Objective To investigate the correlation between sex hormone-binding globulin(SHBG)and lipid metabolism and visceral fat area(VFA)in male adults with different glucose tolerance.Methods A total of 473 male subjects were enrolled from the outpatient clinic and ward in PLA NO.903 Hospital from January 2018 to December 2020.All the subjects were divided into three groups according to OGTT results:normal glucose tolerance group(NGT,n=179),impaired glucose regulation group(IGR,n=90)and newly diagnosed type 2 diabetes mellitus group(T2DM,n=204).Serum SHBG level,abdominal visceral fat area(VFA)and biochemical indexes were compared among the three groups.Results Compared with NGT group,WC,WHR,BMI,HbA1c,FPG,2 hPG,2 hIns,TG and VFA were increased(P＜0.05),while SHBG was decreased(P＜0.05)in IGR group.WC,SBP,DBP,HbA1c,FPG,2 hPG,2 hIns,HOMA-IR,TG,FFA,VFA and VFA/SFA were increased,while HOMA-β,SHBG were decreased(P＜0.05)in T2DM group.Compared with IGR group,SBP,HbA1c,FPG,2 hPG,2 hIns and HOMA-IR were increased,HOMA-β was decreased in T2DM group(P＜0.05).Pearson correlation analysis showed that SHBG was positively correlated with HDL-C(P＜0.01),but was negatively correlated with WC,WHR,BMI,2 hIns,SUA,TG,VFA and SFA(P＜0.05 or P＜0.01).Multivariable linear regression analysis showed that HDL-C,TG and VFA were the influencing factors for SHBG.Conclusion Serum SHBG is closely related to abdominal obesity and lipid metabolism.Increasing the level of SHBG can reduce visceral fat accumulation and improve IR.

Objective:To investigate the effect of high glucose on the release of interleukin (IL)-1β and IL-18 in placental trophoblast by activating NLRP3 inflammasome.Methods:Gestational diabetes mellitus(GDM) placentas and control placentas were collected and the expression levels of NLRP3 and Caspase-1 were determined. Human placental trophoblast HTR-8/SVneo were cultured and divided into control group(5.5 mmol/L glucose), high glucose group(25 mmol/L glucose), DMSO+ high glucose group, and Ac-YVAD-cmk(NLRP3 inflammasome inhibitor)+ high glucose group. The expression levels of NLRP3 and Caspase-1 in cells as well as the contents of IL-1β and IL-18 in the medium were determined.Results:The expression levels of NLRP3 and Caspase-1 in GDM placenta were higher than those in control placenta( P<0.05) and positively correlated with homeostasis model assessment of insulin resistant index(HOMA-IR) and fasting insulin. The expression levels of NLRP3 and Caspase-1 in HTR-8/SVneo cells and the secretion levels of IL-1β and IL-18 in high glucose group were higher than those in control group( P<0.05). Ac-YVAD-cmk significantly suppressed high glucose-stimulated IL-1β and IL-18 secretion( P<0.05). Conclusion:High glucose promotes the release of IL-1β and IL-18 from placental trophoblast via activating NLRP3 inflammasome.

Objective:To observe the effect of dictyophora polysaccharide (DIP) on PINK1/Parkin pathway mediated mitophagy induced by sodium arsenite (NaAsO 2) in human hepatocytes (L-02 cells). Methods:The L-02 cells in logarithmic growth phase and in good condition were divided into control group, NaAsO 2 group (10 μmol/L), DIP group (80 μg/ml), DIP + NaAsO 2 group (80 μg/ml DIP + 10 μmol/L NaAsO 2) , N-acetylcysteine (NAC) group (5 mmol/L), and NAC + NaAsO 2 group (5 mmol/L NAC + 10 μmol/L NaAsO 2). Western blotting was used to detect the expression levels of mitophagy related proteins p62, microtubule-associated protein 1 light chain 3 (LC3)Ⅱ/LC3Ⅰ, PINK1, and Parkin. The mitochondrial stucture and autophagosomes were observed by transmission electron microscope, the fluorescent probe method was used to detect the expression level of intracellular reactive oxygen species (ROS). Results:Compared with the control group, the protein expressions of p62, LC3 Ⅱ/LC3 Ⅰ, PINK1, and Parkin in NaAsO 2 group were higher ( P < 0.05); compared with the NaAsO 2 group, the protein expressions of p62, LC3 Ⅱ/LC3 Ⅰ, PINK1 and Parkin were lower in DIP, DIP + NaAsO 2, NAC, and NAC + NaAsO 2 groups ( P <0.05). According to the transmission electron microscope, compared with the control group, the mitochondria of L-02 cells in NaAsO 2 group were significantly damaged and the number of autophagosomes increased. Compared with NaAsO 2 group, the degree of mitochondrial swelling, vacuolar degeneration and the number of autophagosomes decreased in DIP + NaAsO 2 group. Compared with the control group (33 110.00 ± 2 191.28), the intracellular ROS level in NaAsO 2 group was higher (48 000.00 ± 2 395.31, P < 0.05); the level of intracellular ROS in DIP + NaAsO 2 group (38 670.00 ± 2 620.56) was significantly lower than that in NaAsO 2 group( P < 0.05), and there was no significant change compared with the control group ( P > 0.05). Conclusions:NaAsO 2 can induce PINK1/Parkin mediated mitophagy in L-02 cells. DIP can alleviate NaAsO 2 induced mitophagy. DIP may affect PINK1/Parkin mediated mitophagy induced by NaAsO 2 through the regulation of ROS.

Objective:To explore the mechanism of apoptosis induced by sodium arsenite (NaAsO 2) in human hepatic cells (L-02) through reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, and provide experimental evidence for the mechanism of arsenic poisoning. Methods:L-02 cells were divided into control group, NaAsO 2 group (10 μmol/L NaAsO 2), N-acetylcysteine (NAC) group (5 mmol/L NAC), and NaAsO 2 + NAC group (10 μmol/L NaAsO 2, 5 mmol/L NAC), and were cultured in vitro for 24 h. The intracellular ROS level, mitochondrial membrane potential depolarization ratio and cell apoptosis rate were measured by dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence probe, JC-1 staining and Annexin V-FITC/PI double staining, respectively; the mRNA and the protein of Caspase 3, cytochrome C (Cyt-C) and cytochrome C oxidaseⅣ (COXⅣ) were detected by real time fluorescence quantitative PCR (qRT-PCR) and Western blotting, respectively. Results:There were statistically significant differences in intracellular ROS levels (3 857 392.33 ± 44 928.39, 4 515 288.00 ± 32 660.64, 3 670 150.67 ± 101 987.69, 4 035 235.67 ± 99 995.30), mitochondrial membrane potential depolarization ratios (2.16 ± 0.54, 7.95 ± 0.52, 2.70 ± 0.29, 1.01 ± 0.23) and total apoptosis rates (1.45 ± 0.03, 4.27 ± 0.17, 1.87 ± 0.12, 2.52 ± 0.35) between groups ( F = 62.62, 159.81, 112.70, P < 0.05). There were statistically significant differences in Caspase 3, Cyt-C, COXⅣ mRNA expression levels ( F = 9.20, 7.33, 14.87, P < 0.05) and in cleaved-Caspase 3, Cyt-C, COXⅣ protein expression levels( F = 31.42, 8.01, 83.30, P < 0.05) between groups. Compared with the control group, the intracellular ROS level, mitochondrial membrane potential depolarization ratio and total apoptosis rate were significantly increased ( P < 0.05); Caspase3, Cyt-C mRNA and protein expression levels were significantly increased ( P < 0.05), and COXⅣ mRNA and cleaved-Caspase 3, Cyt-C protein expression levels were significantly decreased ( P < 0.05) in NaAsO 2 group. Compared with the NaAsO 2 group, the intracellular ROS level, mitochondrial membrane potential depolarization ratio and total apoptosis rate of NaAsO 2 + NAC group were significantly decreased ( P < 0.05); the Caspase3, Cyt-C mRNA and cleaved-Caspase 3, Cyt-C protein expression levels were significantly decreased ( P < 0.05), the COX Ⅳ mRNA and protein expression levels were significantly increased ( P < 0.05). Conclusions:NaAsO 2 stimulates L-02 cells to produce excessive ROS, which induces mitochondrial depolarization and further triggers mitochondrial damage, resulting in increased release of Cyt-C and activation of the mitochondrial apoptosis pathway that Caspase 3 protein induces apoptosis in L-02 cells, which may be one of the main mechanisms of arsenic-induced liver injury.

Objective:To explore the role of nuclear factor-E2-related factor 2 (Nrf2) signaling pathway in oxidative damage caused by sodium arsenite (NaAsO 2) in human normal liver cells (L-02), and to provide experimental basis for the study of oxidative damage mechanism of liver damage caused by arsenic. Methods:L-02 cells were cultured in vitro and treated with 0 (control), 25, 50, 75, 100, 125, and 150 μmol/L NaAsO 2, respectively, for 24 h. The half-inhibitory concentration (IC 50) was calculated according to the cell survival rate by CCK8, and L-02 cells were treated with 0, 1/8, 1/4 and 1/2 dose of IC 50 of NaAsO 2, respectively, for grouping experiments. Protein expressions of Nrf2, heme oxygenase-1 (HO-1), NADH quinone oxidoreductase 1 (NQO1) and glutathione peroxidase 1 (GPx1) in L-02 cells and L-02 nucleus were detected by Western blotting. Results:The result of CCK8 showed that the survival rates of L-02 cells in 25, 50, 75, 100, 125, 150 μmol/L NaAsO 2 groups were [(69.53 ± 0.06)%, (41.33 ± 0.08)%, (23.65 ± 0.04)%, (26.51 ± 0.04)%, (31.63 ± 0.01)%, (26.24 ± 0.02)%], which were significantly lower than that of the control group[(100 ± 0.00)%]. The differences were statistically significant ( P < 0.05). The IC 50 calculated by cell survival was 40 μmol/L, and the NaAsO 2 doses used in the experiment were 0 (control), 5, 10, and 20 μmol/L. Western blotting results showed that, compared with the control group, the protein expression levels of Nrf2, HO-1 in L-02 and HO-1 in the L-02 cells nucleus in the 5, 10 and 20 μmol/L NaAsO 2 groups were significantly higher ( P < 0.05). Compared with the control group, the expression levels of GPx1 protein in L-02 cells of 10 and 20 μmol/L NaAsO 2 groups were decreased ( P < 0.05). Compared with the control group, the expression levels of Nrf2 protein in L-02 nucleus in 10 and 20 μmol/L NaAsO 2 groups were significantly increased ( P < 0.05); the expression level of NQO1 protein in L-02 nucleus in 5 μmol/L NaAsO 2 group was significantly increased ( P < 0.05). Conclusion:NaAsO 2 has an effect on the expression of Nrf2 signaling pathway related factors in L-02 cells, and the mechanism of oxidative damage caused by NaAsO 2 in L-02 cells may be related to Nrf2 signaling pathway.

Objective:To investigate the effects of sodium arsenite (NaAsO 2) on the expression of sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator activated receptor α (PPARα) and fatty acid synthase (FAS) in human liver cells (L-02 cells). Methods:L-02 cells were cultured in vitro, and exposed to NaAsO 2 at 0 (control), 2, 4, 8, 16, 32, 64 and 128 μmol/L for 24 h, respectively, and the cell survival rate was determined by CCK-8 method. And a fitting curve was made to calculate the half inhibitory concentration (IC 50), subsequent experiments were carried out with 0, 1/8, 1/4 and 1/2 of IC 50 as arsenic exposure doses. Glycerol phosphate oxidase-catalase (GPO-PAP) method was used to detect the content of triglyceride (TG) in cells; the mRNA expression levels of SREBP-1c, PPARα and FAS were detected by Real-time PCR; and the protein expression levels of SREBP-1c and PPARα were detected by Western blotting. Results:The cell survival rates of 8, 16, 32, 64 and 128 μmol/L NaAsO 2 groups [(92.000 ± 1.414)%, (91.000 ± 0.000)%, (76.500 ± 0.707)%, (53.000 ± 1.412)%, (47.000 ± 1.412)%] were significantly lower than that of the control group [(100.000 ± 0.000)%, P < 0.01]. The IC 50 was 64 μmol/L, and subsequent experiments were conducted with 0 (control), 8, 16 and 32 μmol/L NaAsO 2, respectively. Compared with the control group [(1.000 ± 0.000) mmol/g prot], TG contents of 8, 16 and 32 μmol/L NaAsO 2 groups [(0.691 ± 0.064), (0.474 ± 0.162), (0.184 ± 0.045) mmol/g prot] were significant decreased ( P < 0.01). Compared with the control group, the mRNA expression levels of SREBP-1c, PPARα, FAS, and the protein expression levels of SREBP-1c and PPARα in NaAsO 2 groups were significantly decreased ( P < 0.01 or < 0.05). Correlation analysis showed that NaAsO 2 content was negatively correlated with TG content, SREBP-1c and PPARα protein expression levels ( r =-0.954,- 0.875,-0.965, P < 0.01). Conclusion:NaAsO 2 can reduce the TG content and the expression of lipid metabolism related genes SREBP-1c, PPARα and FAS in L-02 cells, suggesting that arsenic-induced liver injury can cause lipid metabolism disorders.

Objective To investigate the effects of three mitogen-activated protein kinase (MAPK) inhibitors on the expressions of transforming growth factor-β1 (TGF-β1),α-smooth actin (α-SMA) mRNA and protein in human liver stellate cells (LX-2 cells) activated by sodium arsenite.Methods Cultured in vitro LX-2 cells in the logarithmic growth stage were exposed to sodium arsenite at 0.0 (control),2.5,5.0,10.0,20.0,40.0,80.0 μmol/L for 24 h,respectively,and the cell survival rate was determined by CCK-8 assay.According to the results of the study,LX-2 cells were divided into 5 groups:control group,sodium arsenite group,extracellular signal regulation kinase (ERK) inhibition group,c-Jun amino-terminal kinase (JNK) inhibition group,and p38 inhibition group.LX-2 cells were pre-treated with 10.0 μmol/L ERK,JNK,p38 kinase inhibitors (PD98059,SP600125,SB203580) for 30 min in the 3 inhibition groups,and then 20.0 μmol/L sodium arsenite for 24 h.The control group was not treated with sodium arsenite and inhibitors.Sodium arsenite group was not treated with inhibitors.Then mRNA and protein expression levels of TGF-β1 and α-SMA in LX-2 cells were determined by Western blotting and real-time PCR,respectively.Results The survival rates of LX-2 cells in 5.0,10.0,20.0,40.0,80.0 μmol/L sodium arsenite groups were [(92.35 ± 0.92)％,(84.06 ± 0.84)％,(74.27 ± 0.74)％,(59.57 ± 0.60)％,(27.77 ± 0.23)％],which were significantly lower than that of the control group [(100.00 ± 0.00)％,P ＜ 0.05].It was found that the expressions of TGF-β1,o-SMA mRNA and protein of sodium arsenite group were higher than those of the control group (P ＜ 0.01).The expressions of TGF-β1,α-SMA mRNA and protein of the three inhibition groups were lower than those of the sodium arsenite group (P ＜ 0.05).Conclusions Arsenic exposure can cause abnormally high expressions of TGF-β1,α-SMA mRNA and protein in LX-2 cells.Intervention with three MAPK inhibitors can improve the effects of arsenic induced LX-2 cells activation on the expressions of TGF-β1,α-SMA mRNA and protein.

The clinical data of 96 patients with type 2 diabetes mellitus (T2DM) treated in Department of endocrinology of our hospital from January 2016 to December 2017 were retrospectively analyzed. All patients had been treated with metformin ≥1 000 mg combined with sulfonylureas for>12 weeks and their glycosylated hemoglobin (HbA1c) was>7.5%. On the basis of the original scheme, 57 patients received oral sitagliptin (100 mg q.d, sitagliptin group) and 39 patients received insulin glargine injection (insulin group) for 26 weeks. The blood lipid, liver and kidney function were examined before and after treatment. The abdominal visceral fat area (VFA) was measured by CT scan. Results showed that the fasting plasma glucose (FPG) , 2-hour postprandial blood glucose (2 hPG) and HbA1c were significantly lower than baseline levels in both groups(P<0.05). The decrease of VFA in sitagliptin and insulin groups was by 9.6 (1.4,19.6)cm² and by 8.3(-2.2,26.8) cm², respectively; there was significant difference in variation of VFA before and after treatment between the two groups (P<0.05). There was no significant difference in blood pressure, liver function (ALT, AST) and estimated glomerular filtration rate (eGFR) before and after treatment in the sitagliptin group (P>0.05). Additional sitagliptin administration can effectively and safely reduce HbA1c and decrease the abdominal visceral fat content in T2DM patients who failed to metformin and sulfonylureas combined therapy.

Objective To study the effect of hydrogen sulfide on the production of soluble fms-like tyrosine kinase 1 (sFlt-1) through a distintegrin and metalloproteinase 17 (ADAM17) in adipocytes. Methods 3T3-L1 cells were cultured and induced to differentiate into adipocytes, then treated with different doses of sodium hydrogen sulfide (NaHS), L-cysteine or transfected with cystathionine-γ-lyase ( CSE) siRNA, ADAM17 siRNA or treated with ADAM17 inhibitor, monoclonal antibody. 24 hours after treatment, the expression of ADAM17, CSE, and the production of sFlt-1 were determined. Results After the treatment of 10, 25, 50 nmol/L NaHS or 0. 5, 1. 0, 2. 0 μmol/L L-cysteine, the expression of ADAM17 and the production of sFlt-1 in adipocytes were significantly decreased, the higher dose of L-cysteine and sFlt-1, the lower expression of ADAM17 and the production of sFlt-1; the effect of 2.0 μmol/L L-cysteine decreasing the expression of ADAM17 and the production of sFlt-1 were reversed by transfection of CSE siRNA; after the transfection of ADAM17 siRNA and treatment of ADAM17 inhibitor or monoclonal antibody, the production of sFlt-1 in adipocytes were significantly decreased. Conclusion Hydrogen sulfide can reduce the production of sFlt-1 in adipocytes by downregulating the expression of ADAM17.

Objective To investigate the effect of hydrogen sulfide′s donor NaHS on the secretion of interleukin (IL)-1β and IL-18 in adipocytes and its mechanism.Methods 3T3-L1 cells were induced to differentiate into adipocytes and incubated with various concentrations of NaHS or 10 μg/ml caspase-1 inhibitor Ac-YVAD-CMK for 24 hours.The expressions of NLRP3(NLR family, pyrin domain containing 3), ASC(apoptosis associated speck-like protein containing CARD domain), caspase-1, IL-1β, and IL-18 in adipocytes, as well as the content of IL-1 and IL-18 in culture medium were determined.Results 10, 25, and 50 nmol/L NaHS significantly decreased NLRP3, ASC, and cleaved-caspase-1 protein expressions in adipocytes, as well as IL-1β and IL-18 contents in culture medium in a dose-dependent manner.The mature-IL-1β/pro-IL-1β, mature-IL-18/pro-IL-18 ratios in adipocytes and IL-1β and IL-18 contents in culture medium were also reduced by 10μg/ml Ac-YVAD-CMK.Conclusion Donor of hydrogen sulfide NaHS inhibits the maturation and secretion of IL-1β and IL-18 in adipocytes through downregulating the expression of NLRP3 inflammasome.