The Study On The Effect Of San-Cao On Treatment Of Liver Fibrosis And Action Mechanisms

Aims:To screen the active ingredients of the hospital preparation "San Cao Granule" for the therapeutic effect of liver cirrhosis/fibrosis through network pharmacology and to clarify its mechanism, and then to provide a scientific basis for clinical treatment via further validating the effect and mechanism of San Cao Granule on liver cirrhosis/fibrosis.Methods:?Network pharmadcology was used to screen and predict the active ingredients and mechanism of San Cao Granule. ?Liver cirrhosis model was copied by CCL4 in combination with BSA to observe the therapeutic effect of San Cao Granule on liver cirrhosis.?Liver fibrosis model was established by thioacetamide to observe the effect of San Cao Granule on liver fibrosis. ?Liver fibrosis model was performed by porcine serum to observe the effect of San Cao Granule on liver fibrosis. Immunohistochemistry was used to detect ?-SMA and TGF-?1. ?-SMA, TGF-?1, p-Smad3, Smad7, TLR4, MyD88 and NF-?B were determined by western blot. RT-PCR detected TGF-?1, HMGB1 and TLR4.?Mass Spectroscopewas used to identify the active ingredients in San Cao Granule.Results:(1) The prediction of active ingredients and molecular mechanism of San Cao Granule for the treatment of liver fibrosis based on the network pharmacologyWe screened out 68 components and 387 targets of rapamycin,59 protein targets of liver fibrosis related disease,440 interactionalproteinbased on the construction of database. There were 16 components and 51 targets in Rubia cordifolia L.,24 components and 75 targets in Siegesbeckia orientalis L.,2 components and 3 targets in Gentiana macrophylla Pall.,4 components and 82 targets in Salvia miltiorrhiza Bunge., 11 components and 108 targets in Forsythia suspensa (Thunb.) Vahl,11 components and 68 targets in Glycyrrhiza uralensis Fisch. in San Cao Granule.Finally, we screened out 11 components and 16 targets predicted as the direct effect targets through network pharmacology, the nodes with higher values of 'Degree', 'Betweenness'and'Closeness'(above 2 fold of median value)were 3.02,0.41 and 0.07 respectively. There were amyloid beta A4 protein, actin, cytoplasmic 1, transforming growth factor beta-1 and fibroblast growth factor 2 meeting the above conditions.22 KEGG pathways were enriched through ArrayTrack v.3.5.0 pathway enrichment analysis of the treatment of San Cao Granule on hepatic fibrosis. TGF-beta signaling pathway, P53 signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and Chemokine signaling pathwayare either directly/indirectly involved in immune and inflammation or play other important roles in liver fibrosis. TGF-(31 was estimated to participate in many above signaling pathways such as cancer, Pancreatic cancer, Chronic myeloid leukemia, Cytokine-cytokine receptor interaction, Cell cycle, Adherens junction as well as TGF-beta signaling pathway. Therefore, we selected TGF-?1 and its downstream important protein Smad3 and Smad7 for further study.(2) Effect of San Cao Granule on liver cirrhosis induced by CCL4 in combination with BSACompared with normal group, the serum levels of ALT, AST, TB were significantly increased(P<0.01), while the ALB was obviously decreased (P?0.01) in model group. The serum levels of ALT and AST, TB significantly dropped (7><0.05 or P?0.01) in ursodesoxycholic acid (UDCA) and San Cao Granule group with the content of ALB up-regulated obviously (P<0.01) in ursodesoxycholic acid (UDCA) and San Cao Granule group(3.6 and 1.8 g/kg) comparing to the model group. The serum levels of HA, LN and IVC went upward greatly in model group (P<0.01) relative to normal group. And the serum levels of HA, LN and IVC significantly decreased in ursodesoxycholic acid (UDCA) and San Cao Granule treatment groups (P<0.05 orP <0.01) in comparison with model group. San Cao Granulesignificantly reducedthe level of collagen and inflammation of liver. This indicated that San Cao Granule could greatly alleviate the inflammation andlivercirrhosisof rats.(3) Effect of San Cao Granule on TAA-induced liver fibrosisCompared with normal group, the serum levels of ALT, AST, TB were significantly increased(P<0.01), while that of ALB was obviously decreased (P?0.01) in model group.The serum levels of ALT and'AST significantly dropped (P<0.05 or P<0.01) in ursodesoxycholic acid (UDCA) and high and middle dose of San Cao Granule group while the content of ALB were up-regulated obviously (P<0.01) comparing to the model group, while the serum level of TB in ursodesoxycholic acid (UDCA) group andthe three dose of San Cao Granule dose group was significantly reduced (P?0.01). Compared with normal group, the serum levels of HA, LN and IVC went upward greatly in model group (P<0.01). And the serum levels of HA, LN and IVC significantly decreased in ursodesoxycholic acid (UDCA) and San Cao Granule treatment groups ( P<0.05or P<0.01). San Cao Granulesignificantly reducedthe level of collagen and inflammation of liver.(4) Effect of San Cao Granule on porcine serum-induced liver fibrosisCompared with normal group, the serum levels of ALT, AST, TB were significantly increased(P<0.01), while that of ALB was obviously decreased (P<0.01) in model group. The serum levels of ALT and AST significantly dropped (P<0.01) in ursodesoxycholic acid (UDCA) and San Cao Granule group while the content of ALB were up-regulated obviously (P?0.01) comparing to the model group with serum level of TB in San Cao Granule group (3.6 and 1.8 g/kg) significantly reduced (P?0.01). Compared with normal group, the serum levels of HA, LN and IVC went upward greatly in model group (P< 0.01). And the serum levels of HA, LN and IVC significantly decreased in ursodesoxycholic acid (UDCA) and San Cao Granule treatment groups (P<0.05 orP<0.01). San Cao Granulesignificantly reducedthe level of collagen and inflammation of liver. The resultsindicated that San Cao Granule could significantly alleviate the inflammation and hepatic fibrosis of rats.The results of immunohistochemistry showed that the protein of TGF-01 and a-SMAincreased greatly in model group compared with normal group, while the level of TGF-?1 and a-SMAprotein in San Cao Granule-treated group declined significantly relative to model group. Western blotting displayed that the protein of a-SMA, TGF-?1, p-Smad3, TLR4, MyD88, NF-?B (P<0.01) in model group went upward in comparison with normal group, while these proteins dropped after the administration of San Cao Granule (P<0.01). RT-PCR showed that the expressions of TGF-?1, HMGB1 and TLR4mRNA increased in model group comparing to normal group (P< 0.01), while these expressions significantly decreased after San Cao Granule treatment.(5) The identification of main components of San Cao Granule15 components, namely Rubimaillin, Purpurin, Gentiopicroside, Tanshinone IIA, Salvianolic acid B, Danshensu, Glycyrrhizic acid, Glycyrrhetinic acid, cryptotanshinone, alizarin, ferulic acid, Formononetin, orientin, sweroside and swertamarin, were identified through TOF LC/MS/MS after analyzing retention time, molecular weight, first-order spectra and second-order spectra.conclusion:1. Network pharmacology totally predicted 11 constituents which are the possible active compounds of San Cao Granule in treating liver fibrosis,16 direct targets which are the underlying protein targets for the treatment of hepatic fibrosis. TGF-?1/Smad is crucial signaling pathway for San Cao Granule in treating liver fibrosis.2. San Cao Granule, significantly increasing the body weight of rats with liver cirrhosis/fibrosis and attenuating their severity, is able to decrease the level of serum ALT, AST, TB, HALN as well as IVC and rise the content of ALB.3. San Cao Granule can dramatically dip the content of a-SMA, CTGF, HMGB 1, TLR4, MyD88, NF-?B, TGF-?1, p-Smad3, Smad7 in liver of rats with liver fibrosis, and the expressions of TGF-?1, HMGB1 as well as TLR4 mRNA are also declined.4. The main components of San Cao Granule are Rubimaillin, Purpurin, Gentiopicroside, Tanshinone IIA, Salvianolic acid B, Danshensu, Glycyrrhizic acid, Glycyrrhetinic acid, cryptotanshinone, alizarin, ferulic acid, Formononetin, orientin, sweroside and swertamarin.Overall, this study demonstrated that San Cao Granule has potent therapeutic efficiency for liver cirrhosis/fibrosis possibly through HMGB 1/TLR4/NF-?B and TGF-?1/Smad.