The Effects And Mechanisms Of Noradrenaline And Selective α₁-adrenoceptor Subtypes Antagonists On The Activation, Proliferation And Secretion Of Rat Hepatic Stellate Cells

Liver fibrosis is characterized by the pathology of excessive deposition ofextracellular matrix (ECM) due to a variety of pathogenic factors including chronicviral hepatitis, alcoholism, drug interactions, metabolic disorders and so on. Hepaticstellate cell (HSC) is one kind of nonparenchymal cells located between the hepaticsinusoidal endothelial cells and hepatocytes. The activation of the HSC is the centralevent in liver fibrosis, the apoptosis of which is the initiating factor for the reversalprocess.The sympathetic nervous system (SNS) participates to control and adjust theactivity of organism. It can maintain relative equilibrium between organism and theinternal environment as well as the external environment. The SNS also participates inthe liver function regulation. Noradrenaline (NA) as the main neurotransmitter of SNSmay play an important role. The regulative effect of the SNS in hepatic fibrosis and theliver regeneration has been widely proved. It provides solid foundation for further understanding the mechanisms underlying the development of hepatic fibrosis.Previous studies have indicated that there are a variety of adrenoceptor subtypesexpressed in the liver and HSC, but it needs further researches. Some experimentsshowed that NA promotes the proliferation and inhibits the apoptosis of HSC. Ourprevious experiments have confirmed that NA has the effects of promoting HSCproliferation, nonselective α-AR antagonist phentolamine (PTL) can inhibit HSCproliferation in vivo. Our experiments selected three subtypes of α1-AR to investigatethe expressions of adrenoreceptors subtypes in HSC-T6. Then we hope to research theeffects of NA to HSC and NA get through which adrenoceptor subtypes to play the roleand the possible mechanisms of NA to HSC.OBJECTIVETo investigate the expressions of α1-AR subtypes in HSC-T6, we selected threeα1-AR subtypes to study. And to investigate how and whether NA effects HSC-T6, wedetected the activation, proliferation and secretion of ECM in HSC-T6. In order to studythe possible mechanisms of NA effecting HSC-T6, we explored the preliminarysignaling pathway of NA effecting HSC-T6.METHODSCultivated and grouped HSC-T6in vitro. HSC-T6were divided in13groups:Control group; NA (10-4,10-5,10-6,10-7,10-8,10-9mol/L) group; α1B-AR antagonist(Chloroethylclonidine, CEC10-5mol/L) group; α1D-AR antagonist (BMY737810-5mol/L) group; Gα inhibitor (PT10-5mol/L) group; PKC inhibitor (RO-32-043210-5mol/L) group; PI3K inhibitor (LY29400210-5mol/L) group; AKT inhibitor(GSK69069310-5mol/L) group. The distribution and expression levels of α1A-, α1B-andα1D-AR in HSC-T6were detected by immuocytochemistry and RT-PCR. MTT assaywas used to evaluate the cells proliferation. The expressions of HSC activation factors: transforming growth fact β1(TGF-β1) and α-smooth muscle α-actin (α-SMA), theextracellular matrix (ECM) secretion factors: tissue inhibitor of metalloproteinase1(TIMP-1) and collagen-Ι (ColΙ) in HSC-T6were detected by western blot and RT-PCR.The expressions of PKC, PI3K and AKT were detected by western blot and RT-PCR.RESULTS1. The expressions of α1-AR in HSC T6.α1B-AR and α1D-AR were expressed in the membrane of HSC-T6but α1A-AR wasalmost not expressed.2. Effects of NA and the selective α1-adrenoceptor antagonists on the proliferationof HSC-T6.NA significantly induced HSC-T6proliferation in a concentration-dependentmanner, which was inhibited by the antagonists of α1B-and α1D-AR.3. Effects of NA, the selective α1-adrenoceptor antagonists and a variety ofsignaling pathway inhibitors on the activation and ECM secretion of HSC-T6.NA induced the expressions of TGF-β1, α-SMA, TIMP-1and ColΙ, which werereduced by the antagonists of α1B-AR and α1D-AR and the inhibitors of Gα, PKC, PI3Kand AKT respectively.4. Effects of NA and the selective α1-adrenoceptor antagonists on the expressions ofPKC, PI3K and the phosphorylation of AKT on the HSC-T6.NA significantly promoted the expression of PI3K, PKC, and promoted thephosphorylation of AKT. The α1B-and α1D-AR antagonists can significantly inhibit theexpression of PKC, PI3K, and reduce the phosphorylation of AKT.CONCLUSIONS1. α1B-and α1D-AR mainly express in the cell membrane of HSC-T6. And α1B-ARexpressed more than α1D-AR. 2. NA can promote the activation, proliferation and secretion of ECM of HSC-T6in aconcentration-dependent manner, mainly through α1B-and α1D-AR. α1B-and α1D-ARantagonists can down-regulate the function of NA-treated HSC.3. NA significantly induced HSC-T6proliferation in a concentration-dependentmanner and EC50is277nmol/L. α1B-AR antagonist CEC and α1D-AR antagonistBMY7378both reduced HSC-T6proliferation. The role of α1D-AR antagonist is acompetitive antagonist and α1B-AR antagonist is a noncompetitive antagonist.4. NA through α1B-, α1D-AR to activate Gαprotein coupling with PKC-PI3K-AKTsignaling pathway is one of the mechanisms to promote HSC-T6activation,proliferation and secretion of ECM.