The protective effect of puerarin on MIN6 cell injury induced by palmitic acid / 中国药理学通报

Aim To evaluate the effects of puerarin (PR) on pancreatic islet MIN6 cell injury and apopto- sis induced by palmitic acirl ( PA).Methods MIN6 cells pretreated with 2 h different concentrations of PR were then co-cultured with 120 (xmol • L"1 PA for 24 h to establish the cell injury and apoptosis model.MTT, LDH,MDA and GSH were used to determine the dam¬age of MIN6 cells.AOEB fluorescence staining was used to detect the apoptosis of MIN6 cells.Western blot was used to detect the expressions of inflammation- related protein NF-kB , apoptosis-related factors Bcl-2 and Bax.Results Compared with model group, cell viability and GSH activity of puerarin administration groups increased, LDH and MDA contents decreased.the protein expressions of p-NF-KB and Bax were down-regulated, and the protein expressions of Bcl-2 were up-regulated (P <0.05).Conclusions Puerar- in ean improve the function of pancreatic islet cells by inhibiting apoptosis and inflammation, and ameliorate pancreatic islet MIN6 cell injury and apoptosis induced by palmitic acid-induced, alleviate MIN6 cell injury in¬duced by inflammatory factors, which may be achieved by down-regulating the expression of p-NF-KB and Bax proteins,and up-regulating the expression of Bcl-2 pro¬tein.

Mogroside V inhibits H202-induced oxidative damage in MIN6 cells by PI3K/Akt pathway / 中国药理学通报

Aim To investigate the protective effect of mogroside V on hydrogen peroxide ( H,02 )-induced oxidative stress response in mouse islet (3 cells MIN6 and the relation of its mechanism to PI3K/Akt signa¬ling pathway.Methods MIN6 cells were treated with 500 (jimol • L_1 H,(), after mogroside V,and cell via¬bility was detected by MTT.The release of reactive ox¬ygen species ( ROS) and apoptotic percentage of MIN6 cells were determined by flow cytometry.The expres¬sions of apoptosis-related factor Bel-2 , proliferation-re¬lated factor PCNA, protein Akt and p-Akt were deter¬mined by Western blot.Results H,02 restrained the proliferation of MIN6 cells obviously, induced ROS pro¬duction and apoptosis, and reduced the expression of Bel-2 and PCN A.The expressions of protein Akt and p-Akt decreased.After treatment of mogroside V , the release of ROS decreased, and the apoptosis of MIN6 cells was inhibited.The expression levels of apoptosis- related protein Bcl-2 and proliferation-related protein PCN A were reversed.The expressions of protein Akt and P-Akt increased.The viability of MIN6 cells in¬duced by H,0, increased.In addition, mogroside V partly reversed the apoptosis induction and ROS pro¬duction of Akt inhibitor MK2206 (5 jjimol • L"1 ) on MIN6 cells.Conclusions Mogroside V has protec¬tive effect on H202-induced oxidative damage in MIN6 cells and its mechanism is related to PI3K/Akt signa¬ling pathway.

Sangguayin preparation prevents palmitate-induced apoptosis by suppressing endoplasmic reticulum stress and autophagy in db/db mice and MIN6 pancreatic β-cells / 中国天然药物

Sangguayin preparation (SGY-P) is refined from the traditional Chinese medicinal compound Sangguayin, which "clears heat and promotes fluid" and "tonifies kidney and spleen" for "Xiaoke", commonly known as 'Diabetes mellitus' in clinics. Previous studies have shown that SGY-P could reduce insulin resistance and repair damaged pancreas in db/db mice, but the underlying mechanisms were unclear. Here, we investigated whether treatment with SGY-P could protect pancreatic β-cells from apoptosis and uncovered the underlying mechanisms. db/db mice were used to observe the hypoglycemic and islet protective effect in vivo. Apoptosis was induced in mouse insulinoma 6 (MIN6) cells by palmitate, following which the cells were treated with SGY-P for elucidating the anti-apoptotic mechanism in vitro. Cell viability and nuclear morphology were detected by CCK-8 assay and Hoechst 33258 staining. The expression levels of apoptosis-, endoplasmic reticulum (ER) stress-, and autophagy-related proteins were measured by western blot. The results showed that SGY-P reduced fasting blood glucose, pancreatic pathological changes, and islet β-cell apoptosis in db/db mice. Palmitate-induced apoptosis in MIN6 cells was decreased by SGY-P treatment. Hence, SGY-P therapy exhibited a protective effect on pancreatic β-cells by decreasing the expression of cleaved caspase-3, cleaved PARP and Bax, and increasing Bcl-2 by suppressing ER stress (Bip/XBP1/IRE1α/CHOP/Caspase-12) and autophagy (LC3/p62/Atg5) pathways.2/Atg5) pathways.

The protective effect of 1,25-dihydroxyvitamin-d3 on hydrogen peroxide-induced min6 cell apoptosis / 中国药理学通报

Aim To investigate the role and mechanism of VitD in protecting apoptosis of mouse islet (3 cell line MIN6 cells induced by H202. Methods MIN6 cells were treated with H202 after the pretreat-ment of VitD. The proliferation, morphological changes and apoptotic percentage of MIN6 cells were determined by CCK-8, Hoechst 33258 fluorescence staining and flow cytometry respectively. The expressions of ap-optosis-related genes Bel-2, Bax, Caspase-3 and Cleaved caspase-3 were detected by Western blot. Results H202 inhibited proliferation and induced apop-tosis of MIN6 cells through up-regulation of Bax and cleaved caspase-3, down-regulation of Bcl-2, decrease of Bcl-2/Bax ratio,and increase of cleaved caspase-3/caspase-3 ratio. After pretreatment of VitD, MIN6 cell viability was restored by increasing Bcl-2 expression, decreasing Bax and cleaved caspase-3 expression, increasing Bcl-2/Bax ratio, and decreasing cleaved caspase-3/caspase-3 ratio. Conclusions VitD protects MIN6 against H202-induced apoptosis through decreasing pro-apoptotic gene Bax and cleaved caspase-3,and increasing anti-apoptotic gene Bcl-2 expression.

Role of NADPH oxidase in high fat-induced injury in MIN6 islet βcells / 中国药理学通报

Aim To explore the role of nicotinamide adenine dinucleotide phosphate ( NADPH ) oxidase in high fat-induced injury in MIN6 isletβ cells. Methods MIN6 islet β cells were exposed to different concen-trations of palmitic acid ( 0. 1 , 0. 3 , 0. 5 , 0. 8 mmol · L-1 ) for 48 h and different time points of 0. 5 mmol· L-1 palmitic acid(24,48,72,96 h). Cell viability was measured by MTT, the protein expression of NADPH oxidase subunits such as p22 phox , p47 phox , p67 phox and gp91 phox and apoptosis related proteins such as Bcl-2 and Bax were determined by Western blot. Results MIN6 islet cells exposed to palmitic acid at 0. 5 mmol ·L-1 for 48 h showed a decrease in their viability and an increase in the expression of NADPH oxidase sub-units(p22phox、p47phox、p67phox and gp91phox) and Bax(P<0. 05 ) , while Bcl-2 expression was significantly re-duced. the pretreat with NADPH oxidase inhibitor di-phenyliodonium( DPI,10 μmol · L-1 ) significantly in-hibited high fat-induced Bcl-2 and Bax expression( P<0. 05 ) . Conclusion Activated NADPH oxidase might play an important role in the treatment of high fat-in-duced injury in MIN6 islet cells.

Optimization and in vitro validation of EGFP expression controlled by porcine insulin promoter / 中国实验动物学报

Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreaticβ-cells specific transgene expressing pigs.Method Using porcine insu-lin promoter ( PIP, 1500 bp of the 5′UTR from the porcine INS gene including the first exon and the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.

Protective effect of pancreatic β MIN-6 cell on retinal pigment epithelial cell from high glucose-induced damage / 中华实验眼科杂志

BackgroundDiabetic retinopathy (DR) associated with this disease closely approximates the oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose,in recent years,people devote themselves to the protection of RPE cells extensively.ObjectiveTo investigate the protective effect of pancreatic β MIN-6 cells on retinal pigment epithelial(RPE) cells from high glucose-induced damage.MethodsRPE cells were incubated with normal medium for 4 d and divided into 3 groups:normal glucose group,high glucose group and MIN-6 cells group.RPE cell were exposed with 5 mmol/L normal glucose in normal glucose group,exposed with 30 mmol/L high glucose in high glucose group,and exposed with 5 × 104 MIN-6 cells and 30 mmol/L high glucose in MIN-6 cells group.After 24 h,cell viability of RPE cells was determined by MTT cell viability assay.Results More than 95％ cells showed the brown staining by ABC method.After incubation for another 24 hours,the A550 value was 0.44±0.02,0.30±0.01 and 0.41±0.01 in the normal glucose group,high glucose group and MIN-6 cells group respectively with a significant difference among these three groups (F =19.94,P＜ 0.01 ).The A55o value was significantly higher in the normal glucose group and the MIN-6 cells group compared with the high glaucose group (t =6.85,5.62,P＜0.01 ).The survival rate of RPE cells in the normal glucose group was(97.5±3.3 )％,and that in the high glucose group was ( 68.2 ± 4.5 ) ％,showing significant difference between them ( t =11.30,P＜0.01 ).ConclusionsHigh glucose-induced damage of RPE cells is abrogated,and MIN-6 cells can protect RPE cells from high glucose-induced damage.

Reduced Expression of the LRP16 Gene in Mouse Insulinoma (MIN6) Cells Exerts Multiple Effects on Insulin Content, Proliferation and Apoptosis / 华中科技大学学报（医学）（英德文版）

This study assessed the effects of leukemia-related protein 16 (LRP16) on the regulation of pancreatic functions in mouse insulinoma (MIN6) cells.Cells with down-regulated expression of LRP16 were obtained by a shRNA interference strategy.Insulin content and glucose-stimulated insulin secretion (GSIS) were examined by radioimmunoassay.Western blotting was applied to detect protein expression.Glucose-stimulated sub-cellular localization of PDX-1 was immunocytochemically determined.Cell proliferation and apoptosis were detected by flow cytometry.Our results showed that LRP16 regulated insulin content in MIN6 cells by controlling expression of insulin and insulin transcription factors.LRP16 gene silence in MIN6 cells led to reduced cell proliferation and increased apoptosis.The observation of phosphorylation of serine-473 Akt and the localization of PDX-1 to the nucleus under glucose-stimulation exhibited that LRP16 was a component mediating Akt signaling in MIN6 cells.These results suggest that LRP16 plays a key role in maintaining pancreatic β-cell functions and may help us to understand the protective effects of estrogen on the functions of pancreatic β-cells.

Effects of advanced glycation end products on cell viability and level of reactive oxygen species(ROS)in MIN6 cells / 中华内分泌代谢杂志

To explore the effect of advanced glycation end-products(AGEs)on cell viability and level of reactive oxygen species(ROS)in MIN6 cells. After intervention of various concentrations(100,200, and 400 mg/L)of AGEs for some time, cell viability was detected by MTT assay. 2', 7'-dichlorofluorescein diacetate(DCFH-DA)was used as a reactive oxygen species capture agent. The fluorescent intensity of 2', 7'-dichlorofluorescein(DCF), which was the product of cellular oxidation of DCFH-DA, was detected by flow cytometry. The level of ROS and insulin secretion was thus measured. Viability of MIN6 cells was inhibited by AGEs in a dose and time dependent manner(P＜0.05).Intracellular fluorescent intensity of DCF was markedly elevated in the AGEs groups as compared with that in the control group(P＜0.05).Insulin secretion was decreased in the AGEs groups than that in the control group(P＞0.05). The results suggest that AGEs inhibit the viability and induce oxidative stress in MIN6 cells by overproduction of ROS.

Mechanism of lipotoxicity-associated apoptosis mediated by mitochondrial dysfunction in pancreatic β-cells / 中国病理生理杂志

AIM:To investigate the mechanism of lipotoxicity-associated apoptosis mediated by mitochondrial pathway in pancreatic β-cells. METHODS:The pancreatic β-cell line MIN6 cells were incubated respectively in serum-free DMEM medium with or without palmitate (0.1-0.5 mmol/L) for 24 h,followed by Hoechst 33258 staining. The activity of caspase 3 was assayed for determining apoptosis,transmission electron microscope was used for observing mitochondrial structure,and RT-PCR analysis was applied for detection of mRNA expression of bcl-2-associated X protein (bax),B-cell lymphoma 2 (bcl-2),sterol regulatory element binding transcription factor 1c (SREBP1c) and DNA-damage inducible transcript 3 (chop-10). RESULTS:Palmitate induced apoptosis of MIN6 cells and swelling of mitochondria. Palmitate also inhibited mRNA expression of bcl-2,whereas enhanced mRNA expression of bax,SREBP1c and chop-10. CONCLUSION:Palmitate induces apoptosis of pancreatic β-cells by promoting mitochondrial dysfunction,which is associated with abnormal expressions of bax,bcl-2,SREBP1c and chop-10.

Visfatin inhibits pancreatic β-cell line MIN6 apoptosis via PI3K-Akt and MAPK-ERK1/2 signaling pathways / 中华内分泌代谢杂志

To investigate the effects and the mechanism of visfatin on MIN6 cell signaling pathway and apoptosis induced by palmitate.Human recombinant visfatin promotes protein kinase B (Akt) and extracellularsignal regulating kinase (ERK)1/2 phosphorylation in dose-and time-dependent manner,and prevents MIN6 cell from apoptosis induced by palmitate (P<0.05 or P<0.01).The activation of Akt and ERK1/2 signaling pathway may be one of the molecular mechanisms of visfatin.

Palmitate induces cell apoptosis in insulinoma MIN6 cell / 基础医学与临床

Objective To investigate the effect of protein kinase B and its phosphorylation on the apoptosis of MIN6 induced by palmitate.Methods Incubated with different level of palmitate concentration (0 ～ 0.5 mmol/L),MIN6 cells were cultured in high glucose DMEM with or without LY294002 ; the apoptosis of MIN6 cells was detec-ted and quantified with TUNEL technology.Then we inspected the cell ultrastructure through electron microscopy and determined the expression of protein kinase B and its phosphorylation p-PKB (Ser473) by Western blot,fol-lowed by an RT-PCR detection of BAX and BCL-2 expression.Results Apoptosis of MIN6 cells was induced by palmitate in a concentration-dependent correlation and enhanced by LY294002.Palmitate also inhibited phospho-rylation of protein kinase B on Ser473.Finally,we detected a downregulation of BCL-2 mRNA expression and up-regulation of BAX mRNA expression under palmitate-incubated state.Conclusion Palmitate may induce apoptosis of pancreatic β-cells through inhibiting activation of PKB phosphorylation in diabetes.

Microencapsulated pancreatic B cell line as a bioartificial pancreas in agarose/PSSa microbcads / 中华器官移植杂志

Presented in this study was the report of microencapsulated pancreatic B cell line MIN6 in agarose/polystyrene sulfonic acid (PSSa) microbeads as a bioartificial pancreas. One week after subcutaneous xenotransplantation to Lewis rats without use of any immunosup-pressants, the microbeads microencapsulated MIN6 cells were retrieved. The retrieved microbeads were perfectly intact, spherical, smooth and contained viable MIN6 cells. Fibrotic overgrowth and adhesions were rarely observed. The results of the static incubation study revealed that the secretion of insulin from the retrieved microbeads in response to a low glucose concentration was 19.20 ± 1.35μU·100 microbeads-1 /h ; In response to a high glucose stimulation, secretion of insulin was significantly and prominently increased to 51.47±3.82μU·100 microbeads-1/h. The results suggested that this agarose/PSSa microbeads microencapsulated MIN6 cells as a bioartificial pancreas possessed not only an excellent efficacy of immunoisolation, but also a good responsibility of insulin secretion to glucose stimulation.

Transfecting MIN6 cell line and enhancing cell viability by Reg3? cDNA / 西安交通大学学报(医学版)

Objective To create a MIN6 cell line overexpressing murine Reg3? cDNA and to investigate its cell viability character under low glucose concentrations.Methods The full-length murine Reg3? cDNA was inserted into the plasmid pcDNA3.1(-).Then MIN6 cells were transfected with the Reg3?-pcDNA3.1 and pcDNA3.1 vector alone to establish Reg3?-overexpression and empty vector MIN6 cell line.Reg3? protein in the low glucose concentration cell medium was detected by Western blot.Cell viability was evaluated by an MTT reduction conversion assay.Results Three Reg3?-overexpression MIN6 cells were confirmed by real-time PCR and Western blot.Reg3? expression was barely detectable in the cells transfected with the empty vector alone.In contrast,the levels of Reg3? mRNA and protein in three pcDNA-Reg3?-transfected clones were increased by an average 10-and 6-fold,respectively.Western blot also revealed Reg3? protein release into the culture medium.In MTT cell proliferation assay,compared to vector-transfection alone,three clones of Reg3?-overexpression MIN6 cells exhibited 2-fold increases in cell number over 7 days when cultured in the presence of 10 mL/L fetal bovine serum and 5.5 mmol/L glucose.Conclusion The Reg3?-overexpression MIN6 cells have been established.Reg3? protein was detectable in culture medium,supporting an endocrine action,and Reg3? overexpression promoted islet cell growth.