Study On The Mechanism Of Shenling Baizhu Powder In The Treatment Of Metabolic Associated Fatty Liver Disease Based On Network Pharmacology And Proteomics

Objective: To investigate the action mechanism of Shenling Baizhu powder(SLBZP)in the treatment of metabolic associated fatty liver disease(MAFLD)using network pharmacology combined with proteomics,and to validate it through in vivo and in vitro experiments,which is expected to provide theoretical support for clinical applica.Methods:1.Network Pharmacology Research: Network pharmacology was used to explore the active components and therapeutic targets of SLBZP in the treatment of MAFLD.The "herbs-active components-treated targets" complex network and the PPI network were constructed;GO and KEGG pathway enrichment analyses were performed on the therapeutic targets;The active components were molecularly mapped to core therapeutic targets to validate the network pharmacology results.2.Proteomics research: 48 SD rats were randomly divided into a control group(K group)with 8 rats and a model group(MAFLD group)with 40 rats.The MAFLD group feding with high-fat diet(HFD)for 4 weeks and then randomly divided into HFD group,SLBZP low-dose group(SLBZP-L),SLBZP medium-dose group(SLBZP-M),SLBZP high-dose group(SLBZP-H)and Silybin group,8 rats per group.The SLBZP-H,SLBZP-M and SLBZP-L groups were given 22g/kg,11g/kg and5.5g/kg SLBZP solution respectively,and the Silybin group was given 37.8mg/kg Silybin solution by gavage for 3 weeks.(1)To measure the levels of liver lipids,serum lipids and liver function in each group of rats;and to observe the pathological changes in the liver of each group of rats by oil red O staining and HE staining;(2)DIA proteomics techniques were used to identify differentially expressed proteins(DEPs)in each group of livers before and after SLBZP treatment.GO and KEGG pathway enrichment analysis was done for DEPs significantly back regulated by SLBZP,PPI network was constructed,and key core DEPs were validated by Western blotting.3.Palmitic acid(PA)was used to induce HepG2 cells to establish an in vitro model of MAFLD,and the CCK-8 method combined with oil red O staining was used to detect the optimal modeling concentration of PA;The CCK-8 method was used to detect the effect of different concentrations of SLBZP-containing serum on the viability of HepG2 cells and to screen the optimal concentration for subsequent experiments;The experiment was divided into control group(K group),PA group,SLBZP group and Silybin group;Oil red O staining and enzymatic assay were performed to measure the number of lipid droplets and TC and TG levels in HepG2cells;RT-qPCR and Western blotting were performed to detect the mRNA levels of PPARα,CPT1 A,FAS,SCD1,SREBP-1c and AMPK phosphorylation levels in rat liver and HepG2 cells,as well as the protein expression of PPARα,SREBP-1c,SCD1,CPT1 A in HepG2 cells.Results1.The network pharmacology results showed that:(1)160 active components and 265 component targets of SLBZP were collected,as well as 243 disease targets of MAFLD;There were 41 intersecting targets between component targets and disease targets,i.e,which are potential targets of SLBZS to treat MAFLD;(2)The " herbsactive components-treated target" complex network indicated that quercetin,naringenin,lignan and kaempferol were the key active components,and the PPI topology analysis showed that the core targets were mainly ALB,TNF,PPARα and ADIPOQ,etc;(3)The binding energy of all molecular docking results was less than 0,and the optimal affinity was-10.2kcal/mol.(4)GO enrichment analysis showed that SLBZP is involved in regulating lipopolysaccharide response,fatty acid metabolic process and inflammatory response,and KEGG pathway was a key pathway for SLBZP in the treatment of MAFLD.The molecular docking results showed that AMPK and its downstream target SREBP-1c had good docking activity with quercetin and naringenin,with optimal affinity of-8.2kcal/mol and-6 kcal/mol respectively.2.Compared with healthy rats,the hepatic TC,TG and serum TG,AST and ALT levels were significantly increased in the HFD group(P<0.05),and a large number of red lipid droplets were formed between the hepatocytes.The treatment of MAFLD rats with all doses of SLBZP resulted in significant improvements in lipid accumulation,lipid levels and liver function.3.Proteomics results showed that:(1)There were 438 DEPs between the HFD and K groups,and 113 DEPs were significantly reversely regulated after SLBZP intervention.(2)GO enrichment results showed that these 113 DEPs are closely related to lipid metabolism and oxidative stress.SLBZP catalyzes molecular functions such as oxidoreductase activity and binding activity in mitochondria and endoplasmic reticulum to promote oxidative lipid catabolism and maintain oxidative acid metabolism homeostasis.52 signaling pathways,including the PPAR signaling pathway,fatty acid degradation and arachidonic acid metabolism,are involved in the KEGG pathway.(3)PPI topology analysis showed that FABP1,ACSM3,ALDH1A1,PCK1,GOT1,HMGCS2,CYP4A2 and AGXT2 were the core DEPs of SLBZP in the treatment of MAFLD.(4)Western blotting results showed that FABP1 and HMGCS2 expression was increased and PCK1 expression was decreased in the liver of HFD rats.After SLBZP intervention,FABP1 and HMGCS2 expression decreased,while PCK1 expression increased,consistent with the proteomic results.4.Experimental validation results:(1)Results of in vivo experiments: RT-qPCR and Western blotting results showed that compared with the K group,SREBP-1c,FAS and SCD1 mRNA expression was elevated(P<0.001)and PPAR α,CPT1 A mRNA and AMPK phosphorylation were decreased(P<0.001)in the HFD group;Compared with the HFD group,SREBP-1c,FAS and SCD1 mRNA expression was decreased to different degrees in each SLBZP dose group and the Silybin group(P<0.01,P<0.001),PPARα and CPT1 A mRNA expression was increased(P<0.05,P<0.01,P<0.001)and AMPK phosphorylation was significantly increased(P<0.001).(2)Results of in vitro experiments:(1)HepG2 cell viability inhibition and induction of lipid droplet formation by 0.3 mmol/L PA;All concentrations of drugcontaining serum had no effect on the viability of HepG2 cells,with 10% of the drugcontaining serum having the most pronounced effect on the proliferation of HepG2cells;(2)Compared with the K group,HepG2 cells in the PA group showed decreased viability and massive red lipid droplet formation,TC and TG levels were significantly increased(P<0.01);FAS,SREBP-1c and SCD1 mRNA and protein expression were increased(P<0.05,P<0.01,P<0.001),PPAR α and CPT1 A mRNA and protein expression were significantly decreased(P<0.05,P<0.01)and AMPK phosphorylation was reduced(P<0.001);Compared with the PA group,HepG2 cells in the SLBZP and Silybin groups showed significantly reduced lipid droplet formation and decreased TC and TG content(P<0.05);elevated PPARα and CPT1 A mRNA and protein expression(P<0.01),significantly increased AMPK phosphorylation(P<0.001),and FAS,SREBP-1c,SCD1 mRNA and protein expression were decreased(P<0.05,P<0.01,P<0.001).Conclusion1.The network pharmacology study identified ALB,TNF,PPARα and ADIPOQ as the key therapeutic targets of SLBZP for MAFLD,quercetin,naringenin,lignan and kaempferol as the key active components of SLBZP for MAFLD;and all of them have good binding activity to each other;AMPK signaling pathway is the key pathway of SLBZP for MAFLD.2.SLBZP was effective in improving lipid levels and liver function in MAFLD rats.3.Proteomic studies showed that FABP1,ACSM3,ALDH1A1,PCK1,GOT1,HMGCS2,CYP4A2 and AGXT2 are key proteins back regulated by SLBZP,and the PPAR signaling pathway is a key pathway for SLBZP in the treatment of MAFLD.4.SLBZP inhibits lipogenesis and promotes fatty acid oxidation through regulation of the AMPK/SREBP-1c/PPARα pathway and improves lipid accumulation in MAFLD rat liver and HepG2 cells.