| Objective:By establishing the rat model of IGT,to observe the influence of Huayuwendan decoction on IGT rat liver oxidative stress and intestinal flora,further clarify the mechanism of Huayuwendan decoction remedy IGT,to provide experimental basis for clinical application of Huayuwendan decoction.Methods:10 male SD rats were randomly selected from 70 male SD rats as blank group,the rest 60 is only given the high fat and sugar feed for 20 weeks to established the IGT rat model.The stability of the model was identified at the end of 21st week.30 IGT model rats were randomly divided into model group,Huayuwendan decoction group and metformin group,each group of 10.Huayuwendan decoction group was treated with Huayuwendan decoction,metformin group was treated with metformin hydrochloride,blank group and model group were given saline,the total duration of the drug was 4 weeks.Rats FPG and 2hPG were determined on the 25st week,then the serum,liver tissue,and colon feces were collected.The total SOD activity was determined by wst-8 method,the serum CAT,CRP and TNF-α in rats were determined by ELISA,the mRNA expression levels of Nrf2 and HO-1 were detected by Real time qPCR,the protein expression levels of Nrf2 and HO-1 were detected by Western blot,the structure of intestinal flora was detected by High-throughput sequencing.Results:1.In 20 weeks of high-fat and high-sugar diet,41 rats up to standard of IGT model,and the stability was assessed at the end of 21st week,34 IGT models were stable,and the rate of IGT rat model was 56.67%.2.The results of serum FPG and 2hPG showed:On the end of 21st week,compare with blank group,the other three groups were significantly higher in FPG and 2hPG(P<0.01);On the end of 25st week,compare with blank group,model group and Huayuwendan decoction group were significantly higher in FPG and 2hPG(P<0.01);compare with model group,Huayuwendan decoction group and metformin group were decreased in FPG and 2hPG(P<0.05,P<0.01).3.Serum SOD activity determination results showed:compare with blank group,the activity of SOD in model group and Huayuwendan decoction group were decreased(P<0.05,P<0.01);compare with model group,SOD activity was significantly increased in Huayuwendan decoction group and metformin group(P<0.01).4.ELISA assay results showed:compare with blank group,the serum CAT content decreased and CRP content increased in the other 3 groups(P<0.05,P<0.01),serum TNF-α in model group and Huayuwendan decoction group was increased(P<0.05,P<0.01);compare with model group,serum CAT in Huayuwendan decoction group and metformin group was increased(P<0.01);compare with metformin group,the CAT content decreased in Huayuwendan decoction group.5.Real time qPCR and Western blot test results showed:compare with blank group,the expression levels of Nrf2 and HO-1 mRNA in the liver of the other 3 groups were increased(P<0.05,P<0.01),and the expression level of Nrf2 and HO-1 protein was significantly increased(P<0.01);compare with model group,both Nrf2 and HO-1 mRNA and protein expression levels in the liver of Huayuwendan decoction group and metformin group were increased(P<0.05,P<0.01).6.High throughput sequencing for detection of intestinal flora results showed:①The dilution curve showed that OTU increases as the number of sequences increases,inflection point show at the sequence of 4000,it indicates that the amount of sequencing data is reasonable;②α diversity index results showed:compare with blank group,the index of ACE,Chao1,Shannon and Simpson were significantly reduced in the model group(P<0.01);compare with model group,the index of ACE,Chao1,Shannon and Simpson in the group of Huayuwendan decoction group and metformin group was significantly increased(P<0.01);③Rank-abundance curve results showed:The homogeneity of the bacteria and the abundance of the bacteria were from high to low,which were blank group,Huayuwendan decoction group,metformin group,model group;④Anosim analyses results showed that there were differences in intestinal flora structure between the four groups(P<0.05,P<0.01);⑤Metastats analyses results showed that there were differences in the abundance of the intestinal flora between the four groups(P<0.05).Conclusions:1.The model of IGT rat model was successfully prepared by long range high-fat and high-sugar diet,and the model replication rate was 56.67%;2.Huayuwendan decoction could increase the serum SOD activity and CAT in IGT rats,may be related to liver Nrf2,HO-1 mRNA and protein expression level raised,to improve the antioxidant capacity of IGT rats,in order to improve the state of oxidative stress.3.Long range high-fat and high-sugar diet can reduce the diversity and the abundance of intestinal flora in rats with IGT model.4.Huayuwendan decoction can improve the diversity and abundance of intestinal flora in IGT rats model by long range high-fat and high-sugar feeding.5.The improvement of oxidative stress and intestinal flora structure may be one of the mechanisms for treating IGT. |
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