Triptolide Of Angiotensin Ii-induced Neonatal Rat Cardiac Fibroblast Proliferation And Collagen Synthesis And Its Mechanism

Backgrounds:Myocardial fibrosis refers to disproportionate increase of myocardial collagen fibers in a variety of factors. Its main causes include ischemic heart disease (or myocardial disease), hypertensive heart disease, inflammatory cardiomyopathy, metabolic cardiomyopathy, hypertrophic cardiomyopathy etc. Cardiac fibroblasts have the ability of proliferation. The type I, III collagen consists the basic structure of proteins of myocardial interstitial network was mainly secreted by fibroblasts. In the process of myocardial fibrosis, the proliferation of cardiac fibroblast and collagen synthesis increase, that is, myocardial interstitial reconstruction, these changes will result in abnormal myocardial metabolism and function of its increased stiffness, systolic and diastolic dysfunction, eventually lead to heart failure. Triptolide (Triptolide, TP) as a non-specific immunomodulating drug is widely used in clinical medicine to treat rheumatism and other autoimmune diseases for many years. Scholars have found that TP could inhibit human retrobulbar skin fibroblasts and keloid fibroblasts proliferation, but there is no news about its impact on myocardial fibrosis. In previous animal experiments, we have found that TP could inhibit myocardial fibrosis of the spontaneously hypertensive rat, but the exact mechanism was not yet clarified. Therefore, the purpose of this study was to observe whether TP can inhibit cardiac fibroblast proliferation and collagen synthesis, in order to reverse myocardial fibrosis, to further explore the mechanism of reversal of myocardial fibrosis.Objectives:To observe whether TP can inhibit cell proliferation and collagen synthesis of cardiac fibroblasts, so as to reverse myocardial fibrosis and initially explore the possible mechanisms.Methods:Digestion method to obtain and cultivate neonatal SD rat cardiac fibroblasts. The experiment are divided into five groups: control: the culture medium containing DMEM; AngII group: AngII is added into culture medium and the final concentration is 10-7mol/L; TP (100μg/L) groups: the final concentration 100μg/L TP and 10-7mol/L AngII are simultaneously added into the culture medium; TP (10μg/L) groups: the final concentration 10μg/L TP and 10-7mol/L AngII are simultaneously added into the culture medium; TP (1μg/L) groups: the final concentration 1μg/L TP and 10-7mol/L AngII are simultaneously added into the culture medium. MTT colorimetric determination of cell proliferation; Determination of collagen synthesis in hydroxyproline methods. Determination of TGF-β1 secretion by ELISA; Detection of MAPK extracellular signal-regulated kinase 1/2 (ERK1/2) of the content by Western Blot. To observe the influence of AngII on cardiac fibroblast proliferation and collagen synthesis and the intervention of TP at different concentrations.Results:1. The proliferation of CFb is significantly promoted after adding AngII, compared with the blank group (P<0.05 or P<0.01). The 100μg/L TP showed the effect of inhibiting the proliferation of CFb at the first 24hours (P<0.01), reached a peak within 48 hours(P<0.001), started to diminish after 72 hours, indicate the best time to exert effects were at 2 to 3 days.2. With the time increase after adding AngII, collagen synthesis increased, there is significant difference compared with the control group (P<0.05 or P<0.01). After 24h, 48h, 72h of adding TP, the collagen content of each group compared with the AngII group were significantly different. The effect of high concentration TP (100μg/L) reached the peak (P<0.001) at 48 hours (P<0.001).3. After 24hours of adding AngII, TGF-β1 expression was significantly increased (P<0.01). After 24 hours of adding different concentrations of TP, TGF-β1 expression were significantly decreased (P<0.05 or P<0.01).4. After 30min of adding AngII, ERK1/2 phosphorylation increased compared with the negative control (P<0.05). After 30min of adding 100μg/L TP, p-ERK1/2 expression decreased compared with the AngII group (P<0.05). And 1μg/L, 10μg/L TP did not inhibit ERK1/2 phosphorylation caused by AngII. Positive control U0126 significantly inhibited the ERK1/2 phosphorylation (P<0.01). Conclusions: AngII promote neonatal SD rat cardiac fibroblast proliferation and collagen synthesis, the possible mechanism may be the MAPK signal transduction pathway, to promote phosphorylation of ERK1/2 expression and exert promoting cardiac fibroblast proliferation; by increasing the expression of TGF-β1 to promote collagen synthesis. Triptolide significantly inhibited the AngII-induced cardiac fibroblast proliferation and collagen synthesis, with a dose-dependent manner, and the mechanism is probably by inhibiting the ERK1/2 phosphorylation and reducing the expression of TGF-β1.