| Purpose: Alcoholic liver disease(ALD),which is liver damage caused by excessive and uncontrolled alcohol consumption,has become one of the common diseases affecting human health worldwide,and its occurrence and development are closely related to the intestinal microbiota.Studies have confirmed that the spleen tonic Si-jun-zi Decoction exerts its effect of strengthening the spleen and benefiting the qi by regulating the intestinal microorganisms,while fewer studies have explored the scientific connotation of "realizing the spleen to treat the liver" according to the original purpose of the Jin Kui Yao.In this study,we investigated the effect and mechanism of Si-jun-zi Decoction on the prevention and treatment of alcoholic liver injury(ALD)in mice based on the basic pathogenic treatment of "strengthening the spleen to treat the liver" and the changes of intestinal flora.Methods: In this study,36 male C57BL/6J mice were used and randomly divided into the following six groups: normal control group(CG),alcohol model group(MG),Si-jun-zi Decoction low dose group(SL),Si-jun-zi Decoction medium dose group(SM),Si-jun-zi Decoction high dose group(SH),and Silybin group(PS).Except for the control group,all other groups were given gavage of 56 degree white wine for 15 days to establish a model of alcoholic liver disease,and intervention with Si-jun-zi Decoction and Silybin.At the end of the experiment,the feces,serum,spleen and liver of the mice were collected.The serum was analyzed for liver function indexes as well as lipid indexes by automatic biochemical analyzer.The spleen and part of the liver were fixed for histopathological analysis and morphological changes were observed.Serum and some liver were used for enzyme-linked immunosorbent assay and real-time PCR analysis to detect inflammation-related indicators such as IL-6,TNF-α,and LCN2.Fecal samples were analyzed for intestinal flora by 16 S r DNA high-throughput sequencing.Some livers were used for the detection of molecular indicators of AKT/GSK-3β/CREB pathway by protein western blot assay and real-time PCR analysis.The results indicated:1.In terms of liver and spleen injury and inflammation,compared with the model group,the mice in the Si-jun-zi Decoction group gradually returned to normal in terms of hair color,diet,mental status and mouse body weight in a dose-dependent manner.The liver and spleen indices were significantly reduced,among which the liver and spleen indices of SM and SH were reduced significantly with statistically significant differences(P<0.01).Sijun-zi Decoction reduces the levels of liver function indicators ALT,AST,ALP and T-BIL,with the most significant reduction in ALT and AST indicators.The HE staining results showed that the morphology of hepatic cords in liver tissues under Si-jun-zi Decoction treatment was normalized,the necrosis of liver and spleen cells was reduced,and the red marrow and white marrow of spleen tissues were clearly demarcated.ELISA and q PCR results showed that Si-jun-zi Decoction significantly inhibited the expression of protein levels and m RNA levels of inflammatory factors IL-6,TNF-α,and LCN2 in vivo.In the aspect of lipid metabolism,compared with MG,the levels of LDL-C,TC and TG of blood lipid indicators in Si-jun-zi Decoction group were significantly reduced,while the HDL-C level was significantly increased,and the difference was statistically significant(P < 0.05).The results of oil red O staining were consistent with those of blood lipid indicators,suggesting that Si-jun-zi Decoction could reduce lipid accumulation and hepatic steatosis.Among them,SH showed the best effect of reducing lipid accumulation.2.The analysis of intestinal permeability and intestinal microorganisms showed that compared with the model group,Si-jun-zi Decoction significantly reduced the increase in intestinal permeability caused by alcohol(P < 0.05).The Rank-Abundance curve and Rarefaction curve based on Sobs index,Shannon index and Coverage index showed that the sample had good species richness and high coverage at the current sequencing depth,which truly reflected the microbial community status in the sample.Alpha diversity showed a significant decrease in Ace index,Chao index and Shannon index and an increase in Simpson index in the Si-jun-zi Decoction dose group compared to MG,indicating that Sijun-zi Decoction reversed alcohol-induced changes in the abundance and diversity of intestinal microorganisms.PCA and PCo A in Bate diversity showed that the community composition of the high-dose group of Si-jun-zi Decoction was close to that of CG and PS,indicating that Si-jun-zi Decoction improved the structure of intestinal microorganisms and restored the normal state.Meanwhile,Si-jun-zi Decoction also regulates the composition of intestinal microorganisms,increases the abundance of beneficial bacteria such as Muribaculum,Dubosiera,Akkermansia,and Bifidobacterium,and reduces the abundance of pathogenic bacteria such as Helicobacter,Lachnospiraceae＿NK4A136＿group,Mycoplasma,and thereby maintaining the dynamic balance of intestinal flora.3.Combined with the KEGG pathway information predicted by the biological function of16 S r DNA PICRUSt and the network pharmacology target pathway information of Si-junzi Decoction and alcoholic liver disease,it is predicted that Si-jun-zi Decoction can regulate intestinal microorganisms and affect AKT/GSK-3β/CREB signaling pathway to prevent ALD.Further immunohistochemistry and real-time fluorescent quantitative PCR verification showed that compared with the model group,Si-jun-zi Decoction significantly reduced the protein and m RNA levels of AKT and CREB,and significantly increased the protein and m RNA levels of GSK-3β in a dose-dependent manner.SH,CG,MG and PS were selected for protein western blot validation.The results showed that alcohol promoted the expressions of AKT and CREB and decreased the expression of GSK-3β.While the expressions of AKT and CREB in Si-jun-zi Decoction group were significantly decreased,and the expression of GSK-3β was significantly increased.It indicated that Si-jun-zi Decoction could regulate the intestinal flora,reduce the expression of AKT and CREB,increase the expression of GSK-3β,and affect the AKT/GSK-3β/CREB pathway,so as to achieve the effect of treating alcoholic liver disease.Conclusion: Si-jun-zi Decoction adheres to the theory of "activating spleen to treat liver",and achieves the purpose of treating alcoholic liver disease by regulating intestinal microorganisms and affecting the expression of AKT/GSK-3β/CREB signaling pathway.This study has provided new evidence for elucidating the mechanism of SpleenStrengthening Prescription Si-jun-zi Decoction on alcoholic liver injury,as well as new ideas for the scientific connotation interpretation of traditional Chinese medicine prescriptions and the prevention and treatment of alcoholic liver disease. |
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