The Role Of Urotensin Ⅱ In Pathogenesis Of Myocardial Fibrosis And The Intervention Of Ginkgo Biloba Extract

Urotensin II (urotensin II, the UII) is the strongest vasoconstrictorknown in vivo, in addition, it is a strong endogenous mitogen. The recent studyshowed that the UII related closely with pathogenesis of myocardial fibrosis(MF), suggesting that urotensin â…¡may play an important role in MF. Ginkgobiloba extract (GBE) possessing the curative effects of vasodilator,hypolipidemic, anticoagulant, clearing free radicals, anti-inflammatory,analgesic, and anti-tumor, has been widely used in the prevention andtreatment of cardiovascular and cerebrovascular diseases. Studies have shownthat GBE have a role in liver fibrosis, kidney fibrosis, and pulmonary fibrosisprevention, but rare studies were reported about MF and much less studiesclarified from the point of extracellular matrix (typeâ…  and type â…¢ collagenmainly in myocardium) accumulation. The purpose of this study is toinvestigate the role of UII and its possible mechanism in the development ofmyocardial fibrosis, meanwhile, to observe the intervention effect of GBE aswell.Experiment1(Chapter2): The rat myocardial necrosis model wasreplicated by isoproterenol hydrochloride (Iso)(10mg/kg) multi-pointsubcutaneous injection once. Spotty necrosis appear in the model rats'myocardium since12h after Iso injection, ischemic necrosis area significantlyincreasing with time and obvious fibrosis showed up when3weeks.Immunohistochemistry and RT-PCR were used to evaluate UII and GPR14gene and protein expression levels, transforming growth factor β1(TGF-β1),and connective tissue growth factor (CTGF) in the process of MF. Parts ratswere given ginkgo biloba extract GBE,100mg/kg body weight, and the effectto the MF will be obserfed. The results showed that the UII mRNA and protein expression, in particular mRNA, increased since12h after Iso injection, andthis increasing is early significantly than fibrosis change. Although the UIImRNA expression gradually decreased since1week, it has not been restored tonormal levels after three weeks. Furthermore, protein expression levels of UIIincreased steadily with the aggravation of fibrosis. GPR14mRNA and proteinexpression showed the same trend as well, and the increasing is also earlysignificantly than fibrosis change. Expression of UII, transforming growthfactor β1(TGF-β1), and connective tissue growth factor (CTGF) in MF groupincreased significantly. All three indexes in Animals given GBE reduced andMasson staining positive area reduced also.Experiment2(Chapter3): Neonatal rat cardiac fibroblasts were preparedfrom the hearts of1d old Wistar rats and were treated by angiotensin II (AngII)to duplicate the cell model of myocardial fibrosis in vitro. Cell proliferation,collagen I, collagen III, CTGF and TGF-β1contents were detected; theintervention effect of UII inhibitors and GBE were observed meanwhile. MTTassay showed that GBE inhibited Ang II-induced cardiac fibroblastproliferation from6.25g L_-1(6.25g L_-1⁵0g L_-1) significantly ondose-dependent and the UII inhibitor SB657510(10_-7mol L_-1) alleviate AngII-induced cardiac fibroblast proliferation also. ELISA results showed thatcollagen I, collagen III content in supernatant of cardiac fibroblast increasedafter treated by Ang II (10_-7mol L_-1). However, both two contents declinedafter the cells treated with various concentrations of Ginkgo biloba extract,especially collagen I, ginkgo biloba extract can significantly inhibit collagen Icontent on dose-dependent manner since1.56g L-1concentrations group.Also, the UII inhibitor SB657510(10_-7mol L_-1) decreased significantlycollagen I content of the cell treated by Ang II (P <0.05). TGF-β1and CTGFcontent in supernatant of cardiac fibroblast increased after treated by Ang II(10_-7mol L_-1). UII inhibitor SB657510decreased TGF-β1and CTGF levels induced by Ang II, especially TGF-β1(P <0.05). Ginkgo biloba extractinhibited TGF-β1levels induced by Ang II since3.125g L_-1concentrationsand inhibited CTGF levels induced by Ang II since6.25g L_-1concentrations.weaker than the effect of Ang II. Collagen I and collagen III content insupernatant of cardiac fibroblast increased after treated by UII, and both twocontents declined after the cells co-treated by UII inhibitors SB657510. TGF-β1and CTGF content in supernatant of cardiac fibroblast also increased aftertreated by UII. Ginkgo biloba extract can significantly inhibit Cardiacfibroblast proliferation and collagen contents on dose-dependent manner. GBEinhibited significantly UII (10_-8mol L_-1) induced cardiac fibroblastproliferation since12.5g L_-1; inhibited significantly collagen I content since3.125g L_-1. Ginkgo biloba extract inhibited also TGF-β1and CTGF contentinduced by UII.Experiment3(Chapter4), RNA interference technology was utilized oncardiac fibroblasts cultured in vitro to silence TGF-beta and CTGF expression.Collagen I and collagen III expression were detected after gene silence. Normalmyocardial cells show abundance of collagen I expression; UII enhancedcollagen I expression (P<0.01); siRNA interference of TGF-β1decreasedsignificantly collagen I expression induced by UII (P <0.01).siRNAinterference of CTGF also reduced collagen I expression (P <0.05), however,the effects is weaker than siRNA inhibition of TGF-β1. UII also enhancedcollagen III expression; siRNA interference of TGF-β1decreased significantlycollagen III expression induced by UII (P <0.01); siRNA interference of CTGFdecreased collagen III expression on a downtrend, but without statisticalsignificance.Conclusion:1. Urotensin II and its receptor GPR14play a role in the development ofmyocardial fibrosis. 2. Ginkgo biloba extract may alleviate myocardial fibrosis, of which themechanism may be related with the UII, TGF-β1and CTGF.3. TGF-β1, CTGF and UII involved in cardiac fibroblasts fibrosis inducedby Ang II.4. Ginkgo biloba extract may alleviate ECM accumulation induced byAng II.5. UII may induce proliferation and extracellular matrix accumulation oncardiac fibroblast as an independent factor.6. Ginkgo biloba extract may alleviate cardiac fibroblast proliferation,collagen accumulation, and of TGF-β1and CTGF high expression induced bywas UII.7. Extracellular matrix accumulation induced by UII may be TGF-β1andCTGF-mediated and the role of TGF-β1in stronger than of CTGF.Innovation:It is the first time that RNA interference technique was used to explore thepossible mechanism in myocardial fibrosis induced by UII. UII induce cardiacfibroblast proliferation, collagen secretion, lead to ECM accumulation, and thengenerate the occurrence of myocardial fibrosis-like lesions, which mediated byTGF-β1and CTGF.It is the first time to observe the improvement of ginkgo biloba extract onmyocardial fibrosis post necrosis from the point of extracellular matrixaccumulation and its possible mechanisms. Ginkgo biloba extract may inhibitthe occurrence and development of myocardial fibrosis and its mechanism maybe related with the UII, TGF-β1and CTGF. Ginkgo biloba extract has a wideclinical application and has certain advantages to perform the new prevention-myocardial fibrosis as an old drug.