Study On Insulin Resistance And The Mechanism Of Tangnaikang Intervene On ZDF Rats With Spontaneous Type 2 Diabetes Mellitus

Objective: To observe the effects of TNK on the insulin resistance in ZDF rats with spontaneous T2 DM.Study the change of the serum marker metabolites in the ZDF rats based on the 1H NMR,and the alteration of the gut microbiota tructure by the 16 S r DNA,explore the the mechanism of TNK intervenen on the insulin resistance.Methods:（1）Took the 10 male ZDF（fa/+）rats as control group,and 50 male ZDF（fa/fa）rats（the random blood glucose over 11.1mmol/L in different days）were divided into five groups at random according to the weight and random blood glucose: the model group（normal saline 10ml/kg）,metformin group（metformin 0.18g/kg）,TNK high dose group（TNK,3.24g/kg）,TNK medium dose group（TNK,1.62g/kg）,TNK low dose group（TNK,0.81g/kg）.Monitored the body weight,random（RBG）and fasting blood glucose（FBG）,food intake of the rats weekly.Monitored the glucose and insulin tolerance at every three week.At the end of the experiment,measured the FBG before anesthesia;and then tested fasting serum insulin,total cholesterol,triglyceride,free fatty acids,high density lipoprotein,low densith lipoprotein,glutamicpyruvic transaminase,glutamicoxalacetic transaminase,CREA and UREA with the blood,and caculated the area under the curve based on the OGTT,ITT,as well as the HOMA-IR based on the HOMA-IR=FBG×FINS/22.5.（2）Took the six male ZDF（fa/+）rat s as the control group,while the eighteen male ZDF（fa/fa）rats were divided into three groups at random: model group（normal saline,10ml/kg）,metformin group（metformin,0.18g/kg）,TNK high-dose group（TNK,3.24g/kg）,depending on their body weight and RBG（the RBG level≥16.7mmol/L on different date）.Monitored the food intake of the rats weekly,the body weight,RBG,FBG in every two weeks,and monitored the OGTT,ITT in three week.Fed six weeks,the changes of the serum metabolites in ZDF rats were detected by 1H NMR and the metabolic profile were analyzed by multivariate statistical analysis.Identified the potential biomarkers of T2 DM by the differential metabolites,and analyzed the metabolic pathways according to the metabolic network.Detected the levels of serum FINS,TG,TC and FFA of model group and TNK high dose group,calculated HOMA-IR,and collected the fecal in the intestine of the rats.Took three rats from each group for reverse transcription-polymerase chain reaction（RT-PCR）.The changes of 16 S r RNA V4 gut microbiota were analyzed by amplification of sub-pyrophosphate,and the differences between the two groups were analyzed by fluorescence quantitative test.Then analyed the correlation of gut microbiota with FBG,TG,FFA,FINS,HOMA-IR and serum metabolites.Results:（1）The body weight,RBG,FBG of the ZDF（fa/+）rats and ZDF（fa/fa）rats had significant difference（P<0.01）at the begining,while the weight of the ZDF（fa/fa）rats had no significant difference（P>0.05）.After six weeks of the feeding,the food intake,body weight,RBG,FBG,OGTT AUC and ITT AUC level of the ZDF（fa/fa）rats were significantly increased compared with the control group（P<0.01）,while the TC,TG,FFA,LDL,ALT,AST,CREA,UREA,INS,HOMA-IR were significantly increased（P<0.05）,HDL decreased significantly（P<0.05）.In addition to HDL,INS level is on the rise,the rest of the indexes showed a trend of decrease after the intervention of TNK and metformin compared with the model group.While the RBG,FBG,OGTTAUC,ITTAUC,ALT,AST,CREA,UREA level were significantly lower among the TNK high,medium and low dose group and metformin group（P<0.05）,the food intake,body weight,TC,TG,LDL,HOMA-IR level between TNK high dose group and metformin group were significant lower（P<0.05）.Compared with the metformin group,the weight,FBG,six weeks ITTAUC,FFA levels of TNK medium and low dose group increased significantly（P<0.05）,AST,CREA,UREA level of TNK low dose group arised significantly（P<0.05）.At the same time,TNK high-dose group had no difference with metformin group（P>0.05）.（2）Study on the mechanism of IR,（1）compared with the control group,the food intake,body weight,RBG and FBG of ZDF（fa/fa）rats were significantly increased（P<0.01）.Compared with model group,the body weight,RBG and FBG of the ZDF（fa/fa）rats fed with the TNK and metformin were significantly decreased（P<0.05）.Compared with metformin group,there was no significant changes in TNK high dose group（P>0.05）.（2）The results of serum metabolomics showed there were significant different metabolites between the model group and the control group: glucose,latate,acetate,glutamine,creatine,valine,glycine,pyruvate,leucine,isoleucine.The acetate,glutamate,pyruvate,aspartate,betaine,valine,isoleucine,α-ketoglutarate,and proline were significant different in TNK high dose group.And these different metabolites are mainly associated with glucose and amino acid metabolic pathways,according to the analysis of metabolic pathways.（3）Results of the gut microbiota deplayed that Romboutsia and Prevotella₉ were the biomarkers of control and model group.Corynebacterium 1,Prevotella₉ and Lactobacillus were the biomarkers of the TNK high dose group and model group.Compared with the control group,the diversity and abundance of model group were significantly decreased（P<0.01）,the abundance of Bacteroidetes significantly increased（P<0.05）,while Firmicutes decreased significantly in the model group at the phylum level（P<0.05）;the abundance of Prevotella₉ significantly arised while the Romboutsia decreased significantly at genus level（P<0.05）.Compared with model group,the diversity and abundance of the TNK high dose group increased while was no statistical difference（P>0.05）.The abundance of Firmicutes and Proteobacteriaon increased significantly,the Bacteroidetes decreased at the phylum level（P<0.05）,and the Romboutsia and Lactobacillus significantly arised,at the while,the Prevotella₉decreased significantly at the genus level in TNK high dose group（P<0.05）.The result of quantitative PCR showed that there was no significant difference in the abundance of Firmicutes among the three groups,which were different from that of the pyrophosphate sequencing.The aboundance of Prevotella and Lactobacillus were consistent with the results of pyrosequencing.Prevotella₉ was positively correlated with the body weight.Romboutsia,X.Eubacterium._coprostanoligenes_group and Lachnospiraceae_NK4A136_group were negatively correlated with FBG,TG,FFA,Weight,FINS and HOMA-IR.The correlation that Romboutsia with FBG,TG,FFA,FINS,HOMA-IR;X.Eubacterium.with TG,FFA,FINS;Lachnospiraceae_NK4A136_group with TG;Prevotella₉ with weight were statistically significant.The relationship of gut microbiota and the metabolomics: Lactate highly negatively correlated with Firmicutes and Proteobacteriaon,and highly correlated with Bacteroides.Creatine has a high positive correlation with Chloroflexi and Cyanobacteria.Inositol was highly negatively correlated with Firmicutes and Actinobacteria,and highly positively correlated with Bacteroides.Acetate was highly negatively correlated with X.Eubacterium._coprostanoligenes_group.Lactate and inositol were highly positively correlated with Prevotella₉ and Valine also highly positively correlated with Blautia.Conclusion:（1）TNK could regulate the metabolism of glucose and lipid,and improve the insulin resistance of the ZDF rats.（2）The result of metabonomics showed,there were significante differences between ZDF（fa/fa）and ZDF（fa/+）rats in the serum marker metabolites.Glycolysis,gluconeogenesis and amino metabolism may be the mechanism of TNK that improve IR of T2 DM based on the pathway analysis.Above all,IR and the effect of TNK improve could be predicted by the level of branched chain amino acids,which had an instructive role,while the specific mechanisms need to be further explored.（3）The analysis of 16 S r DNA revealed,the structures of gut microbiota significantly different in ZDF（fa/fa）and ZDF（fa/+）rats.The improvement of insulin resistance in ZDF（fa/fa）rats by TNK could be associated with the aboundance of Lactobacillus,Prevotella₉,and Romboutsia.Above all,the main mechanism of TNK that improved the insulin resista nce of ZDF rats maybe associated with the gluconeogenesis,glycolytic and a minoacid metabolism,also related with the Lactobacillus,Prevotella₉,and Romboutsia.