Anti-liver Fibrosis Effect And Mechanism Of Forsythia Suspense Extract And Its Main Component Phillygenin

Objective:Liver fibrosis is the intermediate stage of the transition from liver injury to cirrhosis.Effectively inhibiting the progression of liver fibrosis is the key to preventing the development of liver cirrhosis and even liver cancer.The process of liver fibrosis is accompanied by the occurrence of inflammation.Forsythia suspensa has good anti-inflammatory and anti-liver fibrosis effects.But there is no conclusion about the regulatory relationship between anti-liver fibrosis and anti-inflammatory effects.Besides,there are many ingredients in Forsythia suspensa,and the material basis of its anti-liver fibrosis medicinal effect is not clear.Thus,this paper mainly studies the anti-liver fibrosis effect of Forsythia suspensa and its mechanism,the effective material basis,and whether the anti-liver fibrosis of Forsythia suspensa depends on its anti-inflammatory effect.Methods:（1）Forsythia suspensa extract（FSE）was prepared according to Pharmacopoeia of the People’s Republic of China,and a high-performance liquid chromatography（HPLC）analysis method for the FSE was established to evaluate the quality of FSE and to identify the main components.（2）Rats were subcutaneously injected with 40%CCl₄olive oil solution to establish a liver fibrosis model,and the rats were treated with FSE by gavage.HE staining was used to detect the pathological conditions of rat liver tissues.The results of HE staining were used to grade liver fibrosis according to the Ishak score standard.Sirius red staining was used to observe the deposition of the Extracellular matrix（ECM）.The secretion ofα-SMA and collagen I in liver tissue was analyzed by immunohistochemistry.The expression of serum cytokines ALT,AST,ALP,andγ-GT were detected by biochemical index assay kit.The expression of fibrosis markers HA,LN,PⅢNP,cⅣ,and inflammatory factors IL-6,IL-1β,TNF-αwas detected by ELISA kits.The protein expression of TLR4/MyD88/NF-κB and TGF-β/smads was detected by Western blot.（3）Molecular docking techniques in computer-aided drug design were used to investigate the main active ingredients of FSE with proteins in the TLR4/MyD88/NF-κB and TGF-β/smads signaling pathways.By preparing targets,ligands,and performing Ligand Fit molecular docking,the docking results were evaluated with a consistency score.（4）LPS stimulates LX2 cells to establish an in vitro liver fibrosis model with concomitant prophylactic treatment with Phillygenin（PHI）high,medium,and low dose groups.The proliferation of LX2 cells was detected by MTT method,and the expression of IL-6,IL-1βand TNF-αwas detected by ELISA kits.The protein expression ofα-SMA,collagen I,TLR4/MyD88/NF-κB and TGF-β/smads was detected by Western Blot.MyD88 si RNA was used to silence TLR4/MyD88/NF-κB signaling pathway,and the expression ofα-SMA,Collagen I,TLR4/MyD88/NF-κB and TGF-β/smads was detected.Results:The HPLC analysis method of FSE was successfully established,and the quality of FSE was analyzed following the Chinese Pharmacopoeia.Compared with the reference substance,four components were identified:Forsythiaside A,Phillyrin,（+）-pinoresin-β-D-glucopyranoside（PDG）and PHI.（2）CCl₄ can significantly cause abnormal liver structure,promote the expression of ALT,AST,ALP andγ-GT,inflammatory factors IL-6,IL-1βand TNF-α,and fibrosis markers HA,LN,PⅢNP and cⅣ.FSE can inhibit the significant increase of these indexes and inhibit the deposition of ECM.Western blot results showed that FSE can inhibit the expression ofα-SMA and collagen I and inhibit TLR4/MyD88/NF-κB and TGF-β/smads signaling pathway.（3）The results of computer-aided drug design showed that the docking results of PHI with TLR4/MyD88/NF-κB and TGF-β/smads signaling pathways were better than Forsythiaside A,Phillyrin and PDG.（4）100ng/ml LPS could significantly promote the proliferation of LX2 cells,the secretion of ECM and inflammatory factors.PHI could significantly inhibit the expression of collagen I,α-SMA,IL-6,IL-1βand TNF-αin LX2 cells and inhibit TLR4/MyD88/NF-κB and TGF-β/smads signal pathway.Protein expression of the TGF-β/smads signaling pathway was suppressed when the TLR4/MyD88/NF-κB pathway was inhibited by silencing the MyD88 protein.After further administration of PHI treatment,the expression of MyD88 and its downstream proteins were not significantly downregulated,the expression of proteins in the TGF-β/smads signaling pathway and collagen I andα-SMA was further downregulated.Conclusion:（1）FSE exhibited an anti-fibrosis effect by inhibiting TLR4/MyD88/NF-κB and TGF-β/smads signaling pathways,down-regulating the expression of inflammatory factors and fibrotic markers,and inhibiting HSC activation and ECM accumulation.（2）PHI was able to inhibit LPS-induced activation of LX2 cells and suppress the expression of ECM and inflammatory factors through inhibition of TLR4/MyD88/NF-κB and TGF-β/smads signaling pathways.（3）The anti-fibrosis effect exerted by PHI is partly dependent on its anti-inflammatory effect,that inhibition of the fibrosis signaling pathway TGF-β/smads is associated with inhibition of the inflammatory transduction pathway TLR4/MyD88/NF-κB.