Experimental Study On Taurine Inhibits Proliferation And Its Mechanism Of Myofibroblasts

Myocardial fibrosis （MF） occurs in a variety of cardiovascular disease, for manycardiovascular disease in an important pathological changes. As a result of long-termmyocardial blood supply, myocardial histogenesis dystrophy and atrophy, or large area aftermyocardial infarction, so that normal myocardial tissue structure of collagen fiber excessaccumulation, collagen concentration significantly increased or collagen compositionchange. Modern cell and molecular medicine research confirmed: myocardial fibrosislesions is many heart patients common biggest threats, including rheumatic heart disease,coronary heart disease and myocarditis and other major heart disease, are the basis ofmyocardial fibrosis disease, myocardial fibrosis development would cause a series ofsymptoms, and ultimately to cardiac function complete failure cause patients died. Thepathological process is cardiac function by compensatory period to decompensated phaseshift key, its extinction can restore the myocardial tissue and stroma between balance,ankylose degree gradually softening, which is beneficial to the improvement of heartfunction. So how to the prevention of myocardial fibrosis is the hot spot and focus ofmedical research.Taurine （Tau） is normal exist in the body of the sulfur amino acid, in1827by theTiedemann ox bile from the extraction separation. The human body rich content, mainlyexists in myocardial tissue, has extensive biological effect. Research shows that taurine canmaintain cell osmotic balance, regulating cell calcium steady state, clear oxygen freeradicals, reducing the peroxidation damage and direct membrane stability and function. Inrecent years, the laboratory of taurine regulation of cardiovascular disease pharmacologicalaction and mechanism was studied, confirmed that taurine can inhibit myocardial fibroblastproliferation and reversal myocardial remodeling effect, but the specific mechanism ofaction has not bright.This research is divided into three parts, first research taurine inhibiting effects ofmyocardial fibroblasts proliferation, PKC alpha, iNOS and ERK1/2, and then further studyTau reversal myocardial remodeling of intracellular signal transduction mechanism.1. Tau effects on inhibition proliferation of myoFbs Aim to observe that Tau effects on inhibition proliferation of myoFbs induced byAngiotensinII （AngII） in neonatal rats, determine the Tau inhibition myoFbs proliferationeffect of effective dose. Separation was born1³d big lactation rats myoFbs, culture72hafter subculture2-3generations randomly divided into normal control group, AngII(10-7mol·L^-1) model group and Tau （120,60and30）mmol·L^-1group, continue to cultivate24/48h,observe the change of cell morphology, the application of MTT colorimetric method todetect cell survival, kit test cell medium hydroxyproline （I and III） content, NO content andiNOS activity, ELISA detection cell supernatant fluid of TGF-β1and CTGF content,fluorescent confocal detection ERK1/2nuclear transfer, PKCα membrane inversion andprotein expression, Western blot test p-ERK1/2, p-PKCa and iNOS protein expression.The results show that, compared with controls, myoFbs proliferation live quantityobviously increased in AngII group, myoFbs medium, hydroxyproline （I and III） content,NO content and iNOS activity, TGF-β1and CTGF content, ERK1/2nuclear transfer, PKCαmembrane inversion and protein expression increased （P<0.01）; Compared with modelgroup, Tau （120,60and30）mmol·L^-1group, proliferation of myoFbs quantity reduce（P<0.05, P<0.01）, myoFbs medium, hydroxyproline （I and III） content, NO content andiNOS activity, TGF-β1and CTGF was obviously lower,（P<0.05, P<0.01）. Fluorescentconfocal detection ERK1/2nuclear transfer, PKCα membrane inversion and proteinexpression, Western blot testing p-ERK1/2, p-PKCα and iNOS protein expressionsignificantly reduced. Flow cytometry instrument detection Tau（120,60and30）mmol L^-1group myoFbs apoptosis number no significant reduce.Tau inhibits AngII cause myoFbs proliferation, reduce hydroxyproline （I and III）generation, reduce the TGF-β1and CTGF content, this effect may and NO, iNOS, ERK1/2,PKCα wait for an element to concern, including Tau in dose for60mmol·L^-1effect issignificant.2. Study on mechanism of Tau inhibits proliferation of myoFbs（1） Tau by inhibiting p-PKCα membrane inversion and expression regulation myoFbsproliferation Separation was born1³d rat myoFbs, culture72h after subculture2-3generation randomly divided into normal control group, AngII (final concentration for10-7mol·L^-1) model group, Tau （120,60and30） mmol·L^-1group, D4681(0.625ug·L^-1) group,AngII+D4681group and AngII+D4681+Tau group, continue to cultivate24/48h, applicationMTT colorimetric method to detect cell survival rate, flow cytometry instrument detectioncell cycle distribution, immunocytochemistry method, fluorescent confocal method and Western blot detection p-PKCα membrane inversion and expression. The results show that itcan inhibit the proliferation myoFbs, Tau and D4681after share inhibition effect moreapparent, and reduce the proportion of S phase, rise G0/G1phase rate. At the same time Tauinhibits p-PKCα membrane inversion and expression, D4681and Tau common after useinhibition p-PKCα membrane inversion and the expression effect more apparent, thus it canbe seen that Tau can inhibit proliferation of myoFbs is through the inhibition of p-PKCαmembrane inversion and express the realization of function. Application specific PKCαinhibitor D4681can strengthen Tau effect son inhibition proliferation of myoFbs.（2） Tau can improve activity of iNOS and content of NOSeparation myoFbs were born1³d rat, myoFbs were cultured72h after subculture,2-3generation randomly divided into normal control group, AngII (10-7mol·L^-1) modelgroup, Tau (60mmol·L^-1) group, AG(10μg·L^-1) group, AngII+AG group and AngII+AG+Taugroup, continue to cultivate24/48h, application MTT colorimetric method to detect cellsurvival, measuring cell medium iNOS activity and NO content by flow cytometry, and thedetection instrument cell cycle distribution, application immunocytochemistry method,fluorescent confocal and Western blotting detection in the expression of iNOS cytoplasm.Results show that AngII+AG+Tau group myoFbs proliferation significantly reduced,cell culture fluid iNOS activity decline, NO content lower （P<0.01, P<0.001）, S periodpercentage decline, G0/G1phase proportion increased significantly（P<0.05, P<0.01）, and theexpression of iNOS in cytoplasm increased significantly comparison with the model group,Tau can improve iNOS activity, inhibition proliferation of myoFbs.（3） Tau by inhibiting p-ERK1/2nuclear transfer and expression to inhibit proliferationof myoFbs. Separation myoFbs was born1³d rat, which were cultured72h after subculture2-3generation,then randomly divided into normal control group, AngII (10-7mol·L^-1) modelgroup, Tau(60mmol·L^-1)group, D4681(0.625ug·L^-1)group, AngII+D4681group andAngII+D4681+Tau group, continue to cultivate24/48h, application immunocytochemistrymethod, fluorescent confocal and Western blotting testing p-ERK1/2nuclear transfer andexpression. The results show that Tau inhibits p-ERK1/2nuclear transfer and expression,D4681and Tau common after use inhibition p-ERK1/2nuclear transfer and the expressioneffect more apparent, thus it can be seen myoFbs Tau inhibition of proliferation is throughthe inhibition of p-ERK1/2nuclear transfer and express the realization of function.Application specific PKCα inhibitor D4681can strengthen Tau inhibition p-ERK1/2nucleartransfer and the expression effect. （4） Tau regulate proliferation of myoFbs through ROSResearch now myoFbs found AngII induced proliferation of ROS quantity changes, inorder to further testing whether Tau by reducing the generation of ROS and myoFbsinhibition of proliferation. The myoFbs were cultured in vitro, and application specificantioxidant to detect the content of ROS and the expression of p-PKCα, discuss themechanism of action. Separation myoFbs were born1³d rat, culture72h after subculture2-3generation randomly divided into normal control group, AngII (10-7mol·L^-1) modelgroup, Tau （120,60and30）mmol·L^-1group, NAC (10-4mol·L^-1) and Tau+NAC group,continue to cultivate24/48h, application MTT colorimetric method to detect cell survivalrate, and use the ELISA kit to detecti content of·OH in cell medium, laser confocaldetection the expression of ROS, Western blot detection of p-PKCα expression of myoFbs.Results comparing with control group, AngII group myoFbs proliferation, contentof·OH in cell medium, ROS and p-PKCα expression are increased significantly;Application of antioxidant n-acetyl cysteine （NAC）, compared with the model group, Taugroup and NAC group myoFbs proliferation, content of·OH in cell medium, ROS andp-PKCα expression are all decreased. Therefore, effect of Tau on inhibition of proliferationmyoFbs and reduce the generation of ROS and p-PKCα expression, which can start againstmyocardial fibrosis effect about.To sum up, Tau can inhibit the proliferation of myoFbs and reversal myocardialremodeling, thus to attain the myocardial cell of protection, the intracellular signaltransduction mechanism may be through the activation of PKCα-ROS, ERK1/2and iNOSpath, so as to change the TGF-β1and CTGF of generation and the realization.