| Objective:By establishing hyperlipidemia rat model and using ELISA and 16SrRNA high-throughput sequencing technology,the effects of Huatan Huoxue Recipe on intestinal permeability and intestinal flora of hyperlipidemia rat model were observed.and the mechanism of Huatan Huoxue recipe in lowering blood lipid was discussed.Methods:48 SPF male SD rats were randomly divided into normal control group(NC,n=8)and high fat diet group(n=43).The NC group was given ordinary feed,and the high fat diet group was given high fat feed.Four weeks after feeding,five rats in NC group were randomly selected,and eight rats in HFD group were randomly selected.After fasting for 12 hours,orbital blood was collected and serum was separated.The levels of serum lipids(TC,TG,HCL-C,LDL-C)were measured.After successful modeling of hyperlipidemia,high fat diet was randomly divided into 5 groups:high-fat model group(MD,n=8),Lipitor treatment group(LPT,n=8),Huatan Huoxue Recipe high-dose group(HG,n=8),Huatan Huoxue Recipe group(HZ,n=8),Huatan Huoxue Recipe low-dose group(HD,n=8),which continued to feed on a high-fat diet and gavage,while giving LPT group 0.1800mg·ml-1·d-1 Lipitor,HG group 2.7900g·ml-1·d-1 Huatan Huoxue Recipe,HZ group 1.3950g·ml-1·d-1 Huatan Huoxue Recipe,HD group 0.6975g·ml-1·d-1 Huatan Huoxue Recipe,and NC group and MD group an equal volume of distilled water for 4 weeks.At the end of the 8th week of the experiment,each group of rats was sacrificed,and collected their blood samples,liver tissues,intestinal tissues and rats' feces.Measured the liver wet weight and calculated the liver index.Bying an automatic biochemical analyzer,we measured the blood lipid level that included total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL-C),and high-density lipoprotein(HDL-C),measured Liver function index that included alanine aminotransferase(ALT),aspartate transaminase(AST).Hematoxylin&eosin(HE)and oil red staining(ORO)were used to observe the pathological changes of liver tissues.Pathological changes of colon tissues were observed by HE staining.The levels of serum D-lactic acid(DLA)and serum diamine oxidase(DAO)which reflect changes in intestinal permeability were measured by enzyme-linked immunosorbent assay(ELISA).);the high-throughput sequencing of 16S rRNA gene was used to detect the changes of intestinal microflora in rats.Results:1.Compared with NC group.serum TC,TG and LDL-C levels in MD group were significantly increased(all P<0.01),HDL-C level decreased significantly(P<0.01),serum AST and ALT increased significantly(all P<0.01),and liver index increased significantly(P<0.05).HE and oil red staining in livers showed that MD group existed liver cell steatosis and inflammatory cell infiltration.2.Compared with the NC group,the serum DLA levels and serum DAO activity in MD group were significantly increased(all P<0.05),suggesting that the intestinal mucosa was damaged and the intestinal permeability was increased.The HE staining of the colon suggested that the MD group's mucosal structure continuity was destroyed,intestinal lymphoid tissue hyperplasia which suggested chronic inflammation.3.Compared with the NC group,the distribution of intestinal microflora in the MD group was significantly reduced in species diversity and species abundance.The structure of intestinal microflora showed that the ratio of sclerenchyma to Bacteroides increased,and intestinal pathogenic bacteria such as Verrucomicrobia(P=0.083),Proteobacteria(Proteobacteria),Blautia(Clostridium,P<0.05),Ruminococcus(P<0.05,)Coprococcus(Trichoderma,P<0.05)Romboutsia increased,Beneficial intestinal bacteria such as Bacteroidetes,Tenericutes,Deferribacteres,Actinobacteria,Lachnospiraceae,Bacteroidales_S24-7_group and Lactobacillus decreased in abundance.4.Compared with the MD group:serum TC,TG,LDL-C levels in Huatan Huoxue Recipe groups were significantly lower(P<0.05-0.01),and HDL-C levels were significantly higher(both P<0.01),and the effect of reducing TG was better than that of LPT group;the serum AST and ALT levels in Huatan Huoxue Recipe groups were significantly lower(P<0.05-0.01),and the effect of lowering AST was better than that of LPT group.The liver wet weight and liver index of Huatan Huoxue Recipe groups were significantly decreased(P<0.05-0.01).The results of HE and oil red staining showed that the liver of Huatan Huoxue Recipe groups both cell steatosis and inflammatory cell infiltration were improved.It is indicated that Huatan Huoxue Recipe can reduce the blood lipid level of hyperlipidemia rat model,improve liver function,reduce liver fat deposition and steatosis,and protect liver.5.Compared with the MD group:serum DAO activity and serum DLA levels were significantly lower in the high,middle and low dose groups of Huatan Huoxue Recipe(all P<0.01).HE staining results showed that Huatan Huoxue Recipe groups' tissue structure was clearer,and the mucosa and submucosal glands were arranged in an orderly manner,and the muscle layer and the adventitia were relatively intact,suggesting that the intestinal permeability of the medication group was significantly improved.It is indicated that Huayu Huoxue Recipe can repair intestinal mucosal damage caused by hyperlipidemia,reduce intestinal permeability and protect intestinal mucosa.6.Compared with the MD group:the species diversity and species abundance of the intestinal flora in the high,middle and low dose groups of Huatan Huoxue Recipe increased significantly,and the ratio of Firmicutes to Bacteroidetes was decreased,the conditional pathogens in the intestinal tract of the rats in the medication groups decreased,and the beneficial bacteria increased.The specific performance was as follows:harmful bacteria such as Errucomicrobia(P<0.05),Proteobacteria(P<0.05),Deferribacteres,Blautia(P<0.05)were reduced,while Beneficial bacteria such as Bacteroidetes,Actinobacteria,Tenericumes(P<0.05),Lactobacillus,Roseburia(P<0.05),Lachnospiraceae_NK4A136(P<0.05),Bacteroidales S24-7 were reduced.It is indicated that Huatan Huoxue Recipe can increase the species diversity and species abundance of intestinal flora,enrich the beneficial bacteria,inhibit the pathogenic bacteria,and optimize the structure of intestinal flora destroyed by high-fat diet.Conclusion:1.Huatan Huoxue Recipe can reduce blood lipid levels in high-fat rat model,improve liver function,reduce liver fat deposition and steatosis,protect liver,repair intestinal mucosal damage caused by hyperlipidemia,reduce intestinal permeability,and protect intestinal the mucosa,increases the species diversity and richness of the intestinal microflora,enriches the beneficial bacteria,inhibits the pathogenic bacteria,and optimizes the structure of the intestinal flora.2.The mechanism of Huatan Huoxue Recipe regulating lipid metabolism is related to optimizing intestinal microflora structure.repairing intestinal mucosal barrier and improving intestinal permeability. |
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