Observation Of Curative Effect Of Tangyiping In The Treatment Of Impaired Glucose Tolerance And Its Mechanism Of Regulating Islet β-cell Pyroptosis

Objective:1.Clinical study: A randomized controlled trial was carried to evaluate the effect of Tangyiping on treating patients of impaired glucose tolerance(IGT).2.Experimental study: Tangyiping intervened in high glucose induced Min6 cells in vitro to observe the regulatory effect of Tangyiping on pyroptosis,further explore the role mechanism of Tangyiping in regulating pyroptosis based on the team’sprevious research,so provide an objective basis for the protection of islet β cells by traditional Chinese medicine.Methods:1.Clinical study: According to the estimation results of sample size,100 IGT patients are randomly divided into 2 groups,50 cases in each group.The control group taked lifestyle intervention,and the Tangyiping group was treated with Tangyiping based on lifestyle intervention.The course of treatment was 24 weeks,including 12 weeks of medication and12 weeks of follow-up.The changes in blood glucose,insulin release,insulin sensitivity,pancreatic β-cell function,glycosylated hemoglobin,disease outcome,blood lipids,and TCM syndromes and symptoms were observed in the two groups before and after treatment.2.Experimental study:(1)Experiment 1: Healthy SPF-grade Wistar male rats were gavaged with Tangyiping water decoction to prepare Tangyiping-containing serum with different volume fractions.The optimal concentration and time of Tangyiping intervention in Min6 cells were screened by CCK-8(Cell Counting Kit-8)method,and then the cells were divided into 3 groups:normal group(5.5mmol/L glucose),model group(25mmol/L glucose),TYP group(25mmol/L glucose +optimal intervention concentration of Tangyiping).Cell viability in each group was detected by CCK-8 method,IL1β and IL18 concentrations in cell supernatant were detected by ELISA,cell morphology and pyroptosis were observed by transmission electron microscope and Hoechst33342/PI double staining.The m RNA and protein expressions of NLRP3,Caspase-1 and GSDMD were detected by q RT-PCR and immunofluorescence.(2)Experiment 2: Construct overexpression TXNIP plasmid to transfect Min6 cells,q RT-PCR method to verify the transfection efficiency,add drugs to intervene the cells,and divide them into 5 groups: normal group(5.5mmol/L glucose),model group(25mmol/L glucose),TYP group(25mmol/L glucose+optimal intervention concentration of Tangyiping),TYP+TXNIP-oe group(25mmol/L glucose+optimal intervention concentration of Tangyiping+TXNIP-over expression),TYP+TXNIP-nc group(25mmol/L glucose+optimal intervention concentration of Tangyiping+TXNIP-negative control).Using the same method as Experiment 1,the occurrence of pyroptosis in the overexpression environment of TXNIP was further detected,and the mechanism of Tangyiping in regulating pyroptosis was discussed.Results:1.Clinical study: 3 patients dropped out during the test period,and finally 49 patients in the Tangyiping group and 48 patients in the control group completed the test,and the dropout rates were both <10%.(1)Blood glucose: Compared with the group before the intervention,both groups reduced FPG and 2h PG after the intervention(P<0.05),and the continuous intervention could maintain the hypoglycemic effect.(2)Glucose tolerance:Compared with the group before the intervention,the FPG,1h PG,2h PG,and AUCG of the two groups were decreased after the intervention(P<0.05).After the intervention,the levels of 1h PG,2h PG and AUCG in Tangyiping group were lower than control group(P<0.01),there was no significant difference about FPG(P>0.05).(3)Insulin release: Compared with the group before the intervention,FINS,1h INS,2h INS and AUCI were significantly decreased after the intervention(P<0.01).After the intervention,FINS,1h INS,2h INS and AUCI in Tangyiping group were significantly lower than control group(P<0.01).(4)Islet β-cell function and insulin resistance: ΔI60/ΔG60,HOMA-IR and AUCI/G were decreased in both groups after intervention(P<0.05),while IAI was increased(P<0.05);there were statistical differences between the two groups in terms of HOMA-IR,IAI and AUCI/G after the intervention(P<0.05).(5)Disease outcome: After follow-up,it was found that 28 patients in the Tangyiping group reversed to NGT,16 patients remained on IGT,and 5 patients developed T2 DM.In the control group,25 patients remained on IGT,15 patients reversed to NGT,and 8 patients developed T2 DM.After the treatment,there were significant differences in the NGT reversal rate,IGT maintenance rate and DM progression rate between the two groups(P<0.05).(6)Hb A1c: Compared with the group before the intervention,the two groups could reduce Hb A1 c after the intervention(P<0.05).After the intervention,the Hb A1 c in the Tangyiping group was lower than that in the control group(P<0.01).(7)Blood lipids: Compared with the group before the intervention,TC,TG and LDL-C in the two groups were decreased(P<0.05).After the intervention,the levels of TG and LDL-C in Tangyiping group were significantly lower than control group(P<0.01),the two groups were equally effective in reducing TC.(8)The efficacy of TCM syndromes: After the treatment,the syndrome efficacy of the Tangyiping group was better than that of the control group(P<0.01).(9)The total score of TCM syndromes: Compared with the group before the intervention,the total score of syndromes in the two groups decreased significantly after treatment(P<0.01).After the intervention,the total score of syndromes in the Tangyiping group was lower than control group(P<0.01).(10)Safety: No serious adverse reactions and abnomaly indicators occurred in the two groups of patients during the test period.2.Experimental study:Experiment 1:(1)The optimal intervention concentration of Tangyiping was 10%volume fraction of drug-containing serum,and the optimal intervention time was 48 h.(2)Cell viability: The cell viability of the model group was significantly weaker than that of the control group(P<0.01),and the cell viability of the TYP group was significantly stronger than model group(P<0.01).(3)Concentrations of IL1β and IL18 in the supernatant: The concentrations of IL1β and IL18 in model group were significantly higher than control group(P < 0.01),and the concentrations in the TYP group were lower than model group(P <0.01).(4)Observation under transmission electron microscope: In the control group,the cell morphology was normal,the internal structure was clear;in the model group,obvious cell pyroptosis was clearly seen;in the TYP group,the cell morphology was slightly deformed,and the structure was basically normal,the cell membrane was intact without rupture,and there was no obvious pyroptosis.(5)Hoechst/PI double staining results: The rate of PIpositive cells in model group was higher than control group(P<0.01).The rate of PI-positive cells in TYP group was lower than model group(P<0.05).(6)q RT-PCR results: The m RNA expressions of NLRP3,Caspase-1 and GSDMD in model group were higher than control group(P<0.01),and the m RNA expressions in TYP group were lower than model group(P< 0.01).(7)Immunofluorescence results: The expression of NLRP3,Caspase-1 and GSDMD in model group was higher than control group,and the expression in TYP group was lower than model group.Experiment 2:(1)After the plasmid was transfected into Min6 cells,TXNIP was successfully overexpressed.(2)Concentrations of IL1β and IL18 in the supernatant: Based on the results of Experiment 1,compared with the TYP group,the concentrations of IL1βand IL18 in the TYP+TXNIP-oe group increased(P<0.01),while the concentrations in the TYP+TXNIP-nc group was not significant difference(P>0.05).(3)Transmission electron microscope: Compared with the TYP group,the pyroptosis of the TYP+TXNIP-oe group was obvious;in the TYP+TXNIP-nc group,shape and structure was basically normal,and there was no obvious pyroptosis.(4)Hoechst/PI double staining results: Based on the results of Experiment 1,compared with the TYP group,the rate of PI-positive cells in the TYP+TXNIP-oe group increased(P<0.01),and the rate of PI-positive cells in the TYP+TXNIP-nc group were similar(P>0.05).(5)q RT-PCR results: Based on the results of Experiment 1,the m RNA expressions of NLRP3,Caspase-1 and GSDMD in the TYP+TXNIP-oe group were significantly increased(P<0.01),but there was no significant difference in the TYP+TXNIP-nc group(P>0.05).(6)Immunofluorescence results: Based on the results of Experiment 1,the expressions of NLRP3,Caspase-1 and GSDMD in the TYP+TXNIP-oe group were increased,but there was no significant difference in the TYP+TXNIP-nc group.Conclusions:1.Clinical research: Tangyiping can effectively reduce blood glucose,especially 2h PG,regulate insulin secretion,improve IR,reduce Hb1 Ac,reverse IGT process,improve TCM syndromes and blood lipid,without obvious adverse reactions and adverse events,with good efficacy and safety.2.Experimental research: Tangyiping has a regulatory effect on the pyroptosis of Min6 cells,and can improve the pyroptosis caused by high glucose.Overexpression of TXNIP can cause aggravation of pyroptosis,and Tangyiping may improve islet β-cell pyroptosis by inhibiting the expression of TXNIP,thereby inhibiting the classical pyroptosis pathway of NLRP3/Caspase-1/GSDMD.