| Objective:Gegenqinlian Decoction has the effect of "clearing heat and drying dampness",which is consistent with the pathogenesis of inflammatory reaction.It is clinically used to treat T2 DM.Therefore,in this study,the NLRP3/Caspase-1/IL-1β pathway of inflammatory cytokines was used to investigate the mechanism of Gegenqinlian Decoction in treating T2 DM by regulating the expression of inflammatory cytokines in liver tissue of T2 DM model mice.Methods:1.75 SPF male T2 DM db/db mice were randomly divided into model group,metformin group(0.2 g/kg),Gegenqinlian decoction high-dose,medium-dose and low-dose groups(61.80,30.90,15.45 g/kg)with 15 mice in each group.Another 15 db/m male mice were selected as blank control group.The blank group and model group were given pure water by equal volume intragastric administration,12 weeks after intragastric administration.2.Observe the hair and mental state of mice every day.Body weight,fasting blood glucose(FBG)and glycated hemoglobin(Hb A1c)contents of each group were measured after the end of intragastric administration.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue.NLRP3 and Caspase-1 were detected by IHC.The contents of fasting serum insulin(FINS)and IL-1β and IL-18 in serum were determined by Elisa.The expression levels of NLRP3,Caspase-1,Pro-Caspase-1,IL-1β and IL-18 in liver tissues were detected by Western Blot.m RNA expression levels of NLRP3,Caspase-1 and IL-1β in liver tissues were detected by RT-q PCR.Results:1.General situation: After 12 weeks of intragastric administration,mice in the model group had listless spirit,dull fur,clumping,large urine volume,yellow urine color and adhesional wetting feces.In the administration group,the spirits were fine,the fur was black and smooth,the urine volume was low,the urine color was light,and the feces were normal.2.Body weight and blood biochemical test results: Compared with blank group,body weight in model group was significantly increased at 0,2,4,6,8,10 and 12 weeks(P < 0.01);Compared with model group,body weight of Gegenqinlian decoction high-dose group,medium-dose group and metformin group decreased at 2,4,6,8,10 and 12 weeks(P < 0.01,P < 0.05).Compared with blank group,FBG level in model group was significantly increased at 0,2,4,6,8,10 and 12 weeks(P < 0.01).Compared with model group,FBG level in metformin group and Gegenqinlian decoction high-dose group at week 0 was significantly decreased(P < 0.01,P < 0.05);At the 2nd,8th and 10 th week,the FBG level of mice in each treatment group was significantly decreased(P < 0.01,P < 0.05);At the 4th,6th and 12 th week,the FBG level in metformin group,Gegenqinlian decoction high-dose group and Gegenqinlian decoction medium-dose group was significantly decreased(P < 0.01,P < 0.05).Compared with blank group,the content of Hb A1 c in model group was significantly increased(P < 0.01);Compared with model group,Hb A1 c content in high-dose and medium-dose groups of Gegenqinlian decoction was significantly decreased(P < 0.01).Compared with blank group,serum FINS level in model group was significantly increased(P< 0.01).Compared with model group,serum FINS level in all treatment groups was significantly decreased(P < 0.01).3.Pathological morphology of liver tissue: compared with blank group,liver cells in model group were steatosis,necrosis and blood stasis after HE staining;Compared with the model group,the pathological changes of liver tissue stained by HE were reduced in all treatment groups.4.The results of IHC assay showed that NLRP3 and Caspase-1 were mainly expressed in the cytoplasm and cell membrane of hepatocytes.Compared with blank group,NLRP3 and Caspase-1 protein expressions in liver of model group were significantly increased(P < 0.01).Compared with model group,NLRP3 and Caspase-1 protein expressions in liver tissues of mice in treatment groups were significantly decreased(P < 0.01).The expression trend of related proteins was basically consistent between IHC and WB.5.Elisa assay results: Compared with blank group,serum levels of IL-1β and IL-18 in model group were significantly increased(P < 0.01);Compared with model group,serum levels of IL-1β and IL-18 in treatment groups were significantly decreased(P < 0.01).6.WB assay results: Compared with blank group,the protein expressions of NLRP3,Caspase-1,pro-Caspase-1,IL-1β and IL-18 in liver tissue of model group were significantly increased(P < 0.01);Compared with model group,the protein expressions of NLRP3,Caspase-1,pro-Caspase-1 and IL-18 in liver of mice in high,medium and low dose groups of Gegenqinlian decoction were significantly decreased(P < 0.01),and the protein expressions of IL-1β in liver of mice in high and medium dose groups of Gegenqinlian decoction were significantly decreased(P < 0.01).The protein expressions of NLRP3,Caspase-1,pro-Caspase-1,IL-1β and IL-18 in metformin group were significantly decreased(P < 0.01,P< 0.05).7.RT-q PCR assay results: Compared with blank group,m RNA expressions of NLRP3,Caspase-1 and IL-1β in liver tissues of model group were significantly increased(P < 0.01,P< 0.05).Compared with model group,NLRP3 and IL-1βm RNA expressions in liver of mice in treatment groups were significantly decreased(P < 0.01,P < 0.05),Caspase-1 m RNA expressions in liver of mice in high-dose and medium-dose groups of Gegenqinlian decoction were significantly decreased(P < 0.01).Conclusion:Gegenqinlian Decoction may achieve hypoglycemic effect by inhibiting the expression of NLRP3/Caspase-1/IL-1β signaling pathway inflammatory factors in liver tissue of db/db mice,so as to improve T2 DM. |
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