Study On The Anti-Hepatic Fibrosis Effect And Mechanism Of Total Flavonoids In Mallotus Apelta(Lour.)Muell.Arg Leaf.

Objective:1.To elucidate the chemical constituents of the leaves of the Mallotus apelta and explore the possible constituents of the pharmacological activity.2.To investigate the mechanism of TFM in preventing liver fibrosis by correcting metabolic disorders of the internal environment through metabolomics analysis of collected rat urine samples by nuclear magnetic resonance（NMR）.At the same time,Ingenuity Pathway Analysis（IPA）was carried out on the screened potential biomarkers to find the potential target genes of total flavones in the TFM against liver fibrosis.3.The purpose of this study was to explore the antifibrotic effect of total flavonoids of TFM and its potential mechanism.Methods:1.Total component analysis and separation of total flavones in Mallotus apelta leaf The Mallotus apelta leaf extract was obtained by crushing the Mallotus apelta leaf and extracting with 50%ethanol under reflux.After the samples were filtered,ultra-high performance liquid chromatography-time-of-flight tandem mass spectrometry（UHPLC Triple TOF MS/MS）combined with a self-built standard database was used to analyze the chemical composition of Mallotus apelta leaf.The leaf extract of white dorsal leaf was eluted with 30%ethanol through polyamide column.After concentrated and dried,it was eluted with 40%ethanol by polyamide column.The total flavones of the Mallotus apelta leaf（TFM）were obtained after the eluent was dried.An appropriate amount of TFM was taken to be dissolved and filtered into UHPLC Triple TOF MS/MS analysis.2.Urine metabolomics and IPA analysis in rats Twenty-four male SD rats were randomly divided into three groups（normal group,model group,TFM group）,except for the normal control group,all rats were orally given 2 ml/kg carbon tetrachloride（CCl₄）（olive oil 1:1 mixture）,twice a week for a total of 16weeks.At the eighth week of modeling,two rats in each of the three groups were randomly selected for HE pathological examination.After confirming the successful modeling,from the 9th week,rats in the TFM group were orally administered 100 mg/kg of TFM,and the normal group and the model group were orally administered with an equal amount of solvent once a day for 8 weeks.Twenty-four hours after the last administration,fasting and water were allowed.Urine was collected from three groups of rats in metabolic cages at night and the rats were sacrificed the next day.After anesthesia,the rats were sacrificed by taking blood from the abdominal aorta,and the serum was collected by centrifugation at room temperature to detect the levels of alanine transaminase（ALT）,aspartate aminotransferase（AST）and alkaline phosphatase（ALP）.Part of the liver was placed in formalin solution for subsequent pathological examination.Blood biochemical levels were detected by a fully automatic biochemical analyzer.The pathological changes of liver tissue were observed by HE staining.The dynamic changes of urine metabolites in rats with hepatic fibrosis induced by CCl₄before and after TMF treatment were studied by NMR.IPA was used to find potential target genes for TFM to improve liver fibrosis,and the expression of target genes was verified by real-time fluorescence quantitative PCR（RT-PCR）and Western Blot.3.Study on the pharmacological action of TFM against liver fibrosis and its molecular biological mechanism Hepatic fibrosis was induced by CCl₄in rats.The CCl4-induced rats received intragastric administration of colchicine（0.2 mg/kg per day）,TFM（25,50,100/mg per day）and the equal vehicle was given to normal rats.Pathological evaluation in hepatic tissue were examined by hematoxylin and eosin（HE）staining.And the levels of serum biochemical parameters were detected by automatic biochemical analysis.Meanwhile,the collagen deposition in liver was observed by staining with Masson’s trichrome.Collagenic parameters and inflammatory factors were measured by enzyme-linked immunosorbent assay（ELISA）kits.Additionally,corresponding assay kit was used to estimate the antioxidant enzyme and lipid peroxidation.In order to explore the potential mechanism of anti-fibrotic effects in TFM,the expressions of liver fibrosis related gene and protein were analyzed by real-time quantitative reverse transcription polymerase chain reaction（RT-PCR）and Western blot.Results:1.A total of 63 compounds were identified by comparing with the standard database,of which 18 were flavonoids with the highest area proportion.After elution by polyamide column,the total flavonoids in Mallotus apelta leaf were obtained.2.TFM significantly reduced serum ALT,AST and ALP levels,improved liver steatosis and reduced inflammation.In terms of urine metabolomics,a total of 7 potential biomarkers were found,mainly involving 2 metabolic pathways.IPA analysis showed that TNF may be a potential target for TFM to improve CCl₄-induced liver fibrosis in rats.After TMF treatment,the expression of TNF-protein and m RNA in the liver of the TFM group decreased significantly.3.The CCl₄-induced hepatic fibrosis were inhibited dose-dependently in rats by TFM.The results showed that the key hallmarks of liver injury including aspartate AST,ALT,ALP,albumin（ALB）and total protein（TP）in the serum were reversed in CCl₄-induced hepatic fibrosis rats which were treated by TFM.Furthermore,TFM significantly alleviates collagen accumulation and reduces the contents of hydroxyproline（Hyp）,Type III precollagen（PC-III）,collagen I（Col I）,hyaluronic acid（HA）and laminin（LN）.RT-PCR and Western blot results showed that TFM markedly inhibits liver fibrosis hallmark factorα-smooth muscle actin（α-SMA）expressions in CCl₄-induced hepatic fibrosis rats.Moreover,TFM alleviated the oxidative stress and lipid peroxidation in rats induced by CCl₄.TFM also attenuated the pro-inflammatory cytokines including interleukin-1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）via inhibiting nuclear factor-κB（NF-κB）activation.Meanwhile,transforming growth factor-β1（TGF-β1）/Smad signaling pathway was inhibited by TFM treatment.Conclusion:1.There are abundant flavonoids in the Mallotus apelta leaf,which may be one of the material basis for the pharmacological activity.2.TFM mainly improves CCl₄-induced hepatic fibrosis in rats by correcting the tricarboxylic acid cycle and amino acid metabolic disorder.In addition,this study found that TNF may be a potential target gene for TMF in the treatment of liver fibrosis.3.TFM can alleviate CCl₄-induced hepatic fibrosis in rats,which potential mechanism may be due to its ability of reducing ECM accumulation,improving antioxidant and regulating TGF-β1/Smad signaling pathways and NF-κB-dependent inflammatory response.