The Molecular Mechanism Research Of Fibrosis And Calcification Regulated By Hydrogen Peroxide-mediated Oxidative Stress Injury

Background:Calcific aortic valve disease(CAVD) is a kind of common cardiovascular disease whose incidence increased with age. Fibrosis and calcification is core pathology change of CAVD. Further study found that the process of fibrosis valve include valvular interstitial cells(VICs) changed into myoblast cell, abnormal deposition of extracellular matrix and valve matrix remodeling which caused by express imbalance of Matrix metalloproteinase(MMPs) and Tissue inhibitors of metalloproteinase(TIMPs). The process of valve tissue calcification include calcium deposits based on apoptosis of valvular interstitial cells and bone formation which based on VICs changing into osteoblasts and express osteogenic gene and bone matrix. Oxidative stress injury has become a hotspot in recent years in the pathogenesis study of CAVD. The importance role of hydrogen peroxide (H2O2) mediated oxidative stress damage in the development of CAVD has obtained more and more attention. H2O2maintains at low concentration in physiological state, about0.1-0.2μ M.While in pathological conditions it can reach to100-200μM. Multiple research groups have detected large amounts of H2O2in calcified valves,and large amounts of hydrogen peroxide in vitro vivo calcification model has been found,which prompted that H2O2mediated oxidative stress may play an important role in the process of valvular fibrosis and calcification.Objective:Based on the above theoretical research background,and urrently, physiological features and pathology change research of VICs, especially human species,are still in the preliminary stage. This subject is designed to:1. Establish oxidative stress model of human VICs mediated by H2O2.2. To probe into the molecular mechanism of valve fibrosis induced by H2O2mediated oxidative stress injury.3. To probe into the molecular mechanism of valve calcification induced by H2O2mediated oxidative stress injury.Methods:Therefore, this article will be carried out as the following three parts:1.Establish oxidative stress model of human VICs mediated by H2O2:Two-step enzyme digestion method is used to isolate VICs,then seed VICs into culture plate and cultivate them in the suitable environment.Dynamicly observe the cellular morphology of VICs under optical microscope.VICs are exposed to different consentration of H2O2, observe the apoptotic morphology of VICs under optical microscope after HE staining. MTT assay was used to estimate viability of VICs.2. To probe into the molecular mechanism of valve fibrosis induced by H2O2mediated oxidative stress injury:VICs were exposed to different concentrations of H2O2, Scratch test was used to evaluate the migration ability of VICs.Collect mRNA at different time points (one week, two weeks, three weeks, four weeks).Use qRT-PCR to detect a-SMA, extracellular matrix gene,MMPs and TIMPs mRNA expression after reverse transcription.3.To probe into the molecular mechanism of valve calcification induced by H2O2mediated oxidative stress injury:VICs were exposed to different concentrations of H2O2.Using TUNEL staining method and AnnexinV/PI flow cytometry instrument to detect apoptosis. VICs were exposed to different concentrations of H2O2, then collect mRNA at different time points. qRT-PCR was used to detect apoptotic and calcified related gene expression after reverse transcription.Results:1.Establish oxidative stress model of human VICs mediated by H2O2:Two-step enzyme digestion method can successfully isolate human VICs. Original cells looks like spherical, rod or fusiform, Pseudopodia stretched out a bit, then pseudopodia increased and elongation, and can integrate with each other, at last the VICs looks like star shap. HE staining showed that VICs cytoplasm shrinkage, nucleus pycnosis, nucleoli disappear after exposed to H2O2under the concentration of800μ mol/L. After exposed to different concentrations of H2O2for24hours MTT assay showed the survival rate of VICs rose slightly(p<0.05) when the concentrations were at the range of0-100μ mol/L, then it went down quickly when the concentrations continued to rise, there was an inflection point at which the concentration was800μ mol/L with the survival rate69%(69.8±8.3,n=6), the survival rate decreased expeditiously to14%(14.3±11.0,n=6)when the concentration reached1000μ mol/L.2.To probe into the molecular mechanism of valve fibrosis induced by H2O2mediated oxidative stress injury:Myoblast cell specific marker a-SMA is obviously higher compared with control group(p<0.05).Scratch test showed that cell migration ability was enhanced when the concentration was100μ mol/L compared with control group(p<0.05), cell migration ability of500-800μ mol/L treatment group were obviously weakened(p<0.05). Transcript level of collagen I mRNA was higher compared with control group(p<0.05) at the third week and the forth week. Transcript level of collagen Ⅲ mRNA was also higher compared with control group(p<0.05) at the third week and the forth week. Transcript level of Fibronectin mRNA was higher compared with control group(p<0.05) at the forth week. Transcript level of MMP-3mRNA was declined compared with control group(p <0.05) at the third week and the forth week, and transcript level of TIMP-1was higher compared with control group(p<0.05) at the forth week.3. To probe into the molecular mechanism of valve calcification induced by H2O2mediated oxidative stress injury:TUNEL staining showed that TUNEL staining positive cells (nucleus dyed brown) began to appear when the concentration of H2O2reached500μ mol/L, almost all cells were TUNEL staining positive when the concentration of H2O2reached800μ mol/L. Flow cytometry instrument testing showed that apoptotic rate of control group was2.85%(2.85±0.69%),the300μ mol/L group was6.04%(6.04±0.17%), the500μ mol/L group was6.85%(6.85±0.50%),and the the800μ mol/L group was22.76%(22.76±0.94%).At the early stage, transcript level of c-fos was declined compared with control group(p<0.05),and it positively related to the treatment concentration. At the later stage, transcript level of c-fos, Bax,caspase-3and P53was higher compared with control group(p<0.05), and it also positively related to the treatment concentration. At the later stage, transcript level of Runx2,osteocalcin and osteoponin was higher compared with control group(p<0.05), and the effect of low concentration treatment group(200μ mol/L) is more obvious than high concentration.Conclusion:1. Two-step enzyme digestion method can successfully isolate human VICs, the method is simple and feasible, higher cell can yield, cell morphology homogeneity is better and growth characteristics is stable.2. H2O2has significant impact on viability of VICs, low concentration of H2O2(0-100μmol/L)can elevate viability of VICs,while high concentration of H2O2(>800μ mol/L)can weaken viability of VICs,and can even cause apoptosis and necrosis.3. Hydrogen peroxide mediated oxidative stress can activate VICs, chang it into myoblast cell and raise the expression of its specific markers a-SMA.4. Low concentration of H2O2(0-100μ mol/L)can elevate contraction and migration ability of VICs, high concentration of H2O2(>500μmol/L) inhibit contraction and migration ability of VICs.5. Hydrogen peroxide mediated oxidative stress can chang VICs into myoblast cell and raise the expression of extracellular matrix such as collagen Ⅰ, collagen Ⅲ and fibronectin.6. The decline expression of MMP-3may promote the abnormal deposition of extracellular matrix, and with the extension of processing time,The elevated expression of TIMP-1may collaborative promote the deposition of extracellular matrix.7. H2O2can induce VICs apoptosis, and its induction ability is concentration dependent,the capacity is week below the concentration of500μ mol/L and the capacity is strong when the concentration reached800μ mol/L.8. The molecular mechanism of inducing apoptosis by H2O2may inhibit the expression of proliferation gene c-fos at the early stage.As the processing time is extended, it continue to suppress the expression of c-fos on one hand, and accelerate the expression of pro apoptotic gene such as Bax,caspase-3and P53on another.9. The ability of changing VICs into osteoblasts by200μmol/L treatment group is stronger than400μmol/L treatment group, and is time dependent. With the extension of processing time, the expression of upstream osteogenesis related gene Runx2raised, and then promote the expression of downstream osteogenesis related genes such as osteocalcin and osteoponin.