| Nonalcoholic fatty liver disease(NAFLD),except of alcohol and otherliver damage factors,refers to a sydrome which the main pathological featuresare charactered by excess fat deposition in the liver cells.NAFLD can bedivided into simple fatty liver(SFL),nonalcoholic steatohepatitis(NASH) andrelated liver cirrhosis.NAFLD is thought to be liver metabolic disorders of themetabolic syndrome.Up to now,there are no effective drugs in treatingNAFLD.Physical exercise,diet adjustment,loss weight and improve otherlifestyles are the basic methods in prevention and treatment of NAFLD.The species of intestinal microbiota in the human gut is numberous and various,with the complex functions are very essential for maintaining humanhealth.Recent years, the close relationship between the intestinal microbiotaand obesity, insulin resistance and associated metabolic diseases is more andmore being understood.Research shows that high fat diet can cause pathogenicbacteria increased and intestinal inherent probiotics reduced,which leads totight junction protein expression deficient and elevate intestinalpermeability.The hypothesis of "metabolic endotoxemia" explains well theexistence of chronic inflammation in obese patients.In addition,the intestinalflora can decompose the food which the host can not digest down into shortchain fatty acids(SCFA),promote the storage and absorption offat.Therefore,revealing the mechanism of intestinal microbiota in obesity andits related metabolic diseases is of great value for the prevention and treatmentof NAFLD.Berberine is mainly existed in the Ranunculaceae coptis rhizome,whichcan be extracted from Chinese medicine such as phellodendron,coptis,threeneedles etc.It has significant antimicrobial effect,and is used in the treatmentof gastroenteritis and bacterial dysentery.Recent years,more and morephysiological and pharmacological effects of berberine are beingfound.Studies have found that berberine helps improve insulinresistance,lower cholesterol,anti arrhythmia, treatment of metabolic syndromeand other effects.Several evidences suggest berberine can significantlyinfluence the structure of gut microbiota. Therefore,our study focuses on thegut microbiota,discusses the effect of hydrochloride berberine on NAFLD ratsand NAFLD associated intestinal mucosa injury,so as to provide a theoreticalbasis for the treatment of NAFLD.Objective:To study the protective effect of hydrochloride berberine onthe intestinal barrier of NAFLD rats induced by high fat diet,and to explorethe mechanisms of berberine improving the intestinal mucosal barrierfunctions of NAFLD rats.Methods:24 male Sprague-Dawley rats(weight 180g-200g),after 1 weekadaptive feeding,were randomly divided into 3 groups:Normal control group(CON)(n=8),High-fat diet group(HFD)(n=8),HFD+berberine treatmentgroup(BBR)(n=8).CON was given normal diet,HFD and BBR were givenhigh fat diet.Rats were allowed free access to food and water.After 8 weeks offeeding,BBR were given 150mg/(kg.d) of hydrochloride berberinegavage,while CON and HFD was given the same dose of saline.After 4 weeksgavage,rats were sarcrificed.Complete removal the liver and weighed, calculate the liver index(liverwet weight/body weight×100%);Sudan black B staining to observehistopathological changes of hepatocyte;Analyzed serum ALT,AST and TGlevels by using automatic biochemical analyzer;Observe epithelium villi ofileum mucosa with HE staining;Observed the expression of occludin proteinin rat intestinal mucosa epithelium with immunohistochemical staining;TALmethods to test serum endotoxin levels;Measure serum levels of TNF-α byusing liquid competitive inhibition principle;Test the serum I-FABP levels bydouble antibody ABC-ELISA method;Application of 16 S r DNARealtime-PCR(RT-PCR) to measure copy numbers of gut Faecalibacteriumprausnitzii(F.prausnitzii) and Bacteroides.Results:1 Analysis of body weight(g)and liver index(%).After 12 weeks,ratsbody weight(485.24±50.15)and liver index(3.67±0.22)of HFD weresignificant higher than CON(379.83±61.51)(2.56±0.18);Compared withHFD,body weight(428.60±37.37) and liver index(2.91±0.27) of BBRdecreased significantly.2 Serum ALT(U/L),AST(U/L) and TG(mmol/L) levels.Serum levels ofTG(0.437±0.027),ALT(115.58±22.54),AST(203.35±28.23) of HFD weresignificant higher than those of CON TG(0.344±0.038),ALT(35.96±12.69),AST(125.60±23.76).Compared with HFD,the serum levels of BBR TG(0.377±0.047),ALT(80.94±21.16),AST(155.79±35.95)decreased significantly.3 Hisological analysis:Sudan black B staining showed HFD liver cellsfill with purple lipid deposition and steatosis.Compared with CON,the livercells was normal and without lipid deposition and steatosis.Compared withHFD,the liver of BBR lipid deposition and fatty degeneration showed a lessdegree;The ileum mucosal epithelial villus of CON showed complete structurewith HE staining,intestinal villi arranged in neat rows,HFD arranged indisorder and partial deletion.BBR intestinal mucosa were arranged in order,noobvious atrophy or inflammatory cell infiltration.4 Blood levels of endotoxin(EU/ml),I-FABP(pg/ml) and TNF-α(ng/ml).Serum levels of endotoxin(0.288±0.045),I-FABP(231.22±56.31) ofHFD were significantly higher than CON(0.192±0.049)(136.95±57.52).Compared with HFD,the plasma levels of endotoxin(0.213±0.025) andI-FABP(145.66±52.72) of BBR were significantly decreased;HFD serum levels of TNF-α(1.07±0.11) increased significantly than CON(0.94±0.07),while BBR(1.0±0.07) had no significant decrease compared with HFD.5 Immunohistochemistry analysis.Occludin protein of CON(0.166±0.014) was sustained expressed on the top tight junction of mucosa epithelialcells,while HFD occludin significantly decreased(0.05±0.012). Comparedwith HFD, the entrocytes expression of occludin protein of BBR(0.055±0.009) increased,there was no statistics difference between the two groups.6 Analysis of intestinal microbiota(Copy numbers/g feces).HFDintestinal numbers of F.prausnitzii(7.29±0.47) increased significantlycompared with CON(6.53±0.35) and BBR(6.72±0.34);HFD intestinal numberof Bacteroides(8.70±0.62) reduced significantly than CON(9.49±0.59).Afterberberine hydrochloride gavage,BBR Bacteroides number(9.77±0.87) increased,the difference was statistically significant.Conclusions:1 High fat diet successfully induced NAFLD animal model.2 The presence of intestinal microbiota disturbance within NAFLD rats, numbers of F. prausnitzii increased,Bacteroides deceased.3 Berberine may regulate the intestinal microbiota structure,reduce theendotoxin,I-FABP and TNF-α levels in NAFLD rats,improve the function ofintestinal mucosal barrier. |
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