Effects of compatibility on pharmacokinetics of mangiferin in INS-1 cells and its intracellular distribution / 中国药理学通报

Aim To investigate the effect of compatibility of phellodendron amurense on the pharmacokinetics of mangiferin(MGF) in INS-1 cells, and the distribution of mangiferin in normal and oxidatively damaged INS-1 cells. Methods INS-1 cells were administered in equal doses of mangiferin, anemarrhena and anemarrhena-phellodendron herb pair. LC-MS/MS was used to determine the content of MGF in INS-1 cells. The normal and model groups (INS-1 cells were treated with 140 μmol · L

miR-138-5p protects β cell function by regulating HIF-1α in gestational diabetes mellitus / 中国临床药理学与治疗学

AIM: To study the effect of miR-138-5p on the function of β cell in gestational diabetes mellitus (GDM) and its related mechanism. METHODS: The expression of miR-138-5p in peripheral blood of 15 GDM pregnant women and 15 normal pregnant women were compared by RT-qPCR. miR-138-5p mimic and inhibitor were transfected into INS-1 cells, respectively, and their expression level was over expressed or inhibited. RT-qPCR was used to verify the transfection efficiency.MTT proliferation experiment, Annexin V-FITC apoptosis experiment and insulin release experiment were used to detect the effects of miR-138-5p on INS-1 cell proliferation, apoptosis and insulin release ability. The target gene of miR-138-5p was screened by TargetScan, a miRNA target gene prediction software. The functional rescue experiment confirmed whether miR-138-5p could exert its influence on INS-1 cell proliferation, apoptosis and insulin release ability by targeting its target gene. Western blot was used to detect the molecular signaling pathway of miR-138-5p in INS-1 cells.RESULTS: The expression of miR-138-5p in peripheral blood of GDM pregnant women was significantly lower than that of normal pregnant women. RT-qPCR showed that miR-138-5p mimic and inhibitor could significantly promote or inhibit the expression of miR-138-5p in INS-1 cells. The results of MTT proliferation experiment, Annexin V-FITC apoptosis experiment and insulin release experiment indicated that over expression of miR-138-5p could significantly promote the proliferation of INS-1 cells, inhibit the apoptosis of cells and promote the insulin release ability of cells. However, down-regulating the expression of miR-138-5p could significantly inhibit the proliferation of INS-1 cells, promote apoptosis and inhibit insulin release. HIF-1α was selected as the target gene of miR-138-5p by TargetScan. The double luciferase gene report and Western blot showed that miR-138-5p could inhibit the expression of HIF-1α in INS-1 cells. The functional rescue experiment confirmed that miR-138-5p could affect the proliferation, apoptosis and insulin release of INS-1 cells by regulating the expression of HIF-1α. Western blot showed that miR-138-5p may play a role in INS-1 cells by affecting PI3K, Akt and p-PI3K, p-Akt protein after phosphorylation in PI3K/Akt signaling pathway. CONCLUSION: miR-138-5p may reguLate HIF-1α expression in a targeted manner, thereby affecting the PI3K/AKT signaling pathway, promoting the proliferation and inhibition of the parent cells' apoptosis, and promoting their insulin-releasing ability to protect the function of β cell in GDM.

Obestatin attenuates apoptosis induced by high glucose in INS 1 cells / 基础医学与临床

Objective To investigate the effect of obestatin on the apoptosis of rat pancreatic islet cell line INS1 induced by high glucose .Methods INS1 cells were cultured in different concentrations of glucose .The survival rate and proliferation of INS1 cells were detected by MTT method;Hoechst33258 nuclear staining was used to de-tect nuclear morphology.caspase-3 method was used to study the relationship between the protective effect of obestatin and the PI3K pathway;Finally,using real-time PCR detection of FOXO1 and SREBP1c, Bax, PDX-1 expression, to further clarify the protective effect of obestatin on cells.Results In high glucose condition,obesta-tin promoted the proliferation of INS1 cells at 100 nmol/L,and promoted the proliferation of INS 1 cells significantly ( P<0.01 , compared with the control group and high glucose group ) .Obestatin can reduce high glucose-induced apoptosis(P<0.01).The expressions of FOXO1,SREBP1c,Bax and PDX-1 decreased,while the expression of FOXO1,SREBP1c,Bax and PDX-1 increased in high glucose group .Conclusions OB can attenuate the injury of INS1 cells induced by high glucose in rats .

Inhibition of High Glucose-induced Apoptosis of Pancreatic β-cells by Gallic Acid in Phyllanthus Emblica / 昆明医科大学学报

Objective To explore the protective effect of gallic acid in Phyllanthus emblica on high glucose－induced apoptosis of pancreatic islet β cells, and to provide a reference for the discovery of natural compounds for the treatment of diabetes. Methods In vivo experimental model, wistar male rats were used as in vivo subjects and 50 mg/kg STZ was injected intraperitoneally. After the model was successfully established, 25 mg/kg of gallic acid was given orally, and the positive drug was Sitagliptin. After 4 weeks of administration, the blood was taken and the pancreas was removed for HE staining. Western blot was used to measure the expression of NLRP3 and TXNIP in pancreatic tissue in high sugar state. In vitro model, insulinoma cell line INS－1 cells were used as in vitro targets to establish high levels. In sugar－induced apoptosis model, INS－1 cells were cultured in glucose－free RPMI 1640 complete medium supplemented with 25 mmol/L glucose. Gallic acid was used as the test sample. Experiments were divided into normal controls, high－sugar models, and low, medium and high levels of gallic acid groups. The cell viability was measured by MTT assay. The mRNA expression of NLRP3 and TXNIP in INS－1 cells was detected by QPCR and Western blot, and the expression of NLRP3 and TXNIP protein was detected.Results (1) INS－1 cells were cultured in a medium with glucose concentration of 25mmol/L for 48h, and the apoptosis rate was increased compared with the control group (P＜0.01), indicating that the apoptosis model was established successfully under high glucose conditions. (2) 10, 5, and 2.5 μmol/L GA were used to treat the control group and the high glucose model group cells respectively. The survival rate of the control group did not change significantly (P＞0.05) . Compared with the control group, the expression of NLRP3 and TXNIP in INS－1 cells in the high glucose model group was significantly different (P＜0.05);the protein expression level was significantly downregulated after GA treatment, and there was a statistical difference (P＜0.05) . Compared with the control group, the expression of NLRP3 protein in INS－1 cells in the high glucose model group was statistically different (P＜0.01), and the protein expression level was significantly downregulated after GA treatment (P＜0.01) ; The protein expression level was up－regulated (P＜0.05);the protein expression level after GA treatment was significantly down－regulated (P＜0.05); (4) The expression of NLRP3 and TXNIP mRNA in INS－1 cells was increased in the high glucose model group compared with the control group (P＜0.01) ; The expression of protein was significantly down－regulated after GA treatment (P＜0.01) . Conclusion The cells were cultured for 48 h in glucose－free RPMI 1640 complete medium supplemented with 25 mmol/L glucose. GA has no effect on the proliferation of normal INS－1 cells. GA protects INS－1 cells from apoptosis under high glucose conditions. The mechanism may be related to GA down－regulation of NLRP3 and TXNIP gene expression.

Angiotensin II type 1 receptor autoantibodies induces INS-1 islet β-cell apoptosis by upregulation of autophagy / 中国病理生理杂志

AIM:To explore whether the angiotensin II type 1 receptor autoantibodies(AT1-AA)induces islet β-cell apoptosis and whether autophagy is involved in the process.METHODS:The INS-1 cells treated with AT1-AA at 10-6mol/L for 24 h,and then the apoptosis was analyzed by flow cytometry,Western blot and Hoechst 33258 staining.In addition,the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot.The effects of AT1-AA on the apoptosis,autophagy and viability of INS-1 cells with or without 3-methyladenine(3-MA;a com-mon autophagy inhibitor)or telmisartan(an angiotensin Ⅱ type 1 receptor blocker)pretreatment, were detected by flow cytometry,Western blot and CCK-8 assay.RESULTS: Treatment with AT1-AA at 10 -6mol/L for 24 h significantly re-duced the cell viability(P<0.05).Compared with the negative IgG control group,the apoptotic cells increased after incu-bation with AT1-AA for 12 h,24 h and 36 h,respectively(P<0.05).Moreover,the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time,and the elevation of apoptosis and autophagy were blocked by telmisartan.After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone.CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet βcells by upregulating autophagy via the angiotensin Ⅱtype 1 receptor pathway.

TNF-αpromotes apoptosis of rat islet βcell line / 基础医学与临床

Objective To observe the effect of tumor necrosis factor-α( TNF-α) on islet cell apoptosis and TXNIP expression.Methods INS-1 cells were cultured in vitro, treating with TNF-α(0, 1, 5, 10 and 20 mg/L).We tested the effect of TNF-αon cell viability by CCK-8.INS-1 cells were treated with TNF-α( 5 mg/L, 24 h) for the proper concentration and incubation time; mRNA expression of TXNIP and ChREBP were measured by real-time PCR;in addition, protein levels of TXNIP , ChREBP and FOXO1 were analyzed by Western blot .Results TNF-αdecreased the survival rate of INS-1 cells in a dose-dependent manner ( P<0.05 ) , and induced apoptosis;protein and mRNA expression of TXNIP and ChREBP were significantly higher than that in control group ( P<0.05 );while the expression of protein level of FOXO 1 was down-regulated .Conclusions TNF-αinduces apoptosis in INS-1 cells and aggravates the cells damage .

Effect of salvianolic acid B on intermittent high glucose induced JNK activation and INS-1 cell apoptosis / 中国药理学通报

Aim To investigate the effect of salvianolic acid B (Sal B)on c-Jun N-terminal kinase (JNK)ac-tivation and apoptosis of INS-1 cells induced by inter-mittent high glucose.Methods INS-1 cells were pre-incubated with Sal B for 24 h,followed by exposure to intermittent high glucose (IHG,11.1 mmol·L-1 12 h,33. 3 mmol·L-1 12 h)for 72 h.Cell viability was assessed by MTT assay and cell apoptosis was evalua-ted by flow cytometry.Glucose induced insulin secre-tion capacity and intracellular reactive oxygen species (ROS)contents were measured by enzyme linked im-munosorbent assay (ELISA)and a fluorescent probe DCFH-DA,respectively.Levels of JNK activation and PDX-1 protein expression were determined by Western blot analysis.Results Sal B significantly alleviated IHG-induced cell injury and apoptosis,with glucose induced insulin secretion capacity improved evidently (P<0.05 or P<0.01).Preincubation with Sal B no-tably decreased intracellular ROS and JNK activation in INS-1 cells,while the level of PDX-1 protein was in-creased markedly (P<0.05 or P<0.01 ).Conclu-sion Sal B is capable of ameliorating IHG-induced cell injury and apoptosis in INS-1 cells,which might be derived from suppression of JNK activation and up-regulation of PDX-1 protein expression.

Effect of palmitate on TXNIP expression in INS-1 islet cells / 中国病理生理杂志

AIM:Chronic exposure to elevated levels of free fatty acids (FFAs) in type 2 diabetes patients is toxic to pancreatic β-cells.Thioredoxin (Trx)-interacting protein (TXNIP), an endogenous Trx-inhibiting protein, is up-regulated by glucose and is a critical mediator of hyperglycemia-induced β-cell apoptosis in diabetes.However, the effects of FFAs on TXNIP are unknown.In this experiment we observed the effect of palmitate on TXNIP expression in cultured INS-1 islet cells and the pathways involved were analyzed meanwhile.METHODS:After the full basis of preliminary experiment of incubating INS-1 cells with palmitate at different concentrations for different time, INS-1 islet cells were cultured with 0.5 mmol/L palmitate for 24 h.TXNIP expression, cell apoptosis, and expression of transcription factors related to TXNIP transcriptional regulation were determined.RESULTS:Compared with control group, the expression of TXNIP at mRNA and protein levels in palmitate group was significantly up-regulated (P<0.01).Cleaved caspase-3/caspase-3 ratio was increased in palmitate group (P<0.05), and the apoptosis of the INS-1 cells was also significantly increased (P<0.01).Palmitate enhanced the phosphorylation of nuclear factor-κB (NF-κB) (P<0.01), and the NF-κB inhibitors, PDTC and SN50, both blocked the palmitate-induced up-regulation of TXNIP expression.CONCLUSION:Saturated fatty acid palmitate enhances the expression of TXNIP.The mechanism of palmitate-induced TXNIP expression may be associa-ted with the increase in NF-κB phosphorylation.

Effect of bone marrow mesenchymal stem cells-conditioned medium on the apoptosis of INS-1 cells / 解放军医学杂志

Objective To investigate the effect and mechanism of bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on pro-inflammatory cytokines-induced apoptosis of insulinoma cells (INS-1). Methods-The conditioned medium containing various cytokines and growth factors was collected and condensed from the supernatants of culture medium of BMSC of rats at passage three. After exposed to the medium with a pro-inflammatory cytokines cocktail containing TNF-α, IL- 1β and IFN-γ for 48 hours, INS-1 cells were cultured with BMSC-CM in different concentrations for another 12 hours. INS-1 cells were divided into the following five groups: control group, pro-inflammatory cytokines-treated group, 1× BMSC-CM-treated group, 2× BMSC-CM-treated group and 4× BMSC-CM-treated group. The survival rate of INS-1 cells was assessed by CCK8 assay. The incidence of cell apoptosis was measured using Annexin V/PI staining. Intracellular reactive oxygen species (ROS) production was determined with dichlorofluorescein diacetate (DCFH-DA). Results-Compared with the control group, prolonged exposure to pro-inflammatory cytokines induced significant decrease in the viability of INS-1 cells (75.84%±1.53%) and a great increase in the incidence of cell apoptosis (17.23%±1.77%), and also in intracellular ROS level (34.3%±0.80%) with statistically significant difference (P<0.01). However, BMSC-CM treatment protected INS-1 cells against pro-inflammatory cytokines-induced injury in a dose-dependent manner. Among the BMSC-CM of three different concentrations, 4× BMSC-CM exhibited best protective effects on pro-inflammatory cytokines-treated INS-1 cells, as reflected by increased cell viability (87.7%±2.08%) and significant reduction in the incidence of cell apoptosis (8.67%±1.59%), and also in intracellular ROS production (17.1%±2.14%) with statistically significant difference (P<0.05). Conclusion-BMSC-CM protected INS-1 cells against pro-inflammatory cytokines-induced apoptosis, the possible mechanism underlying this effect might be associated with the reduction of intracellular ROS level.

Optimize multiplicity of infection and selected concentration of antibiotics in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2 / 中国生化药物杂志

Objective To optimize multiplicity of infection ( MOI) and antibiotics ( blasticidin) concentration selecting BSD gene in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2.Methods The INS-1 cells were transfected by fluorescence labeled negative control SGMS2-siRNA lentivirus at MOI of 0, 10, 30, 60 and 120 TU number/cell.The cells were photographed under fluorescent microscopy after 72 h cultivation, then fluorescence ratio and apoptosis rate were calculated to determine optimal MOI.The INS-1 cells were treated by blasticidin with different concentrations of 0, 1, 2, and 3 μg/mL, and the apoptosis rate was observed to acquire optimal concentration of antibiotics.The INS-1 cells were transfected by negative control SGMS2-siRNA lentivirus and SGMS2-siRNA lentivirus (virus titer:1 ×108TU/mL) at optimal MOI and positive-transfected cells were selected by blasticidin at optimal concentration, then mixed cell lines were acquired.The monoclonal cell line was constructed at fluorescence ratio of 90%.Results The optimal MOI was 60 with 100% fluorescence ratio, less than 0.5% apoptosis rate and keep original cellular morphology.The optimal concentration of blasticidin was 2 μg/mL with cell adherence disappear and all cells apoptosis.The Ct value of INS-1-SEMS2 cells detected at the second time was 28.21, which was greater than 27.58 at the first time.The interfering efficiency of siRNA was 77.78% which indicated a successful expression of siRNA and construction of monoclonal stable cell line ( INS-1-SEMS2 ).Conclusion The monoclonal stable cell line was successfully constructed by lentivirus vector-mediated RNA interence silenced gene SGMS2.

Influence of the expression of parathyroid hormone 1 receptor to INS-1 cell function under high glucose condition / 中华内分泌代谢杂志

Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.

Protection of INS-1 cells from STZ injury by gastrodine / 中华内分泌外科杂志

ObjectiveTo study effects of gastrodine (GAS) on insulinoma (INS-1) cells and the protection of INS-1 ceils from steptozotocin (STZ) injury by gaatrodine. MethodsThe experiment was carried out in 5 groups: normal control group ( NC), GAS group (GAS), streptozotocin group (STZ), GAS protection group ( GAS +STZ) and GAS repair group (STZ +GAS). INS-1 cells were cultured, the cell viability was determined by tetrazolium (MTT) assay, insulin concentration was detected by radioimmunoassay, and malondialdehyde (MDA)concentration and total antioxidant capacity (T-AOC) of the culture medium were measured by colorimetry. Results GAS promoted insulin release of INS-1 cells (P＜0.05, P＜O.01). Low-concentration GAS could increase viability of INS-1 cells ( P ＜ 0.01 ). GAS could increase viability of the injured INS-1 cells (P ＜ 0.01 ). High concentration GAS contributed in repair of INS-1 cells injured by STZ and promoted insulin serection ( P ＜ 0.01 ). GAScould decrease MDA concentration (P ＜0.01 ) and significantly increase T-AOC capacity of INS-1 cells injured by STZ (P ＜0.01 ). ConclusionsGAS can increase INS-1 viability, promote insulin secretion of INS-1 cells, alleviate INS-1 cells injury caused by STZ, and strengthen the antioxidant capacity of INS-1 cells injured by STZ.

HIV-1 protease inhibitor nelfinavir decreases insulin secretion from rat insulinoma INS-1 cells / 中华内分泌代谢杂志

Rat INS-1 cells treated with HIV-1 protease inhibitor Nelfinavir for 48 h showed that both basal and glucose-stimulated insulin secretion decreased, the latter was more marked, suggesting that long-term treatment with Nelfinavir may impair ?-cell function.

Construction of mouse PPAR? adenovirus vector and its expression in INS-1 cells / 第三军医大学学报

Objective To construct the recombinant adenovirus vector containing mouse PPAR?,and infect the INS-1 cells.Methods The pCDNA3.0-mPPAR? plasmid was digested with KpnⅠ and EcoRⅤ to obtain full-length coding sequence of mPPAR?,then the mPPAR? cDNA was ligated to the pAdtrack-CMV vector.The plasmid of pAdTrack-mPPAR? was linearized with PmeⅠ,and the fragment was reclaimed and transformed into BJ5183 which contains pAdeasy.After being screened,the extracted plasmid of positive bacteria linearized with PacⅠ was transfected into HEK293 cells with lipofectamine~(TM) 2000 and was identified by the green fluorescence protein(GFP).The harvested virus was amplified in HEK293 cells and infected INS-1 cells.After infection,the expression of PPAR? was proved by GFP expression and Western blotting.Results The recombinant adenovirus vector containing mouse PPAR? was constructed successfully and the titer was 1.5?10~(10) pfu/ml.The PPAR? expression in INS-1 cells infected by the virus was much higher than the controls.Conclusion The constructed recombinant adenovirus vector containing mouse PPAR? provides a potent tool to investigate its biological function in pancreatic islets and other tissues.

Effects of different concentrations of glucose on PPAR? expression in INS-1 cells / 第三军医大学学报

Objectives To observe the effects of glucose at different concentrations on the expression of PPAR? in the rat insulinoma INS-1 cells so as to illustrate the function of PPAR? in the cross-talk of lipid metabolism with glucose metabolism in pancreatic cells. Methods After cultured in RPMI1640 media containing 3, 11 and 20 mmol/L glucose respectively for 1 h, the cells were used to measure the content of intracellular triglyceride. The RNA and nuclear protein of INS-1 cells were extracted, and then the expression of PPAR? mRNA was detected by semi-quantity RT-PCR and the PPAR? protein was analyzed by Western blotting. Results With the increment of the concentrations of glucose, the content of intracellular triglyceride in the cultured cells was elevated, while the expressions of PPAR? mRNA and protein were both down-regulated. Conclusion Glucose promotes triglyceride deposition in INS-1 cells, and the down-regulation of PPAR? presumably contributes to the lipid deposition in INS-1 cells during hyperglycemic states.