Study On Qiyin Granule Inhibiting Ferroptosis And Treating Nonalcoholic Fatty Liver Disease With Liver Depression And Spleen Deficiency By Regulating PPARα/Nrf2/GPX4 Signaling Pathway

Objective:1.Comparing the clinical characteristics and TCM syndrome distribution characteristics under two nomenclatures of fatty liver disease,and clarifying its clinical significance.2.Based on bioinformatics analysis,the key targets of Qiyin granule mediated ferroptosis in the treatment of NAFLD were screened.3.Based on ferroptosis via the PPARα/Nrf2/GPX4 signaling pathway,the therapeutic effect of Qiyin granule on NAFLD patients with liver stagnation and spleen deficiency syndrome was explored.4.Based on ferroptosis via the PPARα/Nrf2/GPX4 signaling pathway,the therapeutic effect of Qiyin granule on NAFLD rats with liver stagnation and spleen deficiency syndrome was explored.Methods:1.Patients admitted to the Department of Hepatology in the Traditional chinese medicine hospital affiliated to Xinjiang medical university,and diagnosed with fatty liver from January 2020 to August 2023,and were selected as the research objects who met the diagnosis of NAFLD and MAFLD,and the differences of clinical and TCM syndromes distribution between the two groups were compared.2.The potential targets of Qiyin granule in the treatment of NAFLD were screened by the network pharmacology,the PPI network of the intersection targets of Chinese medicine and disease was constructed,and the potential targets were analyzed by GO and KEGG enrichment using the DAVID platform.The GEO database was used to screen the common key targets of PPARα with ferroptosis.3.From January 2023 to July 2023,70 patients diagnosed with NAFLD liver stagnation and spleen deficiency syndrome were randomly divided into control group and Qiyin group.Patients in the two groups were given basic treatment such as health education and healthy lifestyle during the study period.Patients in the control group were given basic treatment combined with polyene phosphatidylcholine capsules,and patients in the Qiyin group were given Qiyin granule on the basis of the control group,and the duration of treatment was 12 weeks.Observation indicators of pre-and post-treatment included obesity index(BMI,WC),liver function(ALT,AST,ALT/AST,ALP,GGT,TBIL,DBIL,IBIL),blood lipids(TG,TC,HDL-C,LDL-C,VLDL-C),fasting plasma glucose(FPG),noninvasive liver fibrosis assessment(NFS score,APRI score,FIB-4 index,BARD score,LSM)and liver fibrosis stage,steatosis degree(CAP)and steatosis stage,TCM syndrome score,efficacy evaluation,peripheral serum lipid metabolism(PPARα)and ferroptosis(Nrf2,GPX4)index,safety evaluation.4.The NAFLD rat model with liver stagnation and spleen deficiency syndrome was established by high-sugar and high-fat diet combined with hunger and satiety disorder,tail-clamping stimulation and chronic restraint emergency stimulation.They were randomly divided into normal group,model group,Qiyin granule group,ferroptosis inducer group and ferroptosis inhibitor group.The pathological changes of liver were observed;serum liver function(ALT,AST,GGT),blood lipid(TG,TC,LDL-C,HDL-C)and blood glucose(FPG)were detected by fully automatic biological analyzer.The levels of serum insulin(FINS,HOMA-IR),inflammatory factors(TNFα,IL-1β,IL-6),oxidative stress indicators(ROS,GSH)and liver tissue iron content were detected by ELISA.RT-q PCR and Western blotting were used to detect the m RNA and protein expression levels of lipid metabolism index(PPARα)and ferroptosis index(Nrf2 and GPX4)in liver tissues.The objective indexes of "liver stagnation and spleen deficiency syndrome" were detected and the TCM syndrome scores were evaluated in each group.Results:1.The prevalence of NAFLD and MAFLD were 68.3% and 100%,respectively.Compared with NAFLD group,MAFLD group had a higher proportion of males and obesity,slightly higher BMI,transaminase(ALT/AST and GGT),blood lipids(TG),UA and Cr levels,lower HDL-C,and a higher proportion of smoking and drinking history(P<0.05).Regardless of the nomenclatures of fatty liver disease,the most common TCM syndrome was liver stagnation and spleen deficiency syndrome.2.By network pharmacological analysis,the targets corresponding to the active ingredients of Qiyin granule and NAFLD disease targets were obtained,and 29 core targets were obtained by intersection,including PPARα.KEGG enrichment analysis showed that Qiyin granule played a therapeutic role in NAFLD mainly through PPAR and HIF-1 signaling pathways.Ferr Db platform was used to screen the key targets of PPARα related ferroptosis,including Nrf2.3.Compared with pre-treatment,ALT,ALT/AST,GGT were increased,HDL-C was decreased,LSM and CAP were significantly increased in the control group after treatment;However,after treatment,BMI,WC,ALT,AST,ALT/AST,ALP,GGT,TG,TC,LDL-C,VLDL-C,NFS score,APRI score,FIB-4 index,LSM,CAP,and TCM syndrome score in the Qiyin group were significantly decreased,while HDL-C was significantly increased.The serum levels of PPARα,Nrf2 and GPX4 were significantly higher than those before treatment(P<0.05).After treatment,compared with the control group,the Qiyin group significantly reduced the levels of ALT,AST,TG,VLDL-C and CAP,increased HDL-C,reduced the proportion of moderate to severe fatty liver,and reduced the TCM syndrome score;The levels of PPARα,Nrf2 and GPX4 in serum were increased,and the differences were statistically significant(P<0.05).The TCM syndrome curative effect and steatosis curative effect of Qiyin group were better than those in control group.4.Compared with model group,after treatment with Fer-1 and Qiyin granule,body weight,Lee’s index and liver index of NAFLD rats with liver-stagnation and spleen-deficiency syndrome were decreased,liver steatosis was relieved to varying degrees,and NAS score was decreased;serum liver function(ALT,AST,GGT),glucose metabolism(FPG,FINS,HOMA-IR),inflammatory factors(TNFα,IL-1β,IL-6)and liver tissue iron content decreased;the improvement of blood lipid levels(TG,TC,LDL-C decreased and HDL-C increased),oxidative stress indicators(ROS decreased and GSH increased),the m RNA and protein expression levels of PPARα,Nrf2 and GPX4 in liver tissues increased significantly,and the hippocampal NE,5-HT and urine-Dxylose excretion rate increased,the TCM syndrome score of "liver stagnation and spleen deficiency syndrome" in rats decreased,and the difference was statistically significant(P<0.05).However,Qiyin granule is more effective than Fer-1 in improving the TCM syndrome of "liver stagnation and spleen deficiency".Conclusion:1.Compared with NAFLD patients,MAFLD patients were more likely to be male,obese,have a history of smoking and alcohol use,and have more metabolic comorbidities(such as high BMI,abnormal liver enzymes and lipids,and elevated UA and Cr).The diagnostic criteria of MAFLD fully reflect metabolic dysfunction.Liver stagnation and spleen deficiency are the most common TCM syndromes in both MAFLD and NAFLD.To screen the core targets of Qiyin granule mediated ferroptosis in the treatment of NAFLD by bioinformatics analysis.It is speculated that PPARα/Nrf2/GPX4 signaling pathway is the key signaling pathway of Qiyin granule mediated ferroptosis in the treatment of NAFLD.3.Under the guidance of healthy lifestyle,Qiyin granule can restore liver function in NAFLD patients with liver depression and spleen deficiency syndrome,reduce blood lipid,improve the steatosis degree and TCM clinical syndrome,increase the levels of serum lipid metabolism index(PPARα)and ferroptosis index(Nrf2,GPX4),and speculate that its mechanism may be related to the activation of PPARα/Nrf2/GPX4 signaling pathway to inhibit ferroptosis.4.Qiyin granule have certain therapeutic effect on NAFLD rats with liver depression and spleen syndrome.It can improve liver function injury,regulate glucose and lipid metabolism disorders,reduce the degree of hepatic steatosis,reduce oxidative stress and inflammatory reaction in the body,and improve the syndrome of "liver depression and spleen deficiency" in TCM.The possible mechanism is that Qiyin granule activate PPARα/Nrf2/GPX4 signaling pathway,alleviated systemic oxidative stress effects(increased GSH production,reduced ROS production)and inhibit liver tissue ferroptosis(reduced intrahepatic iron content,increased expression of liver ferroptosis index Nrf2 and GPX4).