| Purpose:The mechanism of action of Jiedu Tongluo Tiaogan Recipe to improve insulin resistance and islet cell protection was observed by animal experiment and cell experiment based on the IRE1/JNK pathway.Methods:1.Animal experiment:Animal model of type 2 diabetes was made by feeding ZDF rats with Purina#5008,and random blood glucose≥11.1mmol/L as a model success criteria.The modeled ZDF rats were divided into model groups,metformin hydrochloride group(0.5·kg-1·d-1),Jiedu Tongluo Tiaogan Recipe high dosage group(5 g·kg-1·d-1),middle dosage group(3 g·kg-1·d-1)and low dosage group(1 g·kg-1·d-1).The rats were sacrificed by feeding the rats drug for 4 weeks and observe the effects on body mass,glycolipid metabolism and other indicators and using Immunohistochemistry,and RT-PCR to observe the changes of P-IRE1、IRE1、P-JNK、JNK,and IRS-1、PI3K、Bax、Bcl-2mRNA in pancreatic tissue.2.Cell experiment:The INS-1 cells were cultured in vitro and the effects of different concentrations of Jiedu Tongluo Tiaogan Recipe on the proliferation of INS-1 cells were detected by MTT.The ERS model was prepared by stimulating INS-1 cells with 5ug/mL tunicamycin for 24h,and interfering with Jiedu Tongluo Tiaogan Recipe（25ug/mL,50ug/mL,100ug/mL）.Cellular protein and mRNA were extracted,and MTT assay,ELASA,Western blotting,and RT-PCR assay were used by observing the changes of IRE1、JNK、IL-6、TNF-α、NF-κb,and IRS-1、PI3K、Bax、Bcl-2mRNA.Results:Animal experiment1.Effects of type 2 diabetes model on body weight and blood glucose in ZDF ratsAt the 11th week of feeding induction,73 ZDF rats were eligible for type 2 diabetes,and the rate of formation was 82.95%.2.Effects of Jiedu Tongluo Tiaogan Recipe on body weight,sugar and lipid metabolism in rats.In terms of body weight,compared with the model group,the weight of the western medicine group and the Jiedu Tongluo Tiaogan Recipe high,middle and low dosage groups decreased,but there was no statistical difference compared with the normal group;In terms of GLU,compared with the model group,there was a difference between the western medicine group and the Jiedu Tongluo Tiaogan Recipe high and middle dose groups（P<0.05）.In terms of TC and TG,compared with the model group,the western medicine group and Jiedu Tongluo Tiaogan Recipe high,medium and low dose groups decreased significantly（P<0.01,P<0.05）.3.Effects of Jiedu Tongluo Tiaogan Recipe on IRE1/JNK conduction pathway in Islet tissueBy immunohistochemical method,compared with the model group,the expressions of IRE1,P-IREI,JNK and P-JNK protein in the cytoplasm of islets of western medicine group and Jiedu Tongluo Tiaogan Recipe high,meddle,and low-dosage groups were significantly decreased（P<0.05,P<0.01）.4.Effects of Jiedu Tongluo Tiaogan Recipe on insulin resistance and apoptosis in Islet tissueUsing RT-PCR,compared with the model group,the western medicine group,the low,middle and high dose groups of Jiedu Tongluo Tiaogan Recipe and significantly improved the expression of IRS-1 and PI3K mRNA（P<0.01）.Both Jiedu Tongluo Tiaogan Recipe highand middle dosage groups and the western medicine group significantly decreased Bax mRNA and increased Bcl-2 mRNA expression（P<0.01）,which significantly reduced the ratio of Bax/Bcl-2<0.01).Cell experiment1.The effect of Jiedu Tongluo Tiaogan Recipe on the survival rate and apoptosis of INS-1cellsCompared with the control group,there was no significant differences in the survival rate of INS-1 cells in the different dose groups of Jiedu Tongluo Tiaogan Recipe from 12.5 to100μg/mL detection by MTT.When the drug concentration reached 150μg/mL,it was cytotoxic compared with the control group.When treated with tunicamycin,the cell viability of INS-1 cells was significantly lower than that of the control group（P<0.01）,and there was no significant difference in the survival rate of INS-1 cells compared with the model group after using the concentration of 25,50 and 100μg/mL of Jiedu Tongluo Tiaogan Recipe（P<0.05,P<0.01）.2.Effects of Jiedu Tongluo Tiaogan Recipe on IRE1/JNK Signaling Pathway of TM Stimulated INS-1 CellsThe expression of IRE-1 and JNK2 was significantly lower than that of the model group under the intervention of 25,50 and 100μg/mL of Jiedu Tongluo Tiaogan Recipe（P<0.05,P<0.01）by Western blotting,and the expression of IRE1 and JNK2 was the most obvious compared with the model group,showing a concentration-dependent manner.3.Effects of Jiedu Tongluo Tiaogan Recipe on NF-κB Signaling Pathway in INS-1 Cells Stimulated by TuntomycinUnder the intervention of Jiedu Tongluo Tiaogan Recipe 25,50 and 100μg/mL,the expression of NF-kB,TNF-αand IL-6 was significantly lower than that of the model group（P<0.05,P<0.01）by Western blotting and ELASA,and when the concentration was 100μg/mL,the expression of NF-κB,TNF-αand IL-6 was the most obvious,which was concentration-dependent..4.Effects of Jiedu Tongluo Tiaogan Recipe on insulin resistance of INS-1 cells stimulated by tunicamycinCompared with the model group,the mRNA expressions of IRS-1 and PI3K-1 in the high,middle and low dose groups of Jiedu Tongluo Tiaogan Recipe were increased（P<0.05,P<0.01）,and it is dose-dependent.Conclusion:The T2DM animal model prepared by 9-week-old ZDF rats fed with Purina#5008 for 11 weeks induction had the highest rate of mold formation,and the model has the best persistence and stability of hyperglycemia.Intervention of Jiedu Tongluo Tiaogan Recipe in ZDF rat type 2 diabetes animal model can significantly reduce blood sugar and regulate blood lipids.In terms of mechanism,Jiedu Tongluo Tiaogan Recipe can reduce the expression of IRE1,P-IRE1 and JNK,P-JNK in Islet tissue,inhibit the IREl/JNK signaling pathway,thereby improving ERS and promoting the balance between anti-apoptotic protein and pro-apoptotic protein,and it achieves islet cell protection and improve insulin resistance in islet tissue of ZDF rats.At the same time,for tunica-stimulated INS-1 cells,the Recipe can reduce the inflammatory factors by inhibiting the NF-κB pathway,thereby reducing the stimulation of ER,inhibiting the IREl/JNK signaling pathway,improving ERS,finally protecting the islet cells and improve IR. |
PDF Full Text Request