Study On The Active Components And Targets Of Total Flavonoids From Litchi Seed In The Treatment Of Liver Fibrosis In Rats Based On Proteomics And Molecular Docking Technology

Objective:The mechanism of the occurrence and development of liver fibrosis（LF）will be explored.At the same time,the effects of Total flavonoids of Lichi Semen（TFL）on LF,active components,target and pathway were investigated.This provides a basis for clarifying the mechanism of TFL in the treatment of LF.Methods:（1）GEO database and iTRAQ proteomics were used to analyze the pathogenesis of LF.SD rats were randomly divided into 0 week model group（0W）,6 weeks model group（6W）,8 weeks model group（8W）and 12 weeks model group（12W）.Except 0W group,the other groups were subcutaneously injected with CCl ₄3m L/kg to induce LF model.At the end of the experiment,half of each sample was used for histopathological analysis,and the other half was used for proteomic analysis and quantitative real-time PCR（q RT-PCR）validation.Hematoxylin eosin staining（HE）,Masson staining and Sirius red staining were used to observe the histopathological changes of LF rats at different modeling time.iTRAQ proteomics was used to analyze the changes of protein expression profile during the development of LF,and to explore the differentially expressed proteins and abnormal signal pathways during the pathological process of LF.LF related transcriptome data sets were downloaded from GEO database to verify proteomic data and bioinformatics analysis,and then biomarkers were mined and verified by q RT-PCR.（2）iTRAQ proteomics was used to analyze the anti LF mechanism of TFL.TFL was extracted by 60%ethanol and purified by AB-8 macroporous resin.The components of TFL were analyzed by UPLC-Q Exactive.The content of proanthocyanidin A2 in TFL was determined by HPLC and the content of total flavonoids was determined by ultraviolet visible spectrophotometer to control the quality of TFL.SD rats were randomly divided into control group（CK）,model group（M）and drug group（TFL）.The LF model was induced by subcutaneous injection of CCl₄3m L/kg.TFL was given 8 weeks after the model was established,and the model was maintained during the administration.After the experiment,the liver was dissected to measure the liver index.HE staining,Masson staining score and Sirius red staining were used to investigate the effect of TFL on LF rats.iTRAQ proteomics technology was used to detect the differentially expressed proteins in rat liver tissue,and bioinformatics technology was used to explore the target and signal pathway of TFL in anti liver fibrosis.Western blot（WB）and q RT-PCR were used to verify the target.The relative contents of retinol and retinoic acid in liver were determined by LC-MS/MS.（3）Virtual screening of active components and targets of TFL.The key proteins of retinol metabolism pathway were selected as receptors.TFL compounds screened by literature search and determined by UPLC-Q Exactive were selected as small molecule ligands.Libdock module of Discovery Studio was used for molecular docking,virtual screening and verification of TFL active components and targets.Results:（1）Histopathology of the rat liver showed that the degree of fibrosis,infiltration of inflammation,necrosis of hepatocytes,and steatosis gradually increased with the passing of time.The iTRAQ proteomic analysis results showed that 689,749 and 585 proteins in the 6W,8W and 12W groups were significantly regulated compared with the 0W group,among which 116differential proteins were consistently up-regulated in the 6W,8W and 12W groups,and 203 differential proteins were consistently down-regulated.The differential proteins were grouped into 5 groups according to their expression patterns,and the most abundant protein expression patterns were gradually down-regulated over time,mainly concentrated in fatty acid metabolism,tryptophan metabolism,cytochrome P450 metabolism of exogenous substances,glycolysis/gluconeogenesis and other metabolic pathways.The results of GEO database combined with proteomic analysis showed that the differential proteins in 8W group could be more verified by liver fibrosis transcriptome dataset,and 30 key proteins related to the occurrence and development of LF were screened out,among which five proteins,Slc37a4、Aldh2、Slc27a5 and Asns,could be used as biomarkers of LF.（2）The content of proanthocyanidin A2 in TFL was 0.923 mg/g,and the content of total flavonoids in TFL was 65%.Eleven components were identified in TFL by UPLC-Q-Exactive.Animal experiments showed that the liver index of rats in group M increased significantly,and liver fibrosis was obvious.After TFL intervention,liver fibrosis was effectively inhibited.Enrichment analysis showed that the differentially expressed proteins were mainly enriched in retinol metabolism,pentose and glucuronic acid mutual transformation,steroid hormone biosynthesis,drug metabolism cytochrome P450 pathway.LC-MS/MS determination of metabolites retinol in liver tissue and the relative content of retinoic acid,the contents of rat liver tissue retinol found M group,was lower than those of CK group,but there was no significant difference,retinoic acid content were significantly lower than that of CK group（P<0.05）,after treatment with TFL retinol levels in rat liver tissue increases slightly,retinoic acid content were significantly increased（P<0.05）,and retinoic acid content of the CK group level.WB and q RT PCR were used to detect the expression of proteins or genes related to retinol metabolism pathway and matrix degradation balance.It was found that after TFL intervention,the expression level of retinoic acid X receptor（Rxra）protein downstream of retinol pathway in LF rats increased,and the expression of Aldh2 protein,Aldh1a7 m RNA and Aox3 related to retinol metabolism process increased.In addition,the expression of matrix degradation balance related enzyme Mmp3 increased significantly（P<0.05）,and Timp1 decreased.（3）In this study,Rxra and Aldh2 proteins related to the retinol metabolism pathway were docked.The results showed that 5 compounds of isoquercitrin,kaempferol,kaempferol,quercitrin and phloperidin in Tf L were closely bound to Rxra receptor protein,and 14 compounds,including proanthocyanidin B3,naringin rutin,proanthocyanidin B2,proanthocyanidin B4 and phloperidin,were closely bound to Aldh2 receptor protein.Conclusion:（1）The occurrence and development of LF is closely related to metabolic abnormalities in fatty acid metabolism,tryptophan metabolism,cytochrome P450 metabolism of exogenous substances,glycolysis/gluconeogenesis and other metabolic pathways.Slc37a4、Aldh2、Slc27a5 and Asns can be used as biomarkers of LF.（2）TFL can reduce inflammation,inhibit collagen deposition and alleviate LF in CCl₄-induced LF rats.The anti LF effect of TFL is related to retinol metabolic pathway.（3）Isoquercitrin,kaempferol,kaempferol,proanthocyanidin B3,rutin and proanthocyanidin B2 may be the main anti-LF active components of TFL.