Study On The Correlation Between Da Chaihu Tang And Its "prescription Elements" And Their Regulatory Effects On Multiple Targets Of The "gut-liver Axis" In NAFLD Model Rat

Nonalcoholic fatty liver disease（non-alcoholic fatty liver diseases,NAFLD）is a worldwide common disease,the disease includes nonalcoholic simple fatty liver（nonalcoholic fatty liver,NAFL）,nonalcoholic fatty hepatitis（nonalcoholic steatohepatitis,NASH）and related liver fibrosis and liver cancer.The main pathogenesis of this disease is not quite clear.As the theory of "enter-hepatic axis" is proposed,the role of intestinal factors in the development of NAFLD is increasingly emphasized.The idea of formula-factors is based on the theory of syndrome-factors.Formula-factors is the component of drugs corresponding to the syndrome-factors and can often embody the therapeutic principles.In the study of compound "multi-target" effect of TCM compound,the formula-factors is also an important connection point for connecting syndrome-factors and disease effect targets.This research is based on the relevant literature research and the theory of"formula-factors" and "correspondence between syndrome and formula factors".Through the establishment of animal models,we observed the effect of dachaihu decoction and various"formula-factors" including in the decotion on the "Im-liver shaft" target in the NAFLD model.First Part-Literature researchIn the first part of the review,we decribed the understanding of Traditional Chinese Medicine on non-alcoholic fatty liver disease in detail including the etiology,pathogenesis and the treatment in TCM.In the second part of the review,we summarized the modern epidemiology,pathogenesis,"im-liver axis" role in the disease and the treatment of NAFLD by regulating the intestinal microecology.Second Part-Theoretical discussionBased on the theory of syndrome-factors,the concept of formula-factors is proposed,which further leads to the principle of "correspondence between syndrome and formula factors".we analyzes the relationship of "correspondence between syndrome and formula factors" "of the dachaihu decoction which is as below:the formula factors of bupleurum,Scutellaria baicalensis,and herbaceous peony is corresponding to the syndrome factors of"hepatobiliary insufficiency",the formula factors of half summer,ginger,date is corresponding to the syndrome factors of "Spleen deficiency phlegm moisture resistance",the formula factors of rhubarb and bitter orange is corresponding to the syndrome factors of"Yang Ming by turbid gas does not fall".At the same time,we found that there was a correlation between the "formula factors" and the pathological target of "im-liver axis",which may provide theoretical basis for the subsequent experimental study.Third Part-Experimental studyObjectiveThe purpose of this study is to observe the effect of dachaihu decoction and various"formula-factors" including in the decotion on the "Im-liver shaft" target in the NAFLD model and to reveal the compatibility law of dachaihu decoction.Methods105 rats were divided into the normal group,the model group,the western medicine group,the Shuganlidan group,the Jianpihuatan group,the Tongfuxiezhuo group and the Dachaihu decoction group.The rats in the model group,the western medicine group,the Shuganlidan group,the Jianpihuatan group,the Tongfuxiezhuo group and the Dachaihu decoction group were feed with high fat and high sugar feed.In the 13th to 16th weeks,rats in the western medicine group,the Shuganlidan group,the Jianpihuatan group,the Tongfuxiezhuo group and the Dachaihu decoction group were respectively filled with the corresponding medicine or Chinese medicine.At the same time,normal and model rats were given equal volume of distilled water.HE staining was used to observe the morphological changes of liver tissue in rats,and the lipid deposition of hepatocytes was observed by oil red O staining,and the fibrosis of hepatocytes was observed by Masson staining.Serum TC,TG,LDL,HDL,AST and ALT were detected in rats.Occludin protein and zo-1 protein expression were detected by Western Blot.Real-time fluorescence quantitative PCR（rt-qpcr）was used to detect the expression of Occludin and zo-1 gene in intestine.Enzyme linked immunosorbent assay（Elisa）was used to detect the contents of TNF-α,TGF-β1,NF-Kb,TLR4 in rat liver tissues and sIgA in rat intestinal tissues.The analysis of intestinal microflora sequencing and bioinformatics in rats was carried out by high throughput IIlumina test system.Results1 Morphology of liver tissue1.1 Hematoxylin and eosin stain（HE stain）The structure of the hepatic lobules in normal group was complete and clear,and the boundary line was clear.The liver cells were normal,and radially distributed around the central vein.The liver sinus was clearly visible.The rat liver cells in the model group were swollen,and some of the cells were balloon-like degeneration,and the nuclei were extruded to one side.Most of the liver cells were found to have different sizes and different amounts of lipid droplet vacuoles.The rats liver cells in the Pioglitazone Hydrochloride group showed a mild fat change and small fat drops were seen in some cells.Liver cells in the Shuganlidan group,the Jianpihuatan group and the Tongfuxiezhuo group show diffuse fat changes in varying degrees,but the degrees of fat change were all less than that of the model group.The diffuse fat changes of liver cells in the rats of the Dachaihu decoction group were significantly improved compared with that of the model group,and some of the cells were normal.1.2 Oil red O stainNo red lipid droplets were found in liver tissues of normal rats.The lipid droplets in rat hepatocytes in the model group were diffuse and granular and fused into sheets.The number of lipid droplets in rat hepatocytes in Shuganlidan group,Jianpihuatan group,Tongfuxiezhuo group and Dachaihu decoction group decreased significantly,and the volume of lipid droplets were relatively small,with a small particle size distribution.1.3 Masson stainThere was no increase in the distribution of collagen fibers in the liver tissues of each group.2.The content of TG,TC,LDL,HDL in serumCompared with the normal group,the serum TG,TC and LDL levels were significantly increased in the model group,and the HDL level was significantly reduced,and the difference was statistically significant（P<0.01）.Compared with the model group,the serum TG,TC and LDL levels in the Pioglitazone Hydrochloride group,Shuganlidan group,Jianpihuatan group,Tongfuxiezhuo group and Dachaihu decoction group were significantly reduced,and the difference was statistically significant（P<0.01）.The serum HDL levels in the Shuganlidan group,Jianpihuatan group and Dachaihu decoction group increased significantly,the difference was statistically significant（P<0.01）.3.The content of AST,ALT in serumCompared with the normal group,the serum AST and ALT levels in the model group were significantly increased,and the difference was statistically significant（P<0.01）.Compared with the model group,the levels of serum AST in the rats of Pioglitazone Hydrochloride group,Shuganlidan group,Jianpihuatan group,Tongfuxiezhuo group and Dachaihu decoction group decreased significantly,and the difference was statistically significant（P<0.01）.Compared with the model group,the levels of serum ALT in the rats of Pioglitazone Hydrochloride group,Shuganlidan group,Tongfuxiezhuo group and Dachaihu decoction group decreased significantly,and the difference was statistically significant（P<0.01 or P<0.05）.4.The content of TNF-α、TGF-β1、TLR4、NF-κB in liver tissueCompared with the normal group,the levels of TNF-α、TGF-β1、TLR4、NF-κB were significantly increased in the rat liver tissues of the model group,and the difference was statistically significant（P<0.01）.Compared with model group,the TNF-α and TLR4 contents in rats of Pioglitazone Hydrochloride group,Shuganlidan group,Jianpihuatan group,Tongfuxiezhuo group and Dachaihu decoction group reduced significantly,and the TGF-β1、NF-kB contents in rats of Pioglitazone Hydrochloride group,Shuganlidan group,Tongfuxiezhuo group and Dachaihu decoction group decreased significantly,the difference was statistically significant（P<0.01 or P<0.05）.5.The protein expression of Occludin mRNA and protein,ZO-1 mRNA and protein in the small intestine tissueCompared with the normal group,the expression of Occludin mRNA,ZO-1 mRNA and Occludin protein,ZO-1 protein in rats in the model group was significantly decreased,and the difference was statistically significant（P<0.01）.Compared with the model group,the expressions of Occludin mRNA,ZO-1 mRNA and Occludin protein,ZO-1 protein in rats in the Pioglitazone Hydrochloride group,Jianpihuatan group,Tongfuxiezhuo group and Dachaihu decoction group were significantly higher,and the difference was statistically significant（P<0.01 or P<0.05）.6.The content of sIgA in the small intestine tissueCompared with the normal group,the content of sIgA in the intestinal tissue of the model group decreased significantly,and the difference was statistically significant（P<0.01）.Compared with the model group,there was a significant increase in sIgA content in rats in the Pioglitazone Hydrochloride group,Jianpihuatan group,Tongfuxiezhuo group and Dachaihu decoction group and the difference was statistically significant（P<0.01）.7.Intestinal flora in fecesIn terms of microflora structure,the intestinal flora of each group was slightly different,but still consisted mainly of three bacteria gates,Bacteroidetes,Firmicutes and Proteobacteria.The bacteria that play an important role in the normal group were Ruminococcaceae,Ruminococcus,Oscillospira,Lactobacillales,Lactobacillaceae,Lactobacillus,Bacilli,TM7,Spirochaetes,Clostridium,Elusimicrobia,Elusimicrobia,Elusimicrobiaceae,Elusi microbiales,Lactobacillus helveticus,Lactobacillus reuteri,Elusimicrobium,RF39,Christensenellaceae;Bacteria that play an important role in the model group was Ruminococcus_gnavus;Bacteria that play an important role in the Pioglitazone Hydrochloride group were Prevotella,Paraprevotellaceae,Blautia;Bacteria that play an important role in the Jianpihuatan group were Bacteroidetes,Bacteroidia,Bacteroidales,Roseburia,Rikenellaceae,Stridium methylpentosum;Bacteria that play an important role in the Tongfuxiezhuo group were Bacteroidaceae,Bacteroides,Porphyromonadaceae,Parabacteroide,4C0d₂,Cyanobacteria,YS2,Parabacteroides_distasonis,Bacteroides_uniformis,Verrucomicrobiae,Verrucomicrobiales,Ver rucomicrobiaceae,Akkermansia,Akkermansia-muciniphila,Coprobacillus,Parabacteroides-gor donii,Blautia-producta,Ruminococcus_torques;Bacteria that play an important role in the Dachaihu decoction group were Erysipelotrichi,Erysipelotrichales,Erysipelothrichaceae.ConclusionsThe results showed that,the "Shuganlidan" formula factors of the prescription mainly regulate lipid metabolism of liver and liver immune function,namely,regulating "hepatic axis",but at the same time,the intestinal mucosal barrier was improved to some extent.The"Jianpihuatan" formula factors of the prescription mainly improves intestinal mucosal barrier function-that is,regulating the "intestinal axis",but also regulates liver lipid metabolism and liver immune function.The "Tongfuxiezhuo" formula factors of the prescription can intervent the occurrence and development of NAFLD mainly by regulating intestinal flora-that is,to adjust im axis,but it can also regulate lipid metabolism,liver immunity and intestinal mucosal barrier.The effect of dachaihu decoction on hepatic lipid metabolism,hepatic immunity and intestinal mucosal barrier was better than that of single prescription factor group.It shows that through the compatibility of the three formula factors,the effect of the combination is enhanced,which reflects the rationality and validity of the compatibility of the dachaihu decoction.Special features and innovation point1.From the perspective of "correspondence between syndrome and formula factors",this paper makes a group analysis of the dachaihu decoction,and provides a new idea for the clinical application and expansion of the application of the dachaihu decoction.2.This paper discusses the advantages of every "formula factors" in the dachaihu decoction on the "Im-liver shaft",which probides research ideas and methods for the development of multi-target action mechanism of TCM compound.