Anti-liver Fibrosis Effect Of Total Flavonoid Extract From Scabiosa Comosa Fisch. Ex Roem. Et Schult And Its Effect On ITGB4/FAK/p38 Pathway

Objective:To study the anti-liver fibrosis effect and its mechanism of the total flavonoid extract?TFS?of Scabiosa comosa Fisch.ex Roem.et Schult,Provide experimental data for the development of anti-fibrotic Mongolian drugs.Methods:Fifty Wistar rats were equally randomized into five groups:a blank control group,a model group,and high-,intermediate-,and low-dose TFS treatment groups.Except for the blank control group,rats in the other four groups were intragastrically administered with CCl₄2 ml/kg to establish the liver fibrosis models.Furthermore,the high-,intermediate-,and low-dose TFS groups were intragastrically given TFS at a dose of 200,100 and 50 mg/kg,respectively.After 10 weeks,the rats were sacrificed,and blood and liver samples were collected.1.Serum ALT,AST,ALP content was measured,liver hydroxyproline content was measured,liver coefficient was calculated.2.HE,Masson staining observed pathological changes in each group.3.Liver tissue was used for transcriptome sequencing.The sequencing results were processed and screened for pathogenesis related to liver fibrosis using GO analysis and KEGG analysis.4.HSC-T6 cells were cultured in vitro,and the growth curve was drawn to grasp the growth characteristics.5.Different concentrations of TFS were used to interfere with HSC-T6 cells at 24h,48h,and 72h,respectively.The inhibition of TFS on the cells of each group was measured by MTT method,and the IC₅₀was calculated to determine the drug concentration and intervention time.6.The liver tissue and HSC-T6 cell RNA of each group were extracted,and the expression levels of?-SMA,Collagen?,ITGB4,FAK,and p38 m RNA were detected by RT-q PCR.7.liver tissue and HSC-T6 cell proteins were extracted from each group,and the expression levels of?-SMA,Collagen?,ITGB4,p-FAK,p38,p-p38 protein were detected by western blot.8.HSC-T6 cells were randomly divided into the control group,TFS high-dose group,TFS intermediate-dose group,and TFS low-dose group,and the corresponding dose of TFS was used for 24 hours to detect the inhibitory effect of TFS on the proliferation of HSC-T6 cells by MTT method.9.HSC-T6 cells were randomly divided into the control group,TFS high-dose group,TFS intermediate-dose group,and TFS low-dose group,and were treated with the corresponding dose of TFS for 24 hours and then stained with Annexin V-FITC,and detected by flow cytometry.Results:1.Compared with the blank control group,the model group had significantly higher ALT,AST,ALP,hydroxyproline,and liver coefficient levels,Compared with the model group,the contents of ALT,AST,ALP liver hydroxyproline,liver coefficient in the high,intermediate,and low-dose groups were significantly reduced?P<0.05?.2.HE,Masson staining results showed that the model group had fibroid lesions and inflammatory cell infiltration compared with the blank group.Compared with the model group,fibroid lesions and inflammatory cell infiltration in the TFS high,intermediate and low dose groups were significantly improved.3.The results of GO analysis and KEGG analysis showed that compared with the blank group,the ITGB4/FAK/p38 signaling pathway in the liver tissue of the model group was significantly overexpressed,and decreased after treatment with TFS.4.The growth curve of HSC-T6 cells showed that the logarithmic growth phase of HSC-T6 cells was from 2 to 5 days.5.Intervention of HSC-T6 cells with different concentrations of TFS after 24,48,and 72 found that the inhibition rate of HSC-T6cells at different concentrations of TFS at the same time increased with the increase of drug concentration?P<0.05?,at different times The inhibition rate of HSC-T6 cells with TFS at the same concentration did not change with time,The IC₅₀was 25?g/ml,so the drug concentration was determined to be 12.5?g/ml,25?g/ml,37.5?g/ml,and the intervention time was determined to be 24 h.6.RT-q PCR of Liver tissue results showed that compared with the blank group,the expression levels of?-SMA,Collagen?,ITGB4,and p38 m RNA in the model group were significantly increased?P<0.05?.Compared with the model group,the expression levels of?-SMA,Collagen?,ITGB4 and p38 m RNA decreased significantly in TFS high,intermediate and low group?P<0.05?,and there was no difference in FAK m RNA expression between groups.The RT-q PCR results of HSC-T6 cells showed that compared with the control group,the expressions of?-SMA,Collagen?,ITGB4,FAK,and p38m RNA in TFS high,intermediate and low group was significantly decreased?P<0.05?.7.Western blot results of liver tissue showed that compared with the blank group,the expression levels of?-SMA,Collagen?,ITGB4,p-FAK,p38,and p-p38protein in the model group were significantly increased?P<0.05?.compared with the model group,?-SMA,Collagen?,ITGB4,p-FAK,p38,p-p38 protein expression levels in the high,intermediate and low dose TFS groups were decreased?P<0.05?.Western blot results of HSC-T6 cells showed that compared with the control group,the expression levels of?-SMA,Collagen?,ITGB4,p-FAK,p38,and p-p38 protein level in the TFS high,intermediate and low dose TFS groups were decreased?P<0.05?.8.The results of MTT experiments showed that compared with the control group,the TFS higher,and the intermediate and low dose groups had different degrees of inhibition on the proliferation of HSC-T6 cells.9.Annexin V-FITC double staining results showed that compared with the control group,the number of apoptotic cells increased in the TFS high,intermediate and low dose groups?P<0.05?.Conculusion:1.Total flavonoid extract of Scabiosa comosa Fisch.ex Roem.et Schult has good anti-fibrosis effect.2.The extract of total flavonoids from Scabiosa comosa Fisch.ex Roem.et Schult can inhibit HSC-T6 cell activation,proliferation,and promote its apoptosis.3.Inhibition of HSC-T6 cell activation,proliferation,and promotion of apoptosis by the total flavonoid extract of Scabiosa comosa Fisch.ex Roem.et Schult may be achieved by inhibiting the ITGB4/FAK/p38 pathway.