT3 expression, therefore, inhibit the proliferation and invasion of glioma cells.

Predictive value of visceral fat area for multiple metabolic risk factors in obese patients undergoing bariatric surgery / 中华消化外科杂志

Objective:To investigate the predictive value of visceral fat area (VFA) on multiple metabolic risk factors in obese patients undergoing bariatric surgery.Methods:The retrospective case-control study was conducted. The clinical data of 146 obese patients undergoing bariatric surgery in the West China Hospital of Sichuan University from June 2015 to May 2020 were collected. There were 57 males and 89 females, aged (33±9)years, with a range from 15 to 65 years. All patients underwent multi-slice spiral computed tomography (CT) examination, and the total fat area (TFA), VFA and subcutaneous fat area (SFA) were measured. Observation indicators: (1) CT findings of abdominal fat area and patients with multiple metabolic risk factors; (2) receiver operator characteristic(ROC) curve of different clinical indicators for predicting multiple metabolic risk factors; (3) relationship between different VFA and clinicopathological features in patients; (4) analysis of factors influencing multiple metabolic risk factors in patients. Measurement data with normal distribution were expressed as Mean± SD, and comparison between groups was analyzed by independent sample t test. Measurement data with skewed distribution were expressed as M (range) and comparison between groups was analyzed by nonparametric rank sum test. Count data were represented as absolute numbers, and comparison between groups was analyzed using the chi-square test. The ROC curve was used to analyze the predictive value of different clinical indicators for multiple metabolic risk factors in patients. Logistic regression analysis was used for univariate and multivariate analysis. Results:(1) CT findings of abdominal fat area and patients with multiple metabolic risk factors. CT findings of 146 patients showed that VFA was (212±122)cm 2, SFA was (419±147)cm 2, visceral-to-subcutaneous fat ratio (VSR) was 0.60±0.54. The levels of systolic blood pressure (SBP), diastolic blood pressure, fasting blood glucose (FBG), triglyceride and highdensity lipoprotein cholesterol (HDL-C) were (131±16)mmHg(1 mmHg=0.133 kPa), (86±12)mmHg, (6.4±2.5)mmol / L, 4.43 mmol /L(range, 1.23-9.99 mmol/L), (1.5±1.3)mmol/L, respectively. Among the 146 patients, 85 had impaired FBG or diabetes, 82 had hypertension, 139 had high triglyceride, 91 had low serum HDL-C; 128 had multiple metabolic risk factors. (2) ROC curve analysis of different clinical indicators for predicting multiple metabolic risk factors: the ROC curve showed that VFA had better ability to predict or diagnose multiple metabolic risk factors in patients ( hazard ratio=0.617, 95% confidence interval as 0.470-0.764, P<0.05). The cutoff value of VFA was 163.52 cm 2 by calculating Yoden index in ROC curve. (3) Relationship between different VFA and clinicopathological features in patients: according to the cutoff value of 163.52 cm 2 in ROC curve, 146 patients were divided into high VFA group (≥163.52 cm 2) and the low VFA group (<163.52 cm 2), with 49 cases and 97 cases respectively. Cases with hypertension, level of SBP, cases with impaired FBG or diabetes, cases with multiple metabolic risk factors were 64, (134±17)mmHg, 63, 90 for the high VFA group, versus 18, (127±13)mmHg, 22, 38 for the low VFA group; there were significant differences in above indicators between the two groups ( χ2=11.309, t=6.916, χ2=5.380, 6.988, P<0.05). (4) Analysis of factors influencing multiple metabolic risk factors in patients: results of univariate analysis showed that VFA was a related factor for multiple metabolic risk factors in patients ( hazard ratio=3.722, 95% confidence interval as 1.341-10.328, P<0.05). Results of multivariate analysis showed that the VFA≥163.52 cm 2 was an independent rsik factcor or multiple metabolic risk factors in patients ( hazard ratio=5.182, 95% confidence interval as 1.441-18.641, P<0.05). Conclusions:VFA is positively correlated with hypertension, systolic blood pressure, impaired FBG and diabetes. VFA≥163.52 cm 2 is an independent predictor for multiple metabolic risk factors in obese patients undergoing bariatric surgery.

Effects of T-2 toxin on expression of hepatocyte growth factor and its receptor in articular cartilage and epiphyseal cartilage of rats under selenium deficiency / 中华地方病学杂志

Objective:To observe the effects of T-2 toxin on expression of hepatocyte growth factor (HGF) and HGF receptor (C-Met) in articular cartilage and epiphyseal cartilage of rats under low selenium condition.Methods:Twenty-four healthy male SD rats weighted 60-80 g were randomly divided into conventional diet group (selenium content of 101.5 μg/kg) and low-selenium diet group (selenium content of 1.1 μg/kg), with 12 rats in each group. After 30 days of feeding, the conventional diet group was further divided into conventional group and T-2 toxin group (100 μg·kg -1·d -1), and the low-selenium diet group was further divided into low-selenium group and low-selenium+T-2 toxin group (100 μg·kg -1·d -1), with 6 rats in each group. After 30 days of feeding, the rats were sacrificed and the cartilage of knee joint was taken, the morphological changes of knee articular cartilage and epiphyseal cartilage were observed by HE staining under light microscope. Immunohistochemical method was used to detect the expression of HGF and C-Met in knee articular cartilage and epiphyseal cartilage, and positive expression rates of HGF and C-Met were calculated. Results:Under light microscope, chondrocytes of articular cartilage and epiphyseal cartilage in low-selenium+T-2 toxin group were sparse, and the necrosis and structural area were found in the deep layer, and the extracellular matrix of chondrocytes in the region was degraded and light stained, and proliferating granulation tissue was visible nearby. The positive expression rates of HGF in articular cartilage [(21.97 ± 6.90)%, (49.41 ± 8.24)%, (76.39 ± 5.88)%] and epiphyseal cartilage [(23.36 ± 12.49)%, (58.43 ± 14.48)%, (66.59 ± 10.83)%] of rats in low-selenium, T-2 toxin and low-selenium+T-2 toxin groups were higher than those in conventional group [(9.13 ± 6.01)%, (11.14 ± 4.67)%, P < 0.05]. The positive expression rates of C-Met in articular cartilage [(25.34 ± 7.53)%, (58.21 ± 12.54)%, (81.46 ± 7.89)%] and epiphyseal cartilage [(35.21 ± 4.71)%, (40.84 ± 2.03)%, (49.41 ± 6.29)%] of rats in low-selenium, T-2 toxin and low-selenium+T-2 toxin groups were higher than those in conventional group [(11.21 ± 5.11)%, (12.12 ± 4.71)%, P < 0.05]. Conclusion:T-2 toxin may affect the expression of HGF and C-Met in articular cartilage and epiphyseal cartilage of rats under low selenium condition.

Relationship between insulin-like growth factor-1 receptor down-regulation and Kaschin-Beck disease / 中华地方病学杂志

Objective To observe the expression level of insulin-like growth factor-1 receptor (IGF-1R) in the cartilage tissue of children with Kaschin-Beck disease (KBD) and T-2 toxin-poisoned rats under low selenium condition,and the effect of IGF-1R inhibitor on apoptosis of human normal chondrocytes (C28/I2 cells),and to investigate the role of IGF-1R in the pathogenesis of KBD.Methods The knuckles of dead children (5 cases) in the KBD areas,car accident death and congenital 6 finger deformity operation children (5 cases) in non-KBD areas in Shaanxi were collected,the expression of IGF-1R in the articular cartilage was detected by immunohistochemistry.Thirty-two male Sprague-Dawley rats with a body mass of 60-80 g were selected,according to the body mass,they were divided into the routine feed group (selenium content:101.5 μg/kg) and the low-selenium feed group (selenium content:1.1 μg/kg) by random number table method,16 rats in each group.After 30 days of feeding,the routine feed group was divided into control group and T-2 toxin group (100 ng·kg-1·d-1),the low-selenium feed group was divided into low selenium group and low selenium + T-2 toxin group,8 rats in each group,the expression of IGF-1R in the articular cartilage of the left knee joint was detected by immunohistochemistry after 30 days of feeding.C28/I2 cells were cultured in vitro and treated with T-2 toxin 0 (control),6,12,and 24 μg/L,and each concentration of T-2 toxin was accompanied with sodium selenite (+ 0.1 mg/L) for 72 h.Meanwhile,IGF-1R inhibitor with 0 (control),250,500,and 1 000 μg/L was treated on C28/I2 cells for 48 h.The expression levels of IGF-1R mRNA and protein in chondrocytes were detected by Real-time PCR and Western blotting,and the apoptosis of chondrocytes was detected by flow cytometry.Results Compared with the control group [(100.00 ± 0.00)％,(100.00 ± 0.00)％],the expression rates of IGF-1R positive cells in articular cartilage surface and middle layers [(72.71 ± 4.75)％,(36.33 ± 4.32)％] of children in KBD group were significantly reduced (t =12.852,32.650,P ＜ 0.01).Compared with control group [(100.00 ± 0.00)％,(100.00 ± 0.00)％,(100.00 ± 0.00)％],the expression rates of IGF-1R positive cells in articular cartilage middle layer [(20.83 ± 2.69)％,(26.45 ± 2.84)％,(20.34 ± 1.82)％],deep layer [(33.55 ± 5.66)％,(48.89 ± 8.39)％,(25.51 ± 7.50)％],and the expression rates of IGF-1R positive cells [(47.50 ± 1.47)％,(28.66 ± 3.58)％,(40.52 ± 6.78)％] in the hypertrophic layer of the metaphyseal plate of rats in low selenium,T-2 toxin,and low selenium + T-2 toxin groups were significantly reduced (P ＜ 0.01).C28/I2 cells were cultured in vitro,compared with the control group,IGF-1R mRNA and protein expression levels in each T-2 toxin groups were significantly reduced (P ＜ 0.05).The expression levels of IGF-1R mRNA (1.95 ± 0.35,2.44 ± 0.17,2.40 ± 0.15) in 6,12,24 μg/L T-2 toxin + 0.1 mg/L selenium groups were significantly higher than those in T-2 toxin groups (0.80 ± 0.08,0.63 ± 0.08,0.61 ± 0.11,t =-12.259,-11.279,-13.371,P＜ 0.05).The expression levels of IGF-1R protein (1.67 ± 0.70,1.07 ± 0.26) in 6,12 μg/L T-2 toxin + 0.1 mg/L selenium groups were significantly higher than those in T-2 toxin groups (0.52 ± 0.05,0.72 ± 0.05,t =-25.977,-10.776,P ＜ 0.05).Compared with the control group [(5.33 ± 0.85)％,(4.03 ± 1.15)％],C28/I2 cells early apoptosis rates [(8.26 ± 1.51)％,(13.00 ± 0.72)％,(13.19 ± 1.05)％] in each of IGF-1R inhibitor groups,and late apoptosis rates [(8.50 ± 0.71)％,(14.21 ± 1.10)％] in 500,1 000 μg/L IGF-1R inhibitor groups were increased significantly (P ＜ 0.05).Conclusions The expressions of IGF-1R in the cartilage tissue of KBD children and T-2 toxin-poisoned rats under low selenium condition are decreased.T-2 toxin decreases the expression of IGF-1R in chondrocytes,and selenium can partly inhibit the effect of T-2 toxin on IGF-1R.Down-regulation of IGF-1R causes chondrocyte apoptosis,and it may play an important role in KBD chondrocyte apoptosis.

Protective effect of mitochondria-targeted antioxidant SS31 on early brain injury following subarachnoid hemorrhage in rats / 中南大学学报(医学版)

Objective:To evaluate protective effects of SS31 on early brain injury (EBI) induced by subarachnoid hemorrhage (SAH) in rats.Methods:A total of 96 Sprague-Dawley rats were randomly divided into 4 groups:A sham group,an SAH group,an SAH+vehicle group (SAH+V),and an SAH+SS31 group.The SAHinduced prechiasmatic cistern rat model was established in this study.Neurological deficit scores were evaluated at 24 h after SAH.The SS31 (5 mg/kg) as well as equal volume of vehicle were administrated intraperitoneally at 2 h after SAH.The neurological scores,brain edema,blood-brain barrier (BBB) permeability,apoptosis,malondialdehyde (MDA),glutathione peroxidase (GPx) activity,superoride dismutase (SOD) activity,and the expression ofcytosolic cytochrome c (Cyt C) and Bax were analyzed at 24 h after SAH.Results:Treatment with SS31 could significantly reduce MDA levels,and restored the activities of GPx and SOD in the cortex following SAH when compared with the SAH+V group.In addition,Bax SS31 trearment increased or decreased the levels of mitochondrial Cyt C or Bax,respectively.Moreover,SS31 treatment ameliorated brain edema and Evans blue dye extravasation,improved neurological deficits,and decreased neuronal apoptosis at 24 h after SAH.Conclusion:SS31 could alleviate EBI after SAH through its antioxidant property and ability in inhibition of neuronal apoptosis.

Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene / 中南大学学报(医学版)

OBJECTIVE@#To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene.
@*METHODS@#Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. 
@*RESULTS@#DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. 
@*CONCLUSION@#An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.

An ER Locating Protein Named RCN2 Interacts With STIM1-Orai1 Complex / 生物化学与生物物理进展

STIM1 is recognized as an ER Ca2+ sensor of calcium release-activated calcium (CRAC) channel that is constructed by membrane protein Orai1, However, this regulatory system may also be regulated by other proteins. Reticulocalbin 2 (RCN2) was purified and identified from STIM1-Orai1 complex. Confocal microscopy revealed that RCN2 co-localized with STIM1 in ER before and after Ca2+ store depletion. Single cell [Ca2+]I measurements of RCN2 EF hands mutant showed slight influence on SOC electrophysiological characters. Furthermore, a novel collar form aggregation of RCN2 surrounding STIM1 clusters suggested that RCN2 potentially plays a role of structure maintenance in STIM1 clustering.

Preparation and Quality Control of Diclofenac Sodium Hollow Suppository / 中国药房

OBJECTIVE: To prepare diclofenac sodium hollow suppository and establish its quality control. METHODS: The hollow type suppository was prepared with mixed fatty glyceride as the base; the content of diclofenac sodium was deter-mined by ultraviolet spectrophotometry. RESULTS: The linear range of diclofenac sodium was 5～25 ?g?mL-1(r=0.999 7),with an average recovery rate of 102.04%(RSD=1.08%). CONCLUSION: The preparation technology of diclofenac sodium hollow suppository is simple and feasible,and the quality of the suppository is stable and controllable.