Study On The Treatment Of Modified Zexie Decoction In NAFLD And The Effect On Intestinal Microbe

Nonalcoholic fatty liver disease (NAFLD) is a kind of clinical pathological syndrome with accumulation of fat in the liver not by alcohol and other liver damage factors, including simple fatty liver (SFL), steatohepatitis (NASH) and liver cirrhosis. The disease presents a high incidence in the global, nearly half of the patients with liver disease in China with NAFLD. It’s along with the high incidence of obesity, metabolic syndrome and diabetes in China.At present; there is no effective medicine with the significant evidence, while traditional Chinese medicine has certain advantages in fatty liver, hyperlipidemia and obesity treatment. Research on safe and effective traditional Chinese medicine of NAFLD and its therapeutic mechanism is of great significance to explore. Non alcoholic fatty liver from the viewpoint of traditional Chinese medicine is spleen deficiency, phlegm resistance, therefore Jianpi Xiezhuo method treatment is applied clinically, and here we have adopted clinical "Jingui" modified Zexie decotion.Objective:1.analysis of the regular drug pattern in treatment of NAFLD by studying the clinical papers published in recent 10 years; 2 evaluating effective, highly repetitive method from different fat contents of the high fat feed; 3 through the low, high dose modified Zexie decoction in the treatment of NAFLD mice, the choice of the optimal dosage regimen; 4 by macro genomics approach to investigate the potential mechanism of intestinal microbes in the modified Zexie Decoction in the treatment of NAFLD in mice; 5 estimating acute toxicity test of modified Zexie Decoction of toxicity; 6 analysis of the basic material in Modified Zexie decoction by UPLC-MS.Methods:the clinical TCM treatment of NAFLD literature were searched from 2004 to 2014, frequency analysis of pathogenesis and herbs was applied; 2 with 45% and 60% fat content and high fat purification feed of NAFLD mouse, evaluated two kinds of feed molding degree by the general condition, blood fat and blood sugar, glucose tolerance test and liver patholog; 3 intervened by low, high doses of Modified Zexie Decoction on NAFLD induced by high fat diet, general condition, blood glucose and blood lipid, glucose tolerance test and pathological evaluation of different doses were estimated;4 after NAFLD mice were treated with Modified Zexie decoction, liver function, hepatic HE and Masson staining, RT-qPCR assay for detection of intrahepatic inflammatory factor expression, immunohistochemistry and Western-blotting detection in ileum colon tight junction protein expression of occludin protein, high-throughput sequencing of 16S rRNAmice feces analysis of fecal microbes were carried out, and potential therapeutic mechanism of Modified Zexie Decoction was researched; 5 male and female mice given the maximum dosage of Modified Zexie Decoction for 7 days, observe death rate, weight and pathology of acute toxicity, assessment of Modified Zexie Decoction; 6 through the UPLC-MS, Modified Zexie Decoction and material basis was analysed, comparison of atractylone, emodin, rhein, aloe emodin and 2,3-acetyl Alisol B control, and the other substances were identified by mass spectrum analysis.Results:1 literature study, research on frequency and medication rule of our clinical literature in recent 10 years of nonalcoholic fatty liver disease, a total of 147 kinds of drugs, and the frequency is 960 times. Drug use is mainly for the high frequency, hawthorn 7.57% Alisma 7.03%, Salvia 5.95%, Atractylodes 5.73%, Poria 4.97%, Radix 4.11%, Bupleuri cassia seed 4.11%, according to the functions attribution, it can classified into diuresis medicine, tonic and promoting blood circulation to remove blood stasis. Non alcoholic fatty liver pathogenisis was confirmed with phlegm resistance, rationality.2 high fat diet intake of different fat content,45% and 60% of fat content of high fat feed, in NAFLD mice can be observed by weight, liver and fat weight, blood lipid, blood glucose abnormalities increase insulin resistance, liver steatosis, it showed that these two kinds of feed could induce NAFLD mice model better, there was no significant difference between 45% fat content, that can meet the need of feed.3 in low, high dose modified Zexie Decoction in the treatment of NAFLD mouse effects, after 4 weeks of treatment, it can significantly reduce the serum of Modified Zexie AST, mouse liver and pathological NAFLD score, a certain improvement trend in body weight, serum ALT, TC, TG, HDL-C, LDL-C, but no significant difference. In the low, middle, high dose group, low dose and mid dose to the whole group treatment result is better. The overall results show that although the modified Zexie decoction has some effect in improving insulin resistance and lipid in NAFLD mice weight, but in improving the effect of inflammatory reaction in the liver, which has significantly guiding to explore the mechanism.4 To investigate the mechanism of Modified Zexie Decoction in the treatment of NAFLD mice, research mainly focus on the inflammatory reaction of gut microbes-intestinal barrier-in the liver. The results show, (1) modified Zexie Decoction reduced the level of serum AST, improve the hepatic tissue inflammatory changes, reduced collagen deposition in the liver, reduce the size of fat cells. (2) reduce the high expression of inflammatory cytokines induced by high fat model within the liver, can reduce the expression of IL-1 beta, TNF alpha and F4/80mRNA, with statistical significance; with lower TLR4, NLRP3mRNA expression trend; no effect on IL-10, IL-18, Caspase-1 expression. (3) has important significance on the intestinal mucosal barrier integrity of tight junction protein expression of occludin, modified Zexie Decoction to change the basic structure of the ileum, colon; in the ileum and colon by immunohistochemistry and Western-blotting in model group, the expression of occludin was significantly decreased after treatment, modified Zexie decoction can recover its expression.(4) on the intestinal microbial 16SrRNA abundance, diversity research, Ace index model group compared with blank group decreased, the drug group compared with the control group was also decreased; Chao index, model group compared with blank group decreased, the drug group compared with blank group decreased; Shannon index model group compared with blank group decreased, the drug group compared with the blank group decreased; Simpson index model group compared with blank group increased, the drug group than the exponential model group declined, suggesting that the model group and drug group compared with the blank group, the intestinal microbial abundance and diversity decline. (5) in the taxonomic level door, the use of high fat diet and modified Zexie decoction can cause intestinal microbial composition in larger; Bacteroidetes (Bacteroidetes) in the model group compared with blank group increased, Modified Zexie Decoction group compared with the blank group Bacteroidetes increased; the Firmicutes (Firmicute) in the model group and to the drug groups were decreased; Proteobacteria (Proteobacteria) appeared in the model group increased, the modified Zexie Decoction intervention decreased proportion, in addition to remove iron bacteria door (Deferribacteres) also appeared to change and as Proteobacteria. (6) in the taxonomy of the genus level, Desulfovibrio (Desulfovibrio) the number of OTU in model group increased significantly, with Modified Zexie decoction after treatment and the blank group decreased to a level consistent; Prevotella genera (Prevotella) increased in the model group number, modified Zexie decoction after treatment can reduce the number of bacteria in the Roche; the genus (Roseburia) and the number of the genus Lachnospiraceae_Incertae_Sedis performance is also consistent with Desulfovibrio, Prevotella.5 through the acute toxicity experiment of modified Zexie decoction, we determine its toxicity reaction, with the greatest concentration of maximum capacity of intragastric administration, continuous observation of 7d. The results show, no animal death, and no toxic reaction, change of liver and kidney pathology. Modified Zexie Decoction maximum dose is 111 times of clinical routine dose, that modified Zexie decoction has no obvious acute toxicity reaction.6 through the study on material basis, modified Zexie Decoction showed, (1) according to the chromatographic peak matching atractylone, emodin, rhein, aloe emodin and 2,3-acetyl Alisol B control products and modified Zexie Decoction of matter in existence, modified Zexie Decoction and emodin, rhein, aloe vera emodin and 2,3-acetyl Alisol B, but did not find the chromatographic peaks of atractylon. (2) combined with mass spectrum analysis, modified Zexie Decoction in addition to the above matter, may also exist of gallic acid, chrysophanol, physcion, Alisol A acetate, Alisol monoacetate C, Alisol B, atractylenolide I, atractylenolide II, atractylenolide Ⅲ. (3) chromatographic peak control products for atractylenolide I, atractylenolide Ⅱ, atractylenolideⅢ, consider the atractylone by oxidation can be transformed into atractylenolide Ⅰ, atractylenolide Ⅱ, atractylenolide Ⅲ.Conclusion:the combination of research and experimental literature, modified Zexie Decoction conforms to NAFLD pathogenesis, the main material basis have proved to be inflammation of the liver lipids and the improvement of the function of certain, modified Zexie decoction has good treatment on high fat induced NAFLD in mice, probably by changing the NAFLD of mouse intestinal microbial composition, improve the intestinal mucosal barrier, reduce inflammatory factors expression in the liver and reduce the inflammation of the liver function.