ALK5 Inhibitor-pyrazole Derivative J-1048 Synthesis And Anti-liver Fibrosis Mechanism

Objective: Approximately 800 million people worldwide suffer from chronic liver disease.Liver disease is one of the causes of morbidity and mortality from human disease.There are no drugs available for the treatment of liver fibrosis.In this experiment,we investigated J-1048 as a small molecule inhibitor of activin receptorlike kinase 5(ALK5),which was induced by thioacetamide(TAA)injection in in vivo experiment;and established fibrosis model by TGF-β stimulation of LX2 cells in in vitro experiment.To investigate the mechanism of action of J-1048 on liver fibrosis and to provide new options for clinical candidates for liver fibrosis.Methods: 1.Chemical synthetic: pyrazole derivative J-1048 was designed and synthesized by us.J-1048 was evaluated for its inhibitory activity against ALK5 and p38α MAP kinase.Positive control drugs were selected as LY-2157299 and EW-7197.2.In vitro experiments: human hepatic stellate cells(LX2)were incubated with J-1048(0-100 μM)for 24 h.MTT assay was performed to detect the survival rate of J-1048 on LX2 cells;TGF-β stimulated LX2 cells for 2 h,and different concentrations of J-1048(0,1.25,2.5,5 μM)and LY2157299(5 μM)were administered.5μM)and LY2157299(5μM)were incubated and treated for 24 h,and total cellular proteins were extracted.α-SMA,Collagen Ⅰ,TGF-βR Ⅰ,Smad2/3,P-Smad2/3 and Smad7 protein levels were detected in cells by Western blot;α-SMA/Smad7 and Collagen Ⅰ/TGF-βR protein levels were detected by immunofluorescence.Collagen Ⅰ/TGF-βR Ⅰ.To observe the association of TGF-β/SMAD signaling pathway with liver fibrosis markers.3.In vivo experiments: C57BL/6 male mice were used.Mice were randomly divided into normal group,thioacetamide group(TAA),low dose group(TAA+1048-12.5mg/kg),high dose group(TAA+1048-25 mg/kg),positive control group(TAA+LY-2157299-50 mg/kg)and drug administration alone group(1048-25 mg/kg).A mouse fibrosis model was established by intraperitoneal injection of TAA(100 or 200 mg/kg)(6 weeks).Serum of mice was collected to detect transaminase levels;histopathological changes in liver were observed by H&E,Masson,Sirius red staining;liver fibrosis indexes(α-SMA,Collagen Ⅰ,TGF-βR Ⅰ,Smad2/3,P-Smad2/3,Smad7)and inflammatory factors were analyzed by Western blot in the liver of mice(P2X7r,NLRP3,IL-1β)protein levels;observation of liver fibrosis factor(α-SMA,Collagen Ⅰ),immune cell(F4/80,MPO)expression by immunohistochemical staining;observation of the relationship between TGF-β/SMAD signaling pathway and liver fibrosis markers by immunofluorescence double staining of α-SMA/Smad7 and Collagen Ⅰ/TGF-βR Ⅰ.Results: Synthetic part: J-1048 had a significant inhibitory effect on ALK5 by enzyme activity assay.In vitro experiments: J-1048 decreased α-SMA,Collagen Ⅰ,TGF-βR Ⅰ,P-Smad2/3 protein levels and restored Smad7 protein levels in activated LX2 cells.In vivo experiments: J-1048 attenuated the liver ALT and AST levels induced by TAA in mice;J-1048 ameliorated liver tissue damage with collagen deposition by H&E,Masson and Sirius red staining;Western blot results showed that J-1048 significantly inhibited the liver fibrosis process in which proteins(α-SMA,Collagen Ⅰ,TGF-βR Ⅰ,PSmad2/3),decreased the release of inflammatory factors such as P2X7 r,NLRP3,IL-1β,and restored the expression level of Smad7;immunohistochemical staining showed that J-1048 reduced liver fibrosis factors(α-SMA,Collagen Ⅰ)due to TAA and decreased the recruitment of immune cells(F4/80,MPO);immunofluorescence double staining of α-SMA/Smad7 and Collagen Ⅰ/TGF-βR Ⅰ showed that J-1048 could restore Smad7 expression and inhibit α-SMA expression;J-1048 treatment could inhibit Collagen Ⅰ and TGF-βR Ⅰ expression levels.Conclusion: Pyrazole derivative J-1048 has significant inhibitory activity against ALK5.J-1048 ameliorates TAA-induced elevation of liver fibrosis markers,and as an ALK5 inhibitor,J-1048 reduces Smad2/3 phosphorylation and restores Smad7 protein levels by inhibiting the TGF-β pathway,and inhibits TAA-induced liver fibrosis in mice via the SMAD signaling pathway.J-1048 could inhibit the production of inflammatory factor IL-1β by inhibiting the P2X7R/NLRP3 signaling pathway,which could play a positive role in the treatment of liver fibrosis.Finally,immunohistochemical and immunofluorescence staining showed that J-1048 reduced TAA-induced macrophage and neutrophil recruitment.J-1048,a small molecule inhibitor of ALK5,may be an anti-fibrotic candidate.