Insulin Resistance Caused By H₂O₂ And Improved By Ginkgo Biloba Extract In L6 Cells

Background:Diabetes is a serious public health problem in the world,and the prevalence rate is rising worldwide.Insulin resistance is an important pathogenesis of type 2 diabetes.Oxidative stress is an important cause of insulin resistance in skeletal muscle.ROS activates NF-?B,which induces stress-sensitive pathways,and affects the conduction of insulin signaling pathway,induce Insulin resistance.NF-?B is an important target of oxidative stress,and it is particularly important to understand and clarify the specific mechanism of NF-?B in oxidative stress-induced insulin resistance.Oxidative stress can be controlled by internal and external factors,and by reducing oxidative stress prevention and treatment of insulin resistance is a new research direction.In this study,oxidative stress models were established by stimulating rat skeletal muscle cells with a certain concentration of hydrogen peroxide,and glucose uptake and insulin resistance were measured.The levels of insulin resistance were observed by using PDTC pretreatment.After understanding the specific mechanism of hydrogen peroxide-induced insulin resistance in skeletal muscle of rats,we wanted to explore whether the extract of Ginkgo biloba extract could improve the insulin resistance of L6 cells and explore the specific mechanism.The study is divided into the following two parts.Part ? Study on hydrogen peroxide-induce d insulin resistance and its mechanismObje ctive :To study the effect of hydrogen peroxide on insulin resistance in L6 cells and to explore the specific mechanism.Me thods:L6 cells were cultured in vitro and induced to differentiate into mature skeletal muscle cells.Cell viability was determined by MTT assay.The concentration and duration of H₂O₂ and PDTC were determined.The cells were divided into 4 groups: normal group,H₂O₂ group,H₂O₂+PDTC group and PDTC group.The production of ROS was detected by DCFH-DA probe,The apoptosis was observed by DAPI,the leavel of glucose uptake was measured b y 2-NBDG method,The production of TNF-?,IL-1?,IL-6 in the supernatant was detected by ELISA.The expression.of Bax,Bcl2,Caspase-8,I?B-a,P-I?B-a,P38-MAPK,P-P38-MAPK,NF-?B,P-NF-?B,IRS1,P-IRS1 Tyr612,PI3 K,GLUT4 were detected by Western Blot.Results:1.L6 cells induced differentiation: induced differentiation of the first 7 days after the cell fusion into a significant muscle.2.Oxidative stress model established: When the concentration of H₂O₂ was 1.0mmol/L,the cell survival rate was about 70%,determine the concentration of H₂O₂ on 1.0mmol/L.When the conc entration of PDTC was 100? mol/L,the cell survival rate was about 80%,which was higher than that of other experimental groups.At 4h,the inhibitory effect of PDTC on cell viability was relatively small,so determine the incubation concentratio n and time of PDTC were 100? mol/L and 4h.DCFH-DA results showed that cells treated with 1.0 mmol/L H₂O₂ for 4h,the intracellular ROS production was significantly increased,oxidative stress model was established.DAPI showed that: normal group showed nucleus integrity,clear edge,chromatin homogeneity;H₂O₂ cells showed typical apoptosis characteristics,such as cell shrinkage,nuclear fragmentation,apoptotic body formation;and H₂O₂+PDTC group showed individual cells apoptosis,most of which are normal.Compared with the normal group,the relative expression of Bax,the ratio of Bax/Bcl2,the relative expression of Caspase-8 and the expression of Bcl2 in H₂O₂ group were significantly lower than those in normal group?P <0.05?.Compared with H₂O₂ group,the relative expression of Bax in H₂O₂+PDTC group had no significant difference?P> 0.05?.The relative expression of Bcl2 was significantly increased,the ratio of Bax/Bcl2 and Caspase-8 were significantly decreased,The difference was statistically significant?P <0.05?.2-NBDG uptake showed that the capacity of 2-NBDG uptake in H₂O₂ group was significantly lower than that in normal group?P<0.05?,and the difference was statistically significant?P<0.05?.Compared with H₂O₂+PDTC group,the capacity of 2-NBDG uptake was significantly increased?P<0.05?.The results of ELISA showed that the levels of TNF-?,IL-1? and IL-6 in H₂O₂ group were significantly higher than those in normal group?P<0.05?.Compared with H₂O₂ group,the levels of TNF-?,IL-1? and IL-6 in H₂O₂+PDTC group were significantly decreased,the difference was statistically significant?P<0.05?.Compared with normal group,the relative expression of I?B-a,P38-MAPK and NF-?B were significantly increased,the level of P-I?B-a/I?B-a,P-P38/P38,P-NF-?B/NF-?B were significantly increased,the relative expression of IRS1,PI3 K,GLUT4 and the relative level of P-IRS1 Tyr612/IRS1 were significantly decreased in H₂O₂ group?P <0.05?.Compared with H₂O₂ group,the relative expression of I?B-a,P38-MAPK and NF-?B and the levels of P-I?B-a/I?B-a,P-P38/P38,P-NF-?B/NF-?B were significantly decreased,The relative expression of IRS-1,PI3 K and GLUT4 and the relative level of P-IRS1 Tyr612/IRS1 were significantly increased in the H₂O₂+PDTC group?P <0.05?.Conclusion:1.H₂O₂ can cause oxidative stress and apoptosis in L6 cells.2.H₂O₂ can lead to increased production of inflammatory cytokines TNF-?,IL-1? and IL-6.3.H₂O₂ can lead to decreased glucose uptake of cells,causing insulin resistance,the mechanism is the activation of NF-?B pathway.4.Application of PDTC can alleviate apoptosis,reduce the production of ROS and inflammatory factors,improve glucose uptake,inhibit the activation of NF-?B pathway,thereby improving insulin resistance.Part ? Effects of Ginkgo biloba extract on hydrogen peroxide-induced insulin resistanceObje ctive :To study the effect of EGB on hydrogen peroxide-induced insulin resistance and its specific mechanismMethods:L6 cells were cultured in vitro and induced to differentiate into mature skeletal muscle cells.Cell viability was determined by MTT assay.The concentration of EGB was determined.The cells were divided into 7 groups: normal group,H₂O₂ group,H₂O₂+EGB group,H₂O₂+PDTC group,H₂O₂+EGB+PDTC group,PDTC group and EGB group.The production of ROS was detected by DCFH-DA probe,the leavel of glucose uptake was measured by 2-NBDG method,The production of TNF-?,IL-1?,IL-6 in the supernatant was detected by ELISA.The expression.of I?B-a,P-I?B-a,NF-?B,P-NF-?B,IRS1,P-IRS1 Tyr612,GLUT4 were detected by Western Blot.Results:1.The improvement of oxidative stress by EGB: MTT results showed that as the increase of EGB concentration,the survival rate of cells increased first and then stabilized,but decreased slightly.At the EGB concentratio n of 200 ? g/m L,the cell survival rate reached a maximum of 108%,determine the pretreatment concentration of EGB was 200 ?g/m L.The intracellular ROS of the H₂O₂ group was significantly higher than that of the normal group?P<0.05?,the oxidative stress model was established.Compared with the H₂O₂ group,The intracellular ROS of H₂O₂+EGB,H₂O₂+PDTC and H₂O₂+EGB+PDTC group were significantly reduced?P <0.05?.The intracellular ROS of H₂O₂+EGB+PDTC group were lower than those in H₂O₂+EGB group and H₂O₂+PDTC group?P <0.05?.2.Effects of EGB on NF-?B activation and insulin signaling: 2-NBDG uptake test showed that the uptake of 2-NBDG in H₂O₂ group was significantly lower than that in normal group?P<0.05?.The uptake capacity of H₂O₂+EGB,H₂O₂+PDTC and H₂O₂+EGB+PDTC group was significantly higher than that of H₂O₂ group?P<0.05?.The uptake capacity of 2-NBDG in H₂O₂+EGB+PDTC group was significantly higher than that in H₂O₂+PDTC group?P<0.05?.The levels of TNF-?,IL-1? and IL-6 in the H₂O₂ group were significantly higher than those in the normal group?P<0.05?.The levels of TNF-?,IL-1? and IL-6 in H₂O₂+EGB,H₂O₂+PDTC and H₂O₂+EGB+PDTC groups were significantly lower than that in H₂O₂ group?P<0.05?.The level of TNF-? in H₂O₂+EGB+PDTC group was significantly lower than that in H₂O₂+PDTC group?P<0.05?.Western blot results showed that the levels of I?B-?,NF-?B,P-I?B-?/I?B-? were significantly higher and the relative expression of IRS1,GLUT4 and P-IRS1 Tyr612/IRS1 were significantly lower in the H₂O₂ group than those in the normal group?P<0.05?.Compared with the H₂O₂ group,the relative level of P-NF-?B/NF-?B was significantly reduced and the relative expression of IRS1,GLUT4 and P-IRS1 Tyr612/IRS1 were significantly increased and in H₂O₂+EGB,H₂O₂+PDTC and H₂O₂+EGB+PDTC groups?P<0.05?.Conclusion:1.EGB can improve the survival rate of L6 cells and reduce the degree of oxidative stress,and the combination of EGB and PDTC in reducing the degree of oxidative stress on cells is better than the application of EGB alone and t he application of PDTC alone.2.EGB can reduce the production of inflammatory cytokines TNF-?,IL-1? and IL-6 in L6 cells,and the combination of EGB and PDTC is superior to PDTC alone alone in reducing TNF-? production.3.EGB can improve the ability of cell uptake of glucose and improve insulin resistance.The specific mechanism is related to the inhibition of NF-?B pathway activation,and the combination of EGB and PDTC is superior to PDTC alone in improving cell uptake of glucose.