Retrospective Study And Mechanism Exploration Of Zhishi Daozhi Decoction In The Treatment Of Non-Alcoholic Fatty Liver Disease

Objective:1.A retrospective study analyzes clinical cases of Zhishi Daozhi Decoction in treating non-alcoholic fatty liver disease(NAFLD)with the pattern of damp-heat accumulation to explore its efficacy.2.Utilize network pharmacology to preliminarily explore the potential mechanisms of Zhishi Daozhi Decoction in treating NAFLD.3.Through a high-fat diet-induced NAFLD mouse model experiment,revealing the protective effects and molecular mechanisms of Zhishi Daozhi Decoction in the treatment of NAFLD,providing experimental evidence for its use in NAFLD treatment.Methods:1.Clinical Study: This study selected patients diagnosed with NAFLD with damp-heat syndrome from the Endocrinology and Metabolic Diseases and Gastroenterology outpatient departments of the Third Affiliated Hospital of Changchun University of Chinese Medicine between March 2020 and October 2023.The patients were divided into two groups based on the treatment method: a control group(Huazhi Rougan Capsule + basic treatment)and an experimental group(Zhishi Daozhi Decoction + basic treatment).The study compared changes in liver function(ALT,AST,GGT),blood lipids(TG,TC,HDL-C,LDL-C),Traditional Chinese Medicine(TCM)syndrome scores,and ultrasound imaging scores before and after treatment between the two groups.2.Network Pharmacology: Using the TCMSP database along with relevant literature searches,the active components of Zhishi Daozhi Decoction were identified and effective compounds meeting the criteria were screened.The Swiss Target Prediction platform was used to predict the targets of these effective compounds.NAFLD disease targets were obtained from the Gene Card,OMIM,and Drugbank disease databases.The targets of Zhishi Daozhi Decoction and NAFLD were mapped and intersected using Venny 2.1,and a Venn diagram was drawn.The intersected targets were imported into the STRING database to obtain the protein-protein interaction network.Cytoscape was used for topological analysis to identify the core proteins involved in the treatment of NAFLD by Zhishi Daozhi Decoction.GO functional and KEGG pathway enrichment analyses were conducted using the Metascape database.3.Experimental Study: Forty-six male C57BL/6 mice,aged 7 weeks,were randomly divided into a control group(Ctrl)(n=13,fed a regular diet)and a high-fat diet group(n=33,fed a high-fat diet).After confirming successful modeling at the 10 th week of feeding,the high-fat diet group mice were further randomly divided into a model group(Model),a Polyene Phosphatidylcholine group(PPC),and a Zhishi Daozhi Decoction group(ZSDZ).Each group received corresponding interventions.The general condition of the mice was observed,and their body weight was regularly measured.At the end of the experiment,HE staining and Oil Red O staining were used to observe the pathological morphology of liver tissue in mice.The NAFLD histological scoring standard was used for evaluation.ELISA was employed to detect levels of triglycerides(TG)and free fatty acids(FFA)in liver tissue.The levels of superoxide dismutase(SOD),malondialdehyde(MDA),tumor necrosis factoralpha(TNF-α),interleukin-1 beta(IL-1β),and interleukin-6(IL-6)were also measured.Western blot analysis was used to detect the expression of Bcl-2,Bax,Caspase-3,Ask1,p-Ask1,JNK,p-JNK,c-Jun,and p-c-Jun proteins in liver tissue.Results:1.Clinical study:(1)This study collected a total of 97 cases that met the inclusion and exclusion criteria,with 44 cases in the control group and 53 cases in the experimental group.There were no statistically significant differences in baseline data such as age and gender between the two groups(P<0.05).(2)Comparison of liver function indicators(ALT,AST,GGT)before and after treatment in patients: After treatment,ALT,AST,and GGT levels in both groups significantly decreased compared to before treatment(P<0.05).The experimental group showed significantly greater improvement in ALT levels compared to the control group(P<0.05).However,there were no significant differences between the two groups in terms of improvements in AST and GGT levels(P>0.05).(3)Comparison of blood lipid indicators(TG,TC,HDL-C,LDL-C)before and after treatment in patients: After treatment,TG,TC,HDL-C,and LDL-C levels in both groups showed significant improvement compared to before treatment(P<0.05).The experimental group showed significantly greater improvement in TG and LDL-C levels compared to the control group(P<0.05).However,there were no statistically significant differences between the two groups in terms of improvements in TC and HDL-C levels(P>0.05).(4)Comparison of TCM syndrome aspects in patients: After treatment,the total TCM syndrome scores in both groups were significantly reduced compared to before treatment(P<0.05).The reduction in scores was significantly greater in the experimental group compared to the control group(P<0.05).In terms of individual TCM syndrome scores,the experimental group showed significant improvement after treatment in symptoms of hypochondriac pain,general heaviness,abdominal distention/pain,sticky or difficult bowel movements,dry and bitter mouth,and sticky sensation in the mouth(P<0.05).There were no significant differences in symptoms of yellowing of the body and eyes or dark-colored urine(P<0.05).In the control group,all symptoms except for dark-colored urine showed significant improvement compared to before treatment(P<0.05).Comparing the two groups after treatment,the experimental group showed significantly greater improvement than the control group in symptoms of abdominal distention/pain,general heaviness,sticky or difficult bowel movements,and sticky sensation in the mouth(P<0.05).There were no significant differences between the groups in symptoms of hypochondriac pain,dry and bitter mouth,yellowing of the body and eyes,and dark-colored urine(P>0.05).(5)Comparison of TCM syndrome efficacy: The overall effective rate in the experimental group was 88.68%,significantly higher than the control group’s overall effective rate of 72.73%.The difference between the two groups was statistically significant(P<0.05).(6)Comparison of ultrasound imaging efficacy: The overall effective rate in the experimental group was 66.04%,compared to 45.45% in the control group.The experimental group showed superior efficacy in ultrasound imaging compared to the control group,and the difference was statistically significant(P<0.05).2.Network Pharmacology:Using the TCMSP database and relevant literature analysis,528 active components of Zhishi Daozhi Decoction were identified.A total of 1989 NAFLD disease targets were retrieved,and 141 intersection targets between Zhishi Daozhi Decoction and NAFLD were found,with 36 core targets identified.The top 10 core targets included AKT1,TNF,IL6,IL-1β,TP53,PTGS2,JUN,PPARG,MMP9,and ESR1.GO enrichment analysis revealed that the primary biological processes involved include responses to exogenous stimuli,hormones,and lipids.The biological components involved include transcription regulator complexes,membrane rafts,and vesicle lumens.Biological functions include DNA-binding transcription factor binding,signal receptor activator activity,protease binding,and oxidoreductase activity.A total of 193 KEGG signaling pathways were identified,involving pathways such as cancer pathways,MAPK signaling pathways,and HIF-1 signaling pathways.KEGG classification analysis showed that the MAPK signaling pathway had the highest number of enriched targets,with apoptosis being the major cellular process involved.3.Experimental Study:(1)General condition of rats: Compared to the Ctrl group,rats in the Model group exhibited greasy fur,reduced activity,fatigue,drowsiness,slow responses,and sticky stools.After treatment with Zhishi Daozhi Decoction(ZSDZ),there were improvements in the rats’ vitality,bowel habits,and fur condition.(2)Body Weight,Liver Wet Weight,and Liver Index: Compared to the Model group,the ZSDZ group showed significant reductions in body weight,liver wet weight,and liver index(P<0.001,P<0.001,P<0.05).(3)Liver tissue pathology: Compared to the Ctrl group,the Model group showed significant fat deposition,while the ZSDZ group was intermediate between the two.NAS scoring results indicated that,compared to the Ctrl group,the Model group had higher mean values in hepatic steatosis,lobular inflammation,and total score(P<0.01).There was a statistically significant difference in hepatocellular ballooning between the Model group and the Ctrl group(P<0.01),but no statistical difference between the ZSDZ group and the Ctrl group(P>0.05).Compared to the Model group,the ZSDZ group had significantly lower mean values for hepatic steatosis,lobular inflammation,and total score(P<0.001,P<0.01,P<0.01).(4)Lipid Metabolism Indicators: Compared to the Ctrl group,the Model group showed significantly increased levels of triglycerides(TG)and free fatty acids(FFA)(P<0.001,P<0.01).The ZSDZ group had reduced levels of TG and FFA(P<0.001,P<0.01).(5)Oxidative stress-related indicators: Compared to the Ctrl group,the Model group showed significantly reduced SOD activity(P<0.01)and significantly increased MDA content(P<0.001).Compared to the Model group,the ZSDZ group significantly increased SOD activity levels(P<0.01)and reduced MDA content(P<0.001).(6)Inflammatory response-related indicators: Compared to the Ctrl group,the Model group had significantly elevated levels of TNF-α,IL-1β,and IL-6(P<0.01).Compared to the Model group,the ZSDZ significantly reduced levels of TNF-α,IL-1β,and IL-6(P<0.01,P<0.01,P<0.001).(7)Hepatocyte apoptosis-related indicators: Compared to the Ctrl group,the Model group showed significantly increased levels of Bax and caspase-3 proteins(P<0.001,P<0.05)and significantly decreased levels of Bcl-2 protein(P<0.001).Compared to the Model group,the ZSDZ group significantly inhibited the expression of Bax and caspase-3proteins(P<0.001)and promoted the expression of Bcl-2 protein(P<0.01),with statistically significant results.(8)Expression of proteins related to the JNK/MAPK signaling pathway: Compared to the Ctrl group,the Model group showed significantly increased ratios of p-Ask1/Ask1,p-JNK/JNK,and p-c-jun/c-jun(P<0.01,P<0.001,P<0.001).Compared to the Model group,the ZSDZ group showed significantly decreased ratios of p-Ask1/Ask1,p-JNK/JNK,and p-c-jun/c-jun(P<0.01,P<0.001,P<0.01).Conclusion:1.Zhishi Daozhi Decoction can reduce liver function and blood lipid levels in patients with NAFLD with damp-heat syndrome,effectively regulating lipid metabolism disorders and slowing the progression of NAFLD.Additionally,it can alleviate symptoms such as general heaviness,abdominal distention or pain,and sticky or difficult bowel movements,showing superior efficacy compared to the control group.2.Zhishi Daozhi Decoction may inhibit the JNK/MAPK signaling pathway,regulating the expression of apoptosis-related proteins Bcl-2,Bax,and caspase-3.It also improves liver lipid metabolism,oxidative stress,and inflammatory responses,thereby slowing or reversing the progression of NAFLD.