| Nonalcoholic fatty liver disease(NAFLD)is a more and more important chronic liver disease in our coutry.The disease spectrum includes simple nonalcoholic fatty liver,nonalcoholic steatohepatitis(NASH),cirrhosis and liver cancer.Most patients with NAFLD accompanies with obesity,type 2diabetes,dyslipidemia and/or high blood pressure.The first treatment for NAFLD is adjusting diet,exercise and changing lifestyle to losing weight.In recent years,the theory of "liver intestine axis" provides a new way of thinking for the pathogenesis and treatment method of liver disease.Hydrochloride berberine is isoquinoline alkaloid extracted from the rhizome of coptis,which is one of the traditional Chinese medicine in our country.Beberine has significant antimicrobial effect,and is used in the treatment of gastroenteritis and bacterial dysentery as over the couter in the clinical.Recent years,Studies have found that berberine has many potential application values,such as improving insulin resistance,lowering cholesterol,anti-arrhythmia and treating metabolic syndrome.The study explores whether berberine can improve the intestinal mucosa function of NAFLD rat through adjusting the gut microbiota,so as to treat the disease of NAFLD.Objective: To study the protective effect of hydrochloride berberine on the intestinal barrier of NAFLD rats induced by high fat diet through adjusting the gut microbiota,and to provide a theoretical basis for the treatment of NAFLD.Methods: Thirty-three healthy male Sprague-Dawley rats(weighted200±10g)were randomized into three groups after adaptive feeding for a week,the normal group(group N),the model group(group M)and the berberine group(group B),11 rats in each group.Normal diets were given to group N and rats in group M and group B were provided with high-fat diets,in which the fat content was 59.8%.The normal diets and the materials of high-fat diets were both provided by the experimental animal center of Hebei Medical University.After 12 weeks later,rats in each group were randomly killed,using HE staining to determine whether the models were successed.Then rats in group B were given 2ml hydrochloride berberine at the level of 150 mg /(kg.d)by intragastric administration.At the same time,rats in group N and group M were also given the same amount of normal saline.In the 16 th weeks,all of the rats were sacrificed in anesthesia by abdominal aorta bleeding.Blood samples of rats were obtained from the abdominal aorta.Level of ALT,AST,TG,TC,endotoxin,D-lactate and I-FABP were assaied.The changes of liver were observed by HE staining.The changes of intestinal mucosa structure were observed by HE staining,scanning electron microscope and transmission electron microscope.The change of gut microbial of the fresh excreta was analyzed through High-throughput sequencing technology.Results:1 When the rats were fed for 12 weeks: Compared with the group N,the weight of rats in group M and group B were significantly higher,the differences were statistically significant(P<0.05),there was no obvious difference between the group M and group B(P>0.05).The HE staining results showed that the liver cells of group M and group B were filled with fatty deposition,and the results showed that the model was successful.2 When the rats were fed for 16 weeks: The body weight in group M was significantly higher than group N,and the difference was statistically significant(P<0.05).Compared with the group M,the weight of rats in group B was decreased,and the difference was statistically significant(P<0.05).3 The blood test results: Compared with the level of ALT,AST,TG,TC in group N,level of ALT,AST,TG,TC in group M was significantly higher,the difference was statistically significant(P<0.05).Compared with group M,level of ALT,AST,TG,TC in group B was decreased,the difference was statistically significant(P< 0.05).Compared with group N,level of endotoxin,D-lactate,I-FABP in group M was significantly increased,the difference was statistically significant(P<0.05).Compared with group M,level of endotoxin,D-lactate,I-FABP in group B was decreased,the difference was statistically significant(P<0.05).4 HE staining of liver: Compared with group N,the structure of liver was disorderd in group M,and liver cells were arranged in disorder,liver cells were filled with the size of fat vacuoles.In group B,the liver cells were less swollen and fat vacuoles,the liver cells arranged more neatly.5 HE staining of intestinal tissue: Intestinal villis were arranged closely in group N.In group M,the intestinal villis became short,sparse,partial lossing.Compared with group M,the intestinal villus of group B was edema mildly,arranged more neatly.6 Scanning electron microscopy observation of intestinal mucosa: Intestinal villus of rats in group N were like broad thick leaves,arranged closely and neatly.The intestinal villis in group M was flat,arranged sparsely,the local villus were fractured.Compared with group M,the intestinal villus in group B were plumper and arranged more regular and neatly.7 Transmission electron microscope observation of intestinal mucosa: The tight junction on the cavity surface between intestinal mucosa epithelial in group N are long and their structure are clear.The tight junction in group M are disappeared.In group B the tight junction are existing.8 The result of High-throughput sequencing technology8.1 Sequence analysis: compared with the group N,the chao1 index and Shannon index in the group M had the tendency of increase,but the difference was not statistically significant.Compared with the group M,the chao1 index and Shannon index in the group B were less than that in group M and group N.The results indicated that the hydro-chloride berberine was important to the change of intestinal bacteria.8.2 Species accumulation curve: the curve in the experiment was rising slowly,witch indicated that the number of samples was enough and could estimated the aboundance of intestinal bacteria.8.3 Bacteroidetes The change of fecal microbialAt phyla level,The most abundant sequences obtained were the Firmicutes and Bacteroidetes constituting more than 90% of population in the group N and group M.The two bacteria in the group B constituted about 73%.Compared with the group N,the abundance of Firmicutes and Proteobacteria was higher and the abundance of Bacteroidetes and Cyanobacteria was less in the group M.Compared to the group M,a higher abundance of Bacteroidetes and Proteobacteria and a less abundance of Firmicutes and Cyanobacteria was observed in the group B.At genus level,the 20 most predominant genera consisted of 92.5% of the entire genera in the group N.The 20 most abundant genera of bacteria constituted 94.3% of all bacteria in the group M.the 20 most predominant genera consisted of 92.8% of the all genera in the group B.The 3 most predominant genera were Lactobacillus,Prevotella and unknown genera belonging to phylum Firmicutes,order_Clostridiales in the group N.The 3most predominant genera were two unknown genera belonging to phylum Firmicutes,order Clostridiales and one unknown genera belonging to phylum Bacteroidetes,family S24-7 in the group N.The 3 most predominant genera were Bacteroidetes,one unknown genera belonging to phylum Bacteroidetes,family S24-7,one unknown genera belonging to phylum Firmicutes,family Lachnospiraceae in the group B.Compared to the group N,the abundance of Lactobacillus and Prevotella was reduced,and the abundance of unknown genera belonging to order Clostridiales and unknown genera belonging to family Lachnospiraceae and unknown genera belonging to family S24-7 were higher in the group M.Compared to the group M and group N,the abundance of Bacteroides was higher.Compared to the group M,the abundance of unknown genera belonging to family S24-7 and unknown genera belonging to Lachnospiraceae was less in the group B,but it was higher compared to group N.In conclusion,the high fat diets can change the intestinal flora.The change of abundance of the flora is primary.The change of kind of microbia is slight.Hydro-chloride berberine can decrease the diversity of the intestinal flora,enrich the amount of bacteroides and inhibit the growth of the unknown bacteria genus in the family of lachnospiraceae and S24-7.Conclusions:1 High fat diets can induce the model of NAFLD in rats,change the structure of intestinal flora and damage the intestinal mucosal mechanical barrier.2 Hydro-chloride berberine can reduce the level of endotoxin and blood lipid,the body weight and hepatic steatosis and improve the liver function in NAFLD rats.3 Hydro-chloride berberine can reduce the permeability of intestinal mucosal in NAFLD rats and repair intestinal mucosal mechanical barrier.4 Hydro-chloride berberine can decrease the diversity of the intestinal flora,enrich the amount of bacteroides and inhibit the growth of the unknown bacteria genus in the family of lachnospiraceae and S24-7. |
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