益气活血化瘀片抑制氧化应激与调控mTOR/SREBP-1通路治疗代谢功能障碍相关脂肪性肝病的作用机制OA

Objective To investigate the mechanism of Yiqi Huoxue Huayu Tablets(YHHT)in treating metabolic dysfunction-associated steatotic liver disease(MASLD)via inhibiting oxidative stress and regulating the mammalian target of rapamycin(mTOR)/sterol regulatory element-binding protein-1(SREBP-1)pathway.Methods(1)Network pharmacology was used to predict the potential targets of YHHT against MASLD.A"drug-compound-target-disease"network was constructed to screen key active components.A protein-protein interaction(PPI)network of potential targets was built to identify core targets.GO functional and KEGG pathway enrichment analyses were performed on potential targets.Molecular docking validation was conducted between key active components and core targets.(2)db/db mice were randomly divided into control group,model group,metformin group(0.31 g·kg-1),and low-and high-dose YHHT groups(0.615,1.23 g·kg-1),with 10 mice in each group.MASLD mouse model was established by high-fat diet feeding.Drugs were administered by gavage once daily for 8 consecutive weeks.After the last gavage,body weight,wet liver weight and liver index were measured,and serum and liver tissue samples were collected.Serum levels of total cholesterol(TC),triglyceride(TG),free fatty acid(FFA),alanine aminotransferase(ALT),aspartate aminotransferase(AST),fasting plasma glucose(FPG)and fasting insulin(FINS)were measured,and homeostatic model assessment for insulin resistance(HOMA-IR)was calculated.Pathological changes of liver tissue were observed by HE staining.Lipid deposition in liver tissue was assessed by Oil Red O staining.Hepatic MDA content was detected by TBA colorimetry,SOD activity by WST method,and GSH/GSSG ratio by enzymatic cycling colorimetry.Mitochondrial ROS(mtROS)level in liver tissue was measured by DCFH-DA fluorescent probe.Mitochondrial ultrastructure was observed by transmission electron microscopy.mRNA expression levels of mTOR,SREBP-1,FASN and SCD1 in liver tissue were detected by RT-qPCR.Protein expression of mTOR,p-mTOR,SREBP-1,FASN and SCD1 was measured by Western Blot.Expression of p-mTOR and SREBP-1 protein in liver tissue was detected by immunohistochemistry.Results(1)A total of 328 potential targets of YHHT against MASLD were identified.Key active components included quercetin,kaempferol,calycosin,aloe-emodin and rhein.mTOR was identified as an important core target.Biological processes mainly involved oxidative stress response,regulation of apoptotic signaling pathways and steroid metabolic processes.PI3K-Akt,HIF-1 and mTOR signaling pathways were identified as key pathways.The binding energies between key active components and the core target mTOR were all≤-8 kcal·mol-1,indicating strong binding activity.(2)Compared with the control group,the model group showed significantly increased body weight,wet liver weight and liver index(P＜0.001);elevated serum TC,TG,FFA,ALT and AST levels(P＜0.001);increased FPG,FINS and HOMA-IR(P＜0.001);and enlarged Oil Red O-positive area(P＜0.001).Hepatic mtROS and MDA levels were significantly increased(P＜0.001),while SOD activity and GSH/GSSG ratio were decreased(P＜0.001).Hepatocyte mitochondria showed swelling,cristae rupture,reduced matrix density and vacuolar degeneration.mRNA expression of mTOR,SREBP-1,FASN and SCD1 was significantly upregulated(P＜0.001),and protein expression of FASN,SCD1,p-mTOR/mTOR and n-SREBP-1/m-SREBP-1 was increased(P＜0.05,P＜0.01,P＜0.001).Compared with the model group,the high-dose YHHT group showed significantly reduced body weight,wet liver weight and liver index(P＜0.01,P＜0.001).All treatment groups exhibited significantly decreased serum TG,FFA and ALT levels(P＜0.001),and the high-dose group also showed reduced TC and AST levels(P＜0.05,P＜0.001).FPG,FINS and HOMA-IR were all significantly decreased in all treatment groups(P＜0.001),and the number and area of hepatic lipid droplets were markedly reduced(P＜0.01,P＜0.001).Hepatic MDA levels were significantly decreased(P＜0.001)and SOD activity increased(P＜0.01,P＜0.001)in all treatment groups;mtROS was reduced and GSH/GSSG ratio elevated in the high-dose group(P＜0.001).Mitochondrial morphology was relatively preserved with intact membranes and dense cristae in all treatment groups.mRNA expression of mTOR,SREBP-1 and SCD1 was significantly downregulated(P＜0.01,P＜0.001),and FASN mRNA was decreased in both low-and high-dose YHHT groups(P＜0.01).Protein expression of FASN and p-mTOR/mTOR was significantly downregulated in all treatment groups(P＜0.05,P＜0.01),SCD1 and n-SREBP-1/m-SREBP-1 were reduced in low-and high-dose YHHT groups(P＜0.05,P＜0.01).Immunohistochemical results were consistent with Western Blot findings.Conclusion YHHT may ameliorate dyslipidemia and hepatic lipid deposition in MASLD mice by inhibiting oxidative stress and downregulating the mTOR/SREBP-1 pathway and its downstream proteins FASN and SCD1.