Mechanism Of Cyclocarya Paliurus Triterpenoids In Treating NAFLD Based On Network Pharmacology And Experimental Verification

Background: Non alcoholic fatty liver disease(NAFLD)is a chronic liver disease prevalent worldwide,and its incidence rate is increasing year by year.NAFLD patients usually have risk factors for metabolic syndrome related diseases such as obesity,type 2 diabetes and hyperlipidemia.The course of NAFLD can be divided into four stages: fatty liver,fatty hepatitis,liver fibrosis,and cirrhosis.Severe cases can develop into liver cancer.Currently,the pathogenesis of NAFLD is still unclear,but it is generally believed to be related to factors such as lipid metabolism disorders,intestinal flora imbalance,chronic inflammation,and oxidative stress.Due to the slow course of NAFLD and its unclear clinical symptoms,it is often overlooked,leading to a large number of patients missing the best opportunity for treatment.Currently,there is still a lack of effective NAFLD treatment drugs in addition to non drug treatments such as diet adjustment,exercise,and weight management.Therefore,the research on the pathogenesis and treatment of NAFLD has become a hot spot in the clinical and scientific research fields.In recent years,many studies have shown that Traditional Chinese Medicine and natural drugs can play an important role in the treatment of NAFLD,and become one of the research directions in the treatment of NAFLD.Cyclocarya paliurus(Batal)Iljinskaja(C.paliurus)is a common traditional Chinese medicine,also known as Maoshu bark,hickory,and so on.It is a deciduous tree native to China and widely distributed in Asia and Europe.The leaves,bark,fruits and other parts of C.paliurus are widely used in the preparation of Traditional Chinese Medicine preparations.Because it contains a variety of bioactive ingredients,it is considered to have many pharmacological effects,including anti diabetes,anti-inflammatory,antioxidant,anti-tumor,etc.In addition,C.paliurus is also used in food processing and health product manufacturing.Its fruit has a unique taste and a delicious taste,and is rich in nutrients such as polyphenols,proteins,vitamins,and minerals.This article will review the chemical constituents,pharmacological effects,and health benefits of C.paliurus,and explore the development direction of its future research.The main chemical components of C.paliurus include triterpenoids,flavonoids,glycosides,phenolic acids,and other compounds,among which triterpenoids are the main active components.Studies have shown that the triterpenoids of C.paliurus s have many pharmacological effects,such as anti-diabetes,blood lipid lowering,antioxidant,anti-inflammatory,anti-tumor and so on.Although many studies have reported the pharmacological and health effects of C.paliurus triterpenes,their mechanisms and specific pathways of action still need further research and exploration.Therefore,future research should focus on the extraction and purification of the active components of C.paliurus,the exploration of the mechanism of action,and its application in drug research and health product development.Objective: To study the therapeutic effects and potential mechanisms of C.paliurus triterpenes on NAFLD through network pharmacology,molecular docking,and experimental validation.Methods:(1)This study selected the methods of network pharmacology and molecular docking to explore the mechanism of action of the triterpenoid components of C.paliurus in the treatment of NAFLD.Firstly,through the analysis of drug compound databases and gene databases,targets related to NAFLD were selected,and a target pathway network was constructed using network analysis tools to analyze the interaction relationship between the triterpenoid components of C.paliurus and NAFLD targets.KEGG analysis and GO analysis were used to predict the NAFLD pathway of C.paliurus triterpenes.Then,through molecular docking,the interaction between the selected triterpenoid components and the predicted NAFLD target was simulated to evaluate their affinity and stability.The results showed that multiple triterpenoids in C.paliurus have good interaction ability with multiple NAFLD targets,which mainly involve multiple biological processes such as liver lipid metabolism,inflammatory reaction,oxidative stress,and cell apoptosis.(3)Cell experiment: Cultivate AML12 cells and determine the concentration of oleic acid(OA)solution induced AML12 cell modeling 24 hours after intervention through cytotoxicity experiments.Finally,treat AML12 cells with OA solution(200 m M)to construct a NAFLD model.After 24 hours of intervention with green willow triterpenoids,cell proliferation was detected using MTT assay.The subsequent experimental groups were set as blank group(basic culture medium),model group(200 m M concentration OA),and low dose,medium,and high dose groups of C.paliurus triterpenoid(62.5% μg/m L,125 μg/m L,250 μg/m L).We used a reagent kit to detect the secretiond of total cholesterol(TC)and triglycerides(TG),performed oil red O staining and observe pathological changes in mouse liver cells.Western blot method was used to detect TLR4,NF-κB and NLRP3 protein expression levels in AML12 cells.(4)Animal experiment: After feeding C57BL/6 mice with high fat and high sugar feed for one month,they were randomly divided into:(1)control group,(2)model group,(3)CPST-25 mg/kg,(3)CPST-50 mg/kg,(4)CPST-100 mg/kg blank group,and the other groups were given high fat and high sugar feed;The mice were continuously administered for 6 months,and their food intake and body weight were recorded daily.They were administered orally according to their body weight.After the last administration,all mice fasted for 12 hours and were euthanized.The liver mass of the mice was measured,and the liver index was calculated by dividing the liver mass by the body mass.Collect liver tissue,perform H&E staining,and observe pathological changes in liver tissue.We used a reagent kit to detect multiple indicators such as TC and TG in liver tissue;Western blot method was used to detect the expression protein levels of TNF-α,IL-6,AKT1,TLR4,NF-κB,NLRP3 in mouse liver tissue.Result:(1)Through network pharmacology and molecular docking,31 triterpenoid active components from C.paliurus were screened,and 93 common targets with NAFLD were identified.Through network topology analysis using PPI networks,46 key targets were obtained.PPI networks showed that C.paliurus might act on NAFLD through proteins such as TNF,IL-6,AKT1,and NLRP3.Meanwhile,the KEGG pathway enrichment analysis results show that the NF-κB signaling pathway is closely related to the pathogenesis of NAFLD.(2)Through in vitro experimental verification,it was found that the triterpenoid components of C.paliurus could significantly reduce multiple indicators such as TG and TC in the liver tissue of the NAFLD mouse model,improve inflammatory infiltration of liver cells,and reduce lipid deposition in liver cells.In addition,the triterpenoids of C.paliurus could significantly reduce the protein expression levels of TNF-α,IL-6,AKT1,TLR4,NF-κB and NLRP3 in the high-dose group.(3)Through in vivo experimental verification,it was found that the triterpenoids of C.paliurus could promote the proliferation of AML12 cells,reduce fat deposition and lipid content in mouse liver cells,and reduce the protein expression levels of TLR4,NF-κB and NLRP3,confirmed its protective effect was relation to regulating TLR4/NF-κB/NLRP3 signaling pathway related protein expression.Conclusion: Network pharmacology has found that the mechanism of the treatment of NAFLD with C.paliurus triterpenoids mainly acts on cellular inflammation,lipid metabolism,insulin resistance,and other cellular signaling pathways.Animal experiments have found that C.paliurus triterpenoids can treat NAFLD,mainly by improving liver inflammatory response and lipid regulation.The C.paliurus triterpenoids might alleviate inflammatory response by regulating targets such as TNF,IL-6,AKT1,TLR4,regulate the TLR4/NF-κB/NLRP3 signaling pathway to affect inflammatory response,thereby play a role in treating NAFLD.Cell experiments have found that C.paliurus triterpenoids could promote the proliferation of AML12 cells,reduce fat deposition and lipid content in mouse liver cells,and reduce the expression levels of TLR4,NF-κB and NLRP3.This might be one of the mechanisms of its therapeutic effect on NAFLD.