The In Vitro And In Vivo Study Of A Pyrazole Derivative,J-1063,as A Novel Candidate Anti-liver Fibrosis Agent:Synthesis,Biological Evaluation,and Mechanistic Analysis

OBJECTIVE: The liver is an important central digestive organ of the human body responsible for storing glycogen,removing toxins,and participating in protein synthesis to maintain metabolic homeostasis.Due to direct contact with external toxins,the liver is easily damaged under stress conditions.Currently,chronic liver disease with liver fibrosis,regardless of etiology,including chronic parenchymal damage,persistent activation of inflammatory responses,and abnormal deposition of extracellular matrix(ECM),has caused significant morbidity and mortality worldwide,with Upward trend.Although it has been reported that early liver fibrosis is reversible,the specific mechanism of liver fibrosis reversal is still unclear,and there is currently no effective treatment for liver fibrosis.Therefore,research and development of anti-fibrotic drugs remains a top priority.ALK5 is mainly expressed in the inner layer of blood vessels and also plays an important role in vascular smooth muscle cells.Meanwhile,ALK5 is also a key factor for fibrotic protein expression in hepatic stellate cells(HSCs)and liver fibrosis-related cells.In recent years,more and more studies have found that chemically synthesized drugs have unique advantages in the treatment of liver diseases,and many potential small-molecule compounds have gradually received more and more attention in the treatment of liver fibrosis.Therefore,this study also designed and synthesized a small molecule inhibitor of ALK5,J-1063,and explored the specific mechanism of J-1063’s treatment of liver fibrosis by inhibiting ALK5 in vitro and in vivo experiments.Methods:1.In this study,we designed and synthesized the pyrazole derivative J-1063,and compared it with the current ALK5 inhibitor LY-2157299,which has excellent efficacy in terms of synthetic route,to explore the synthetic advantages of J-1063.The kinase inhibitory activity of J-1063 against activin receptor-like kinase 5(ALK5)and p38α mitogen-activated protein(MAP)was also evaluated in enzymatic assays,with LY-2157299 and EW-7197 selected as positive control drugs.In addition,the docking study of J-1063 and ALK5 kinase protein was carried out using Vina 1.1.2 software.2.In vitro experiments,we detected the effect of J-1063 on transforming growth factor-β(TGF-β)-induced activation of human hepatic stellate cells(LX2)and the release of fibrosis-related inflammatory factors.First,different concentrations of platelet-derived growth factor-BB(PDGF-BB)and TGF-β were used to stimulate the starved LX2 cells for 24 hours,and then the total protein and total RNA of the activated LX2 cells were extracted for western blotting and fluorescence quantitative reverse transcription polymerization.Enzyme chain reaction(Reverse Transcription PCR,RTPCR)experiments to detect and analyze the protein and gene expression of α-smooth muscle actin(α-SMA)and type I collagen(Collagen-1)to determine the in vitro model of TGF-β stimulus concentration.Then,the effect of different concentration gradients of J-1063(0-80 μM)on the viability of LX2 cells was detected by MTT method,and the concentration of J-1063 in vitro was screened out.An in vitro experimental model of hepatic stellate cell LX2 activation induced by TGF-β(5 ng/ml)was established,and LX2 cells were randomly divided into six groups,namely the normal group and the model group stimulated with TGF-β(5 ng/ml).,TGF-β(5 ng/ml)+J-1063(2.5 μM)administration group,TGF-β(5 ng/ml)+J-1063(5 μM)administration group,TGF-β(5ng/ml)+J The-1063(10 μM)administration group and the TGF-β(5 ng/ml)+ TGF-βR1 inhibitor LY2157299(5 μM)positive control group were subjected to normal culture in vitro for 24 hours and starvation culture for 24 hours.Then,the model group and the administration group were replaced with media containing J-1063(2.5 μM,5μM,10 μM)and LY2157299(5 μM),respectively,and 1 hour later,TGF was added to the medium of the model group and the administration group.-β(5 ng/ml)was cultured for 24 hours to induce activation of LX2 cells.The levels of inflammatory factors interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the collected cell supernatants were determined by Elisa method.Detection of TGF-β/Smads and Nucleotide-binding domain-(NOD-)-like receptor protein 3(NLRP3)/cysteinecontaining aspartate proteolytic enzyme(Caspase1)The protein expression of signaling pathway-related proteins and α-SMA,Collagen1,high mobility group box protein B1(HMGB1),receptor for glycation end products(RAGE)and related inflammatory factors,and finally by RT-PCR and immune cells Fluorescence staining experiments were further verified.3.In vivo experiments,we explored the regulatory mechanism of J-1063 on inflammation in the microprocess of liver fibrosis.Male C57BL/6 mice were randomly divided into four groups,namely normal group,high-dose J-1063(50 mg/kg)administration group,medium-dose J-1063(25 mg/kg)administration group,and lowdose J-1063(25 mg/kg)administration group.1063(12.5 mg/kg)administration group,3 mice in each group.After 1 week of adaptation,a 2-week pre-experiment of J-1063 in vivo administration concentration screening was carried out.During the preexperiment,the death and body weight changes of the mice were observed and recorded every day.After the experiment,pictures of the liver were collected,and the serum of the heart blood of the surviving mice in each group after centrifugation was collected to measure the content of related biochemical indicators aspartate aminotransferase(AST)and alanine aminotransferase(ALT)to determine the content of thioacetamide(TAA)in vivo.)-induced liver fibrosis formal experimental dosing concentration.Male C57BL/6 mice were randomly divided into six groups after 1 week of adaptation,including normal group,TAA group,TAA+J-1063(6.25 mg/kg)low-dose administration group,TAA+J-1063(12.5 mg/kg)group.kg)high-dose group,TAA+TGF-βR1 inhibitor LY2157299(50 mg/kg)positive control group,J-1063(12.5mg/kg)high-dose group alone,6 mice in each group.Except for the normal group and the high-dose J-1063(12.5 mg/kg)single-dose group,the other groups were given intraperitoneal injection of TAA three times a week for 6 weeks to establish TAAinduced liver fibrosis in male C57BL/6 mice Model.During the experiment,the changes in the body weight of the mice were observed and recorded every day.After the experiment,photos of the liver were collected,and the serum of the heart blood of each group of mice after centrifugation was collected to determine the content of related biochemical indicators AST and ALT;and RNA extraction.The paraffin tissue sections were subjected to immunohistochemical staining,Sirius Red staining(Sirius Red),trichrome collagen staining(Masson),apoptosis staining(TUNEL),and hematoxylineosin staining(H&E),respectively,to observe the liver tissue.Pathological changes such as the recruitment of fibrosis-related proteins,collagen and inflammatory factors.The expression levels of fibrosis and inflammation-related proteins and genes were further detected by western blotting and RT-PCR experiments.Immunohistofluorescence staining was performed using frozen tissue sections to detect the expression of macrophage and neutrophil markers.Finally,the gene expression levels of chemokine 1(CXCL1)and chemokine 2(CXCL2)were determined by realtime fluorescent quantitative reverse transcription polymerase chain reaction(Quantitative Reverse Transcription PCR,q RT-PCR)experiments.Results: 1.Compared with LY-2157299,the synthetic route of J-1063 is shorter,the cost of the reagents used in the synthesis is low,the procurement is convenient,and the synthesis operation does not require excessive temperature conditions,which is easy to operate.The enzymatic assay results show that J-1063 has significant ALK5 inhibitory activity and no effective inhibition of p38α MAP kinase,with a selectivity index of 208,which is better than the positive control drug LY-2157299,similar to EW-7197,indicating that it is effective ALK5 inhibitors.In addition,the docking results show that J-1063 can bind to the ATP site of ALK5 in a relatively stretched conformation,and the software also gives a high binding affinity score,which once again shows that the binding mode is reliable,and J-1063 has a high activity potential for ALK5.2.In vitro experiments,TGF-β(5 ng/ml)can obviously induce the activation of LX2 cells,which is manifested as the production of extracellular matrix-related proteins(α-SMA and Collagen1)and inflammatory factors.After treatment with three doses of J-1063(low(2.5 μM),medium(5 μM)and high(10 μM)and the positive control drug LY2157299(5 μM),the TGF-β/Smads signaling pathway-related protein(TGF-βR1)was significantly inhibited,Smad2/3,p-Smad2/3,and Smad4),as well as inhibiting the expression of ECM-related proteins and genes,J-1063 protected LX2 cells from TGF-β(5 ng/ml)-induced myogenesis Fibroblast differentiation,meanwhile,the protein expression of HMGB1 and RAGE and the related protein and gene expression of the NLRP3/Caspase1 inflammasome pathway were also inhibited.In addition,the expressions of inflammatory factors IL-1β,IL-18 and TNF-α released after activation of LX2 cells were also significantly decreased.3.In the in vivo pre-experiment,the survival rate of the mice in the J-1063(12.5mg/kg)administration group was 100%,and the body weight kept rising steadily.There was no significant difference between the content and the normal group,and it was suitable to be set as the high-dose administration group in the formal experiment of TAA-induced liver fibrosis.In the formal experiment of TAA-induced liver fibrosis in mice,the model group had significant ECM deposition and abnormal expression of inflammatory factors in the liver fibrosis microenvironment,and the body weight of the mice decreased significantly during the modeling period.The fibrosis was obvious,and the contents of related biochemical indexes AST and ALT were significantly increased.Immunohistochemical staining,Sirius Red staining,Masson staining,TUNEL staining,and H&E staining showed abnormal expression of fibrosis-related proteins in paraffin sections of the model group,resulting in a lot of collagen deposition and obvious inflammatory cell infiltration.As well as the expression of ECM and inflammationrelated proteins and genes in the liver fibrosis microenvironment of the model group were also significantly increased.However,administration of J-1063 and LY2157299 positive control group significantly improved the abnormal microenvironment in mouse liver fibrosis,reduced ECM deposition,apoptosis of hepatocytes,infiltration of inflammatory cells and release of related factors.Conclusion: The pyrazole derivative J-1063 designed and synthesized in this study has significant ALK5 kinase inhibitory activity and high selectivity,and may be an effective ALK5 inhibitor.In the molecular docking experiments,the kinase domain of J-1063 has obvious similarity with the kinase domain of ALK5,which may have a high activity potential for ALK5.J-1063 inhibits the canonical TGF-β/Smads signaling pathway,significantly reduces the activation of hepatic stellate cells(HSCs)and the deposition of extracellular matrix(ECM),and plays a crucial role in the reversal of liver fibrosis.In addition,in the progression of liver fibrosis,J-1063 significantly inhibited the activation of NLRP3 inflammasome and the release of HMGB1 danger signals,reduced the release of inflammatory factors under liver injury and various stimuli,and improved liver fibrosis.inflammatory microenvironment.At the same time,J-1063 significantly reduced the expression of macrophage and neutrophil markers,and exerted a certain inhibitory effect on the harmful recruitment of inflammatory cells in a TAA-induced liver fibrosis model.ALK5 small molecule inhibitor J-1063 is expected to be a potential candidate for anti-hepatic fibrosis drugs.