Study On The Efficacy And Material Basis Of Shenling Jianpiwei Formula In The Treatment Of MAFLD

BackgroundMetabolic associated fatty liver disease(MAFLD),once known as non-alcoholic fatty liver disease,mainly manifests as triglyceride cholesterol aggregation,causing disorders of lipid metabolism,is one of the most common chronic liver diseases in the world,with a global prevalence of over 25%.Shenling Jianpiwei formula composed of ten herbs,including Glehnia littoralis,Poria cocos and Atractylodes macrocephala,which is based on the Song Dynasty’s "Taiping Huimin and Pharmaceutical Bureau" Panax ginseng replacing Glehnia littoralis,adding Citrus sinensis,and removing the Platycodonis radix.It is named after two herbs,namely,Glehnia littoralis and Poria cocos,which highlight the expertise of this formula in strengthening the spleen.The effect of this formula is to nourish the spleen and stomach,and to promote dampness,which is effective in the treatment of spleen and kidney deficiency and liver depression and phlegm obstruction.Its clinical indications are: failure to eat and drink,vomiting and diarrhea,thinness and atrophy caused by weakness of the spleen and stomach.The main clinical pathology of MAFLD is the damage to the liver,and it is written in Zhang Zhongjing’s Jin Kui Yao Lue: "Those who treat the disease before the disease see the disease of the liver and know that the liver transmits the spleen,so we should first strengthen the spleen".Based on the basis of Chinese medicine,"Knowing that the liver transmits the spleen,we should first strengthen the spleen" is the main theoretical basis for treating MAFLD from the spleen,and the main pathogenesis is the loss of spleen health and phlegm.Lipid metabolism can be better treated by "dispersing essence through spleen qi" to transport lipids from water and grain to all limbs and bones,thus reducing the accumulation of lipids around the liver and harmonizing liver qi.However,whether Shenling Jianpiwei Formula can relieve MAFLD and the related mechanism of action are still unclear,which has the research prospect of further exploring the clinical use mechanism,and also provides the basis for the secondary development of Yunnan characteristic best-selling Chinese patent medicine.ObjectTo investigate the efficacy and mechanism of action of Shenling Jianpiwei formula in alleviating MAFLD using cellular and animal experiments.And established an accurate and rapid method to identify the chemical components of Shenling Jianpiwei formulaMethod1.Drug efficacy evaluationCell experiment: Liver L-02 cells were divided into normal group,model group,fenofibrate group and Shenlingjian Piwei formula extract low-medium-high-dose groups(10μg/m L,50 μg/m L,100 μg/m L),all of which were modeled and administered for 24 h.The cells were collected and the activities of TC,TG,ALT and AST were detected.Animal experiments: Seventy male SD rats were randomly divided into normal control group(normal saline),model group(normal saline),fenofibrate group(20 mg/kg),Shenlingjian Piwei formula extract low-dose and high-dose groups(5.25g/kg,15.75g/kg),and intragastric administration every day for 12 weeks.Except for normal group,rats in other groups were fed high fat diet for 12 weeks to induce MAFLD rat model.In order to block intestinal flora,they were divided into ampicillin group(0.24g/kg),ampicillin Shenling Jianpiwei Granules high-dose group(0.24g/kg,15.75g/kg),except for ampicillin Shenling Jianpiwei prescription high-dose group was intragastric administration of Shenling Jianpiwei prescription extract,and both groups were fed high-fat diet for 12 weeks to induce high-fat model.Ampicillin group was given discontinuous intragastric administration in the last 4 weeks to maintain diarrhea.The rats were killed and serum and organs collected.The levels of TC,TG,HDL-C,LDL-C,AST,ALT and FFA in serum were detected.Viscera indexes(heart,liver,spleen,lung,kidney,perirenal white fat,inguinal white fat and brown scapular fat)were calculated;The histopathologic changes of liver were observed by HE staining.The activities of BCA,TC,TG,AST,ALT,HDL-C,LDL-C,MDA,GSH and SOD in liver were detected.2.Sequencing analysis of 16 S r DNA amplicon and culture of significant flora of cecal contents in rats16Sr DNA sequencing analysis:Extraction of experimental rat cecum content DNA,PCR amplification,purification,purification recovery,library construction and up-sequencing.Comprehensive analysis of intestinal microecological data based on high-throughput sequencing results,to evaluate the differences in intestinal microecological diversity,functional gene differences and their dynamic changes between groups by KEGG and COG analysis,and to evaluate the differences in intestinal microbial functional gene abundance and changes in metabolic differences in rats with lipid metabolism disorders before and after drug administration.Significant bacterial community culture:Single colonies were selected,activated bacterial solution was incubated in a 37℃ shaker for 12 h.10-80.1m L diluent was coated in AGAR solid medium and RSM liquid medium prepared by adding different concentrations of Shenling Jianpiwei granule extracts in advance for culture.3.The effect of Shenling Jianpiwei formula on the chronic low-grade inflammatory state in the liver of rats with disorders of lipid metabolismThe serum of rats was collected,and the kit was applied to determine the changes of IL-1,LPS,IL-6,NF-KB and TNF-α,the changes of LPS,the determination of IL-1,IL-6,NF-κB,TNF-α,L-FABPF and FATP4 in rat liver and FATP4 in colon level changes,WB detected the expression of TLR4 in the liver and occludin in the colon.4.Determination of the content of Shenling strengthening spleen and stomach,analysis of its chemical components and analysis of its components into blood of MAFLD ratsUsing phenol-concentrated sulfuric acid system,the absorbance value was determined at485 nm by UV spectrophotometer;according to the reaction color development of vanillin-glacial acetic acid-perchloric acid system,the absorbance value was determined at540 nm by UV spectrophotometer,and the content was determined;the extract of Shenling Jianpiwei formula was sonicated,and the chemical composition in Shenling Jianpiwei formula was analyzed by UHPLC-Q-TOF-MS;in In rat serum,the protein was removed by methanol,centrifuged at low temperature,and the supernatant was nitrogen-blown,re-solubilized by methanol and detected by UHPLC-Q-TOF-MS.ResultsIntervention effect of Shenling Jianpiwei Formula on L-02 cells of fatty milk liver and MAFLD rats: After Shenling Jianpiwei Formula was administered to fatty milk induced liver L-02 cells,the contents of TC and TG in cells were significantly decreased(P<0.001),and ALT and AST were decreased to a certain extent.After Shenling Jianpiwei formula extract was administered to MAFLD rats,the abnormal body weight and serum TG content of MAFLD rats were significantly decreased(P<0.001,P< 0.01),the serum HDL-C was significantly increased(P<0.001),and the levels of TC,ALT,AST,LDL-C and FFA were significantly decreased;It improved the abnormal enlargement of organs,significantly reduced the accumulation of white fat of testis and white fat finger around kidney(P<0.001),and increased the accumulation of brown scapular fat(P<0.001).The inflammatory vesicles in the liver were significantly relieved,the liver cells were arranged neatly,the TC,TG,ALT,MDA and GSH in the liver were significantly decreased(P<0.05,P<0.01,P< 0.001),and the SOD activity was significantly increased(P<0.05).Regulating effect of Shenling Jianpiwei Fang on intestinal microflora in MAFLD rats: Shenling Jianpiwei Fang can significantly increase the number of microflora OTU,and restore the microflora close to the normal group.At the door level,the richness of Firmicutes/Bacteroidetes was reduced and tended to the normal group.For Actinobacteria and Proteobacteria,compared with normal group,the relative abundance of MOD group was decreased,while fenofibrate group and Shenling Jianpiwei Fang extract group were significantly increased.At the genus level,there were higher levels of Lactobacillus and lower levels of Faecalibaculum and Methanobrevibacter.On the species level,Lactobacillusreuteri,Lactobacillusreuteri and Lactobacillusgasseri under Lactobacillus were significantly increased.It is worth noting that the composition and abundance of the flora were more simple after the administration of ampicillin,and the flora richness was restored to a certain extent after the administration of Shenling Jianpiwei Fang extract.By Pearson correlation analysis,TC is negatively correlated with Proteobacteria at the serum and flora level of rats(P < 0.05),while Lactobacillus is positively correlated with HDL at the genus level(P < 0.05).Prevotellaceae UCG-003 was negatively correlated with TC(P < 0.05),and Collinsella and Methanobrevibacter were positively correlated with TG,AST and LPS(P < 0.05).[Eubacterium]coprostanoligenesgroup was positively correlated with LPS(P < 0.05),and Allobaculum was negatively correlated with AST(P < 0.05).On the second level,Anaerobiospirillumsp.3 j102 and TC,AST negative correlation(P < 0.05),Escherichiacoli and IL-1 into positive correlation(P < 0.05),IL-1 was negatively correlated with Lactobacillusgasseri(P < 0.05).At the level of liver and microflora in rats,Spirochaetes were positively correlated with IL-1 and NF-k B(P < 0.05),Tenericutes and Verrucomicrobia were positively correlated with ALT(P < 0.05).In the genus level,Lactobacillus was positively correlated with HDL-C(P < 0.05).Collinsella was positively correlated with IL-1,NF-k B,TNF-α and MDA(P < 0.05).Faecalibacterium was positively correlated with IL-1,NF-k B and MDA(P < 0.05).Methanobrevibacter and Allobaculum were positively correlated with TNF-α.At the species level,Lactobacillus gasseri was positively correlated with SOD(P < 0.05),Chlamydia trachomatis and others were positively correlated with ALT(P < 0.05).Shenling Jianpiwei can regulate cell signal transduction,information storage and processing,metabolism,cell cycle control,cell division,chromosome zoning,nucleotide transport and metabolic function.Significantly increased the number of colonies and bacterial fluid OD600 in vitro,respectively,with Lactobacillus murinus and Lactobacillus reuteri.Effects of Shenling Jianpiwei Formula on chronic low-grade inflammation of liver in MAFLD rats: After intragastric administration of Shenling Jianpiwei formula in MAFLD rats,FATP4 content was significantly decreased(P<0.001),serum LPS of rats induced by high fat diet was significantly decreased,liver lipid TLR4 protein expression was down-regulated,Occludin protein expression was up-regulated.The levels of LPS,IL-6 and NF-k B induced by high fat diet were significantly decreased(P<0.01,P<0.001).It is worth noting that after the administration of ampicillin destroys intestinal flora,the body weight of rats is decreased,the degree of inflammatory cell infiltration in liver sections is aggravated,the cell arrangement is more disordered,and the serum HDL-C content is increased.Decreased the levels of TC,TG,AST,ALT,LDL-C and FFA(no significant effect),increased the content of MDA and decreased the content of GSH in liver,and decreased the number of OTU.Down-regulation of cell signaling,information storage and processing and metabolism,cell cycle control,cell division,chromosome zoning,nucleotide transport and metabolic function;LPS content in serum was increased,TLR4 protein expression in liver was up-regulated and Occludin expression in intestinal mucosa group was down-regulated,while NF-k B,IL-1 and IL-6 had no significant changes.After Shenling Jianpiwei Formula was given,the above conditions were improved.Determination of Shenling invigorating Spleen and stomach,analysis of chemical components and analysis of components into blood of MAFLD rats: The total polysaccharide content of Shenling Jianpiwei Recipe was 110.7mg.g-1,and the total triterpene content was 9.8mg.g-1.37 kinds of chemical components were identified from Shenling Jianpiwei Recipe,and 24 kinds of prototype drug-derived components were identified from Shenling Jianpiwei recipe into MAFLD rat serum.Conclusion:Shenling Jianpiwei can regulate lipid metabolism in MAFLD rats by alleviating lipid accumulation,relieving oxidative stress and regulating lipid metabolism.Its mechanism may be to restore the abundance of beneficial bacteria,reduce the abundance of pathogenic bacteria,restore intestinal flora disorder,reduce intestinal mucosal permeability,and reduce the entry of inflammatory factors into the liver to alleviate the lipid metabolism disorder of MAFLD.At the same time,we identified 24 components of Shenling Jianpiwei formula into the blood of MAFLD rats,which may enter the blood to regulate the drug-induced components of MAFLD rats.The results will provide important scientific basis for the clinical application and further development of Shenling Jianpiwei formula.