| Objective:In this study,we explored the mechanism of inhibiting HSC cell activation by regulating sodium-taurobile acid cotransport polypeptide(NTCP)pathway,aiming to provide a new target and basis for inhibiting HSC cell activation.Methods:Part 1:Identification of Traditional Chinese Medicine Components and Blood Ingredient in the Medicated Serum of Bielong Ruangan TabletsThe rat tail venous blood was taken,the protein was precipitated by solvent method,and the sample was injected to establish the LC fingerprint of Bielong Ruangan Tablets;Using reference materials and LC-MS/MS method to identify the blood entering components of Bielong Ruangan Tablets,and using network pharmacology methods to explore the mechanism of inhibiting HSC cell activation by Bielong Ruangan Tablets.Part 2:Mechanism of Bielong Ruangan Tablets Inhibiting HSC Activation through NTCPThe effect of Bielong Ruangan tablets on the activation of HSC cells;Gene silencing technology inhibits the expression of NTCP;RT-PCR,Western blot detected the changes in NTCP,α-SMA signal axis expression and the expression of antibodies TLR4,NF-κB,NLRP3,TGF-β,The expression of IL-10,MMP-9,and FXR.Resulst:1.A method was established for the analysis and determination of serumsamples from rats after administration,and compared with the retention time and mass spectrometry fragment ion information of blank plasma,Fangfengdeco-ction,and control samples.A total of 12 compounds derived from Bielongruangan tablets were identified in rat serum,including alkaloids(10.32%),amino acids and derivatives(16.13%),flavonoids(10.05%),lignin and coumarins(2.58%),and lipids(14.75%),Nucleotidesand derivatives(5.9%),organic acids(7.37%),others(11.24%),phenolic acids(14.1%),quinones(0.83%),tannins(0.37%),terpenoids(6.36%).2.Based on network pharmacology methods,258 key action targets of Bielong Ruangan Tablets were obtained.KEGG and GO enrichment analysis was conducted on the key targets,and the results showed that biological processes such as inflammatory response,immune response,and cellular response to lipopolysaccharides played a key role.In the "drug active ingredient key target disease" network,the degree values were sorted.The top active ingredients,such as quercetin,kaempferol,linolenic acid,myristic acid,dihydrocapsaicin,etc.,coming from Bupleurum chinense,Poria cocos,Dilong,Biejia,Paeonia lactiflora and Rhizoma curcumae,suggesed that Bielong ruangan tablet had a material basis for the treatment of liver fibrosis.3.Hepatic stellate cell model:Compared with the normal group,the number of hepatic stellate cells in the HSC activation model group was higher than that in the normal group.PDGF-AA had an effect on the proliferation of HSC cells,P=0.013,and TGF-β had an effect on the proliferation of HSC cells,P=0.009.4.Western Blot:The HSC activation model group(2 group)compared to the normal HSC group(1 group),the HSC activation model+siRNA NTCP group(5 group)compared to the HSC+siRNA NTCP group(4 group),and the HSC activation model+siRNA NTCP control group(8 group)compared to the HSC+siRNA NTCP control group(7 group),all of which significantly incresed indicated that the secretion of inflammatory factors increases after activation of hepatic stellate cells.The HSC activation model+drug-containing serum(3 group)compared to(2 group),HSC activation model+siRNA NTCP+medicated serum(6 group)compared to HSC activation model+siRNA NTCP(5 group),HSC activation model+siRNA NTCP+medicated serum control group(9 group)compared to HSC activation model+siRNA NTCP control group(8 group),all of which significantly reduced,indicated that Bielong Ruangan Tablet medicated serum has a inhibitory effects on TLR4,NF-κB,NLRP3,TGF-β,IL-10,MMP-9,FXR,and NTCP.HSC activation model+siRNA NTCP(5 group)compared with(8 group),the expression of NF-κB was decreased,indicating that silencing NTCP after HSC activation may inhibit the secretion of NF-κB;(4 group)compared with(7 group),the expression of NLPR3 and TGF-β were decreased,suggesting that silencing NTCP may inhibit the expression of NLPR3 and TGF-β;(5 group)showed an increase in MMP-9 expression compared to(8 group),indicating that silencing NTCP after HSC activation may promote MMP-9 secretion;compared with(group 4),the expression of FXR increased in(group 1),indicating that silencing NTCP may promote the secretion of FXR.In the comparison group,P<0.05.5.PCR:(2 group)compared to(1 group),(5 group)compared to(4 group),(8 group)compared to(7 group),the NTCP/α-SMA content increased,indicating an increase in NTCP/α-SMA secretion after activation of hepatic stellate cells.(3 group)compared to(2 group),(6 group)compared to(5 group),(9 group)compared to(8 group),the NTCP/α-SMA content decreased,indicating that Bielong Ruangan Pian medicated serum has an inhibitory effect on NTCP/α-SMA.In PCR detection of α-SMA factor,(5 group)showed no significant difference compared to(8 group),while(6 group)showed a difference compared to(9 group),indicating that after silencing the NTCP gene,the intervention of drug-containing serum on cell α-SMA reduction was better than the HSC activation model+siRNA NTCP+drug-containing serum control group.In the comparison group,P<0.05.Conclusions:1.This study comprehensively elucidated the chemical composition of Bielongruangan tablets and preliminarily analyzed their blood entering components,providing a scientific basis for further research on the pharmacological substance basis and quality control of this formula.2.The mechanism of HSC activation is closely related to the abnormal expression of TLR4,NF-κB,NLRP3,TGF-β,IL-10,MMP-9,FXR,NTCP and energy metabolism disorders.3.Inhibiting the expression of NTCP can inhibit NF-κB,NLRP3,and TGF-βsecreting,while promote MMP-9 and FXR secreting.4.Bielong Ruangan Pian can improve the degree of HSC activation in rats.The main mechanism of action is:(1)inhibiting the activation of hepatic stellate cells by reducing the expression of NTCP,thereby inhibiting the progression of liver fibrosis;(2)Reducing the expression of NTCP can simultaneously inhibit the expression of α-SMA and reduce inflammatory response;(3)By regulating TLR4,NF-κB,NLRP3,TGF-β,the expression of IL-10,MMP-9,and FXR can effectively inhibit the activation of hepatic stellate cells,thereby inhibiting the progression of liver fibrosis... |
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