| Background of the study:Liver fibrosis is a pathological process in which the liver is damaged by various pathogenic factors,resulting in diffuse overdeposition of extracellular matrix(i.e.collagen,proteoglycans and glycoproteins,etc.),causing scar repair,and is a key step in chronic liver disease cirrhosis.It is mainly manifested by the abnormalities of liver parenchymal and non-parenchymal damage and repair functions,proliferation activation of hepatic stellate cells,and accumulation of extracellular matrix.Activation of hepatic stellate cells is the key link of liver fibrosis,and induction of hepatic stellate cell apoptosis is one of the effective ways to improve liver fibrosis.Currently,there are limited drugs available for the treatment of liver fibrosis,therefore,it is important to seek innovative drugs in Chinese medicine to improve liver fibrosis,further block the progression of liver fibrosis to cirrhosis and reduce the morbidity and mortality rate.Kaempferol-3-O-rutinoside is a natural active ingredient and the active component of the Chinese medicine Curcuma longa.Studies have shown that it has anti-inflammatory,antioxidant,and hepatic fibrosis alleviating potential.Since there are no experimental studies related to the improvement of hepatic fibrosis by kaempferol-3-O-rutinoside,this experiment aims to investigate the therapeutic effects and mechanisms of kaempferol-3-O-rutinoside on hepatic fibrosis in vivo and in vitro,in order to seek innovative drugs against hepatic fibrosis.Research methods:Firstly,we established a model of liver fibrosis in juvenile zebrafish by using zebrafish as the object of in vivo experimental study,and combined with HE staining,Sirius red staining and immunofluorescence staining to observe the effect of thioacetamide on liver fibrosis in juvenile zebrafish to establish the success of the model.Secondly,the toxicological and pharmacodynamic experiments were performed based on kaempferol-3-O-rutinoside to clarify the safe dosing range and optimal concentration,and then combined with HE staining,Sirius red staining and immunofluorescence staining to observe the intervention of low,medium and high kaempferol-3-O-rutinoside dosing concentrations on liver fibrosis in juvenile zebrafish.Finally,HSC-T6 was used as the in vitro experimental study object,combined with WB,CCK-8 proliferation assay and Tunnel staining to explore the molecular mechanism of apoptosis induced by kaempferol-3-O-rutinoside in HSC-T6 cells.Results of the study:1.A model of liver fibrosis in zebrafish larvae was successfully constructed by immersing zebrafish larvae in 0.06%thioacetamide solution 2 days after fertilization,combined with irregular morphology and bubble-like lesions in the liver of zebrafish larvae as seen by HE staining,obvious collagen fiber deposition in the liver of zebrafish larvae as seen by Sirius red staining and high expression of a-SMA by immunofluorescence staining.2、The in vivo experiments were based on the model of liver fibrosis in juvenile zebrafish,and the toxicological experiments of kaempferol-3-O-rutinoside showed that the maximum safe dosing concentration range was<100μM,and the pharmacodynamic experiments of kaempferol-3-O-rutinoside showed that 25μM,50μM and 100μML kaempferol-3-O-rutinoside could reduce the deposition of liver fibrosis in juvenile zebrafish of which 100μM was the best concentration for administration.3、In vitro experiments were conducted on HSC-T6 cells,and the low,medium and high administration concentrations of 12.5μM,25 μM and 50 μM were established by CCK-8 toxicity assay,and then the proliferation assay,Tunnel staining and immunofluorescence of kaempferol-3-O-rutinoside could inhibit HSC-T6 cell proliferation and promote HSC-T6 cell apoptosis,combined with WB It was concluded that kaempferol-3-O-rutinoside could induce apoptosis of HSC-T6 cells by regulating the Bcl-2/Bax/Caspase-9/Caspase-3 signaling pathway,thus improving liver fibrosis.CONCLUSION:In conclusion,kaempferol-3-O-rutinoside has the effect of improving liver fibrosis in juvenile zebrafish by modulating the Bcl-2/Bax/Caspase-9/Caspase-3 signaling pathway to induce apoptosis of hepatic stellate cells to improve liver fibrosis. |
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