Study On The Mechanism Of Improving The IR Of Active Components From Agrimony By Nrf2-ARE Signal Pathway

Diabetes mellitus is a metabolic disease of endocrine disorders,which mainly accompanied by low glucose tolerance,positive urine and persistent high blood sugar and other pathological features.At present,it is mainly classified as type 1 diabetes(T1DM)and type 2 diabetes(T2DM).In fact,T2 DM has attracted global widespread attention,because it accounts for more than 90% of all diabetics.Insulin resistance(IR)is caused by oxidative stress through multiple pathways and multiple targets acting on insulin signaling pathways,and it is the primary cause of T2 DM.The oxidative stress signaling pathway as a targeting pathway,which has great significance meaning on the development of new drugs of diabetes in researching and exploiting of improved IR drugs.Therefore,we will further explore the specific mechanism of FC and TC components in Agrimony to alleviate the effect of IR by anti-oxidation pathway for clarify the specific pathways,and signal pathways of improve IR in these active components of Agrimony.These works based on the active components FC and TC have antioxidant activities in our previous studies,and it will lay the foundation in the development of active components of as antidiabetic drug.The main contents and conclusions of this paper are listed as follows:(1)A stable model of IR in vitro by high glucose induced HepG2 cells has been established.(1)The HepG2 cells have been treated with different concentrations of high glucose culture medium,and we have been observed the situation of glucose uptake.The results have been shown that the fluorescence value of uptake 2-NBDG in the intervention group of high glucose(55mM)was(0.49)is the lowest in all the high glucose intervention group,and is half of the value of normal control group.In the sugar consumption experiment,there are significant differences between the intervention group and the normal control group after 12 hours,and the glucose consumptions were 1.65 and 4.12 mM,respectively.After 24 hours of intervention,the significant differences are still existed,and the glucose consumptions were 6.38 and 3.50 mM,respectively.Therefore,we can be sure that the optimal conditions of modeling is the high glucose(55mM)and intervention 24 h,and the model can maintain a stable IR state within 24 h.(2)The results of levels of ROS in the cells showed that the high glucose(55mM)intervention group was significantly higher than other groups about the average fluorescence.Consequently,the oxidative stress level increased significantly in the model of IR,which indicated that oxidative stress plays a key role in the pathogenesis of IR.(2)Studied on the effects of FC and TC on glucose metabolism in IR-HepG2 cells.(1)We respectively got the mean fluorescence values of relative to the normal control group were 0.84 and 0.75 by normalization,when the concentration of FC and TC was 2.5 μg/m L.Compared with the model group(0.51),the fluorescence values were increased by 33% and 24%,respectively.There were significant differences between the FC group,TC group and the model group,which indicated FC and TC could improve the uptake of 2-NBDG in IR cells.(2)FC and TC glucose consumption were 5.69 and 5.17 mM at a concentration of 2.5μg/mL,but the normal control group and the model group were 6.43 and 3.45 mM.Compared with the model group,the glucose consumption was significantly increased in FC and TC group.These results indicated that the glucose metabolism in IR cells can be significantly improved when FC and TC reached a certain concentration.(3)Studied on the effects FC and TC on oxidative stress in IR-HepG2 cells by using biochemical method and q-PCR technology.(1)In the IR group,the intracellular ROS content decreased gradually accompanied with the increase of FC and TC concentration.When the FC and TC concentration reached 2.5μg/m L,the average fluorescence values of ROS were 80403.3 and 107841.6 respectively,and the normal control group and model group were 76767.1 and 142026.8 respectively.Compared with the model group,the intracellular ROS concentration in FC and TC groups reduced significantly.FC and TC have the similar regulation effect on the oxidative stress damage markers MDA.Indicating that both of FC and TC can effectively reduce the content of ROS and MDA in cells of IR.Meanwhile,they can enhance the activity of SOD(superoxide dismutase).(2)The relative expression of antioxidant gene Nrf2 increased with the increase of FC and TC concentration.FC and TC can significantly enhance the relative expression of gene Nrf2 at the concentration of 2.5μg/m L.(3)The expression of antioxidant enzyme gene of GSH-PX,NQO1,HO-1 and γ-GCS increased with the increase of FC and TC concentration.Compared with the model group,FC and TC can significantly enhance the relative expression of antioxidant enzyme gene at the concentration of 2.5 μg/m L.The above results suggested that components of FC and TC can effectively improve the oxidative stress in cells of IR-HepG2 by activating the Nrf2-ARE antioxidant signaling pathway to initiate the expression of downstream antioxidant enzymes gene.(4)Studied on the effects FC and TC on JNK and IRS-1 / PI3 K / Akt signaling pathways in IR-Hep G2 cells by using q-PCR and Western blotting technology.(1)The expression of gene JNK was decreased accompanied with the increase of FC and TC concentration,showing dose-dependent.Compared with the model group,FC can significantly reduce the relative expression of gene JNK at the concentration of 2.5μg/mL.(2)The relative expression of gene IRS-1 increased with the increase of FC and TC concentration in a dose-dependent manner.Compared with the model group,FC and TC can significantly enhance the relative expression of gene IRS-1 at the concentration of 2.5μg/m L.Both of them have similar effects on the relative expression of gene Akt.(3)Compared with the normal control group,the phosphorylation level of JNK(Thr183 / Tyr185)and IRS-1ser307 were significantly increased in the cell of model group.FC and TC can significantly reduce the phosphorylation of JNK(Thr183 / Tyr185)and IRS-1ser307 at the concentration of 2.5μg/m L.The above results suggested that FC and TC can upregulate the expression of gene IRS-1 and decrease the phosphorylation level of IRS-1ser307 by down-regulating the expression of gene JNK and decreasing the phosphorylation of JNK(Thr183 / Tyr185)at a certain concentration,so that the insulin signal to normal transmission,and ultimately to improve IR.