Objective To evaluate the effect of hydrogen peroxide (H2O2) on autophagy in melanocytes,and to explore its possible regulatory mechanisms.Methods Normal human melanocytes at exponential growth phase were divided into several groups:blank control group receiving no treatment,positive control group treated with 100 nmol/L sirolimus solution,and experiment groups treated with H2O2 solution at different volume fractions of 10-7-10-3 respectively.After 4-hour treatment,cell counting kit-8 (CCK-8) assay and flow cytometry were performed to evaluate the cellular proliferative activity and detect apoptosis of melanocytes respectively.Acridine orange staining was performed to detect autophagosome formation,transmission electron microscopy to observe ultrastructural changes of autophagosomes,and Western blot analysis to measure the expression of autophagy-specific protein Beclin 1 and microtubuleassociated protein 1 light chain 3B (LC3B).A total of 84 autophagy-related genes were analyzed by RT2 Profiler PCR Array,so as to screen differentially expressed autophagy-related genes.Results After the treatment with H2O2 at different volume fractions of 10-3,5 × 10-4,10-4,5 × 10-5,10-5,5 × 10-6 and 10-6,experiment groups showed significantly decreased cellular proliferative activity,but significantly increased apoptosis rate compared with the blank control group (F =286.95,301.23,respectively,both P ＜ 0.05).With the increase in volume fractions of H2O2,the cellular proliferative activity was significantly gradually decreased (P ＜ 0.05),while the apoptosis rate showed an opposite trend (P ＜ 0.05),except that the 5 ×10-6 H2O2 group showed no significant differences in the apoptosis rate compared with the 10-5 H2O2 group and 10-6 H2O2 group.Acridine orange staining and electron microscopy showed autophagosome formation in the 10-5 H2O2 group,10-6 H2O2 group and positive control group.Western blot analysis revealed that Beclin1 expression and LC3B-Ⅱ/LC3B-Ⅰ ratio were significantly higher in the 10-5 H2O2 group,10-6 H2O2 group and positive control group than in the blank control group (all P ＜ 0.05).RT2 Profiler PCR Array showed significant up-regulation of ATG12,ATG3,ULK1,PIK3CG,PTEN and PIK3C3 genes and significant downregulation of EIF2AK3 gene in the 10-5 H2O2 group,10-6 H2O2 group and positive control group compared with the blank control group.In the 10-5 H2O2 group and positive control group,the mTOR gene was significantly up-regulated,and the ULK2 gene was significantly down-regulated.The 10-6 H2O2 group showed no obvious changes in the expression of mTOR gene,but significant up-regulation of AMPK and JNK1 genes.Conclusion H2O2 at volume fractions of 10-5 and 10-6 can induce autophagy in melanocytes,likely by influencing the expression of some related signaling molecules.

Objective To study the protective effect of Astragalus membrance on mice infected with tachyzoites of Toxoplasma gondii. Methods ICR mice were infected intraperitoneally with 10 5, 10 3, 10 2 tachyzoites of virulent RH strain of Toxoplasma gondii, and the mice were orally treated with Astragalus membrance 0 075 g/d per mouse or Azithromycin [150 mg/(d?kg)] each day starting from day 1 post-infection for 10 days. The survival rate and period were investigated. The parasite loads of livers and lungs of the mice infected with 10 2 tachyzoites were determined by fluorescence PCR methods at 4 day-post-infection (dpi) and 8 dpi. Results When infected with 10 5, 10 3 tachyzoites, treated with Astragalus membrance, the average survived days of the mice were 5 57 days and 6 23 days, and treated with azithromycin were 6 96 days and 8 12 days respectively. The azithromycin group but not the astragli group survived significantly longer than the control(P

Objective To evaluate the effect of the treatment with low-concentration hydrogen peroxide (H2O2) on the adhesive function of and autophagy in human melanocytes,and to screen long noncoding RNAs (lncRNAs) related to autophagy.Methods Melanocytes were isolated from foreskins of healthy males after circumcision,and subjected to cultivation.Melanocytes at exponential growth phase were divided into 3 groups:control group receiving no treatment,H2O2 group treated with 400 μ mol/L H2O2,and H2O2 + NAC group pretreated with 4 mmol/L NAC for 2 hours followed by the treatment with 400 μmol/L H2O2.After 5-day treatment,immunofluorescence study was performed to determine the expression of Ecadherin,microtubule-associated protein 1 light chain 3 (LC3)and p62,and Western blot analysis to determine the expression of autophagy-related protein LC3 and p62.Cell structures and autophagosomes were observed by transmission electron microscopy,and autophagy-related lncRNAs were screened using gene chip technology.Statistical analysis was done with Graphpad Prism 6 software using one-way analysis of variance for comparison among groups,and Tukey's test for multiple comparisons.Results Under the confocal microscopy,the H2O2 group showed significantly decreased fluorescence intensity of E-cadherin and LC3 in the melanocytes and decreased number of autophagosomes in melanocytes,but significantly increased fluorescence intensity of p62 compared with the control group and H2O2 + NAC group.Western blot analysis showed that the LC3-Ⅱ/LC3-Ⅰ ratio in the melanocytes was significantly lower in the H2O2 group (0.604 ± 0.012) than in the control group (1.200 ± 0.081,q =7.718,P ＜ 0.01) and H2O2 + NAC group (1.017 ± 0.062,q =5.076,P ＜ 0.05),while the p62/β-actin ratio in the melanocytes was significantly higher in the H2O2 group (0.881 ± 0.079) than in the control group (0.456 ± 0.121,q =4.847,P ＜ 0.05) and H2O2 + NAC group (0.492 ± 0.049,q =4.439,P ＜ 0.05).There were no significant differences in the LC3-Ⅱ/LC3-Ⅰ ratio or p62/β-actin ratio between the H2O2 + NAC group and control group (P ＞ 0.05).Gene chip technology showed that 18 autophagy-related lncRNAs were associated with premature senescence of melanocytes and differentially expressed in the H2O2 group compared with the control group,and the autophagy-related lncRNA NONHSAT190308.1 (＞ 10-fold increase) was screened out.Conclusion Lowconcentration H2O2 can decrease the expression of E-cadherin and the level of autophagy in melanocytes,and can up-regulate the expression of autophagy-associated lncRNA NONHSAT190308.1.

AIM:To investigate the effects of Panax notoginseng compound formula(PN)on endometrial fibro-sis by regulating inflammatory reaction and intestinal flora(IF)in a rat model of intrauterine adhesion(IUA).METHODS:The rat IUA model was established by following the mechanical injury method.A total of 50 rats were randomly divided in-to sham group,model group,low-dose(210 mg/kg)PN group,medium-dose(420 mg/kg)PN group and high-dose(840 mg/kg)PN group.After 8 weeks of intragastric administration,the uterus was collected to observe morphological changes with naked eye.The degree of uterine tissue damage and fibrosis was evaluated through hematoxylin-eosin(HE)and Mas-son staining.The collagen type Ⅰ(Col Ⅰ)was detected by immunohistochemistry.The interleukin-6(IL-6)and IL-10 pro-tein expression was detected by Western blot.The levels of IL-6,IL-1β,tumor necrosis factor-α(TNF-α)and vascular endothelial growth factor B(VEGFB)were detected by enzyme-linked immunosorbent assay(ELISA).The IF diversity and population structure were observed by 16S amplicon.RESULTS:Compared with the sham group,the uteruses of rats in the model group showed:reduced elasticity,accompanied by congestion and edema;decreased number of glands and blood vessels,and thinned endometrium(P<0.01);increased collagen fibers and Col Ⅰ protein expression(P<0.01);sig-nificantly increased IL-1β,IL-6,TNF-α and VEGFB levels in the uterine tissue(P<0.01);decreased IL-10 level(P<0.01);and reduced IF diversity(P<0.05).Compared with the model group,the drug intervention groups exhibited:re-covered elasticity of the uterus and relieved congestion and edema;increased number of endometrial glands and blood ves-sels(P<0.05);decreased collagen fibers and Col Ⅰ protein expression(P<0.01);reduced IL-1β,IL-6,and TNF-α lev-els to varying degrees in the uterine tissue(P<0.05);elevated IL-10 level(P<0.01);and improved IF diversity(P<0.05).CONCLUSION:The PN is able to significantly improve the endometrial tissue fibrosis in IUA rats.The under-lying mechanisms may be related to the inhibition of IL-6,IL-1β and TNF-α expression,up-regulation of IL-10,and im-provement of IF diversity.