Endogenous Myocardial Protective Mechanism Research Of RhoE

Chapter 1 IntroductionWe hypothesized that RhoE plays a myocardial protective role in myocardial infarction by regulating NF-κB signal.Based on RhoE,mice with RhoE haploinsufficiency and overexpression were established.Based on the success of gene engineering mice,mice in vivo MI model was established to observe the effects of different expression level of RhoE on the post-MI heart from the angles of inflammation,infarct size,heart function and survival rate.After that,the interaction between RhoE and NF-κB signals was observed in vivo MI model and in vitro adult mouse myocardial cell culture experiments.Thus,the protective mechanism of RhoE and NF-κB signal crosstalk in MI is clarified to provide scientific basis and theoretical support for CVD treatment.Chapter 2 Effect of RhoE defect on Myocardial InfarctionObjective: To investigate the effects of RhoE deficiency on inflammation,heart infarct size,cardiac function and survival rate after MI in mice.Methods: RhoE haploinsufficiency mice were constructed and in vivo MI model was established.Western blotting was used to detect RhoE protein level and inflammatory cell markers in the mouse hearts on day 3 post MI.Meanwhile,qRT-PCR was used to detect inflammatory factor level,TTC staining was used to detect myocardial infarction area,and ultrasound was used to detect cardiac ejection fraction value.Survival rate was observed in the first week post MI.Results: RhoE protein expression in the heart of RhoE deficient mice was significantly reduced.The expression of F4/80 and Ly-6G in myocardium was significantly increased after RhoE deficiency.IL-1β,TNF-α,MMP2 and MMP9 genes were also increased.Myocardial infarction area was enlarged,cardiac ejection fraction value was reduced,and the survival rate was lower in the first week post MI.Impression: RhoE deficiency can aggravate myocarditis after MI,enlarge infarct size,reduce cardiac function and short-term survival rate.Chapter 3 Protective effect of RhoE overexpression on myocardial infarctionObjective: To investigate the effect of RhoE overexpression on inflammation,infarct size,cardiac function and survival rate after MI in mice.Methods: Heart-specific RhoE transgenic mice were constructed and mice in vivo MI model was established.Western blotting was used to detect RhoE protein level and inflammatory cell markers on day 3 post MI.Meanwhile,qRT-PCR was used to detect inflammatory factor level,TTC staining was used to detect myocardial infarction area size,and ultrasound was used to detect cardiac EF value.Survival rate was observed in the first week post MI.Results: The expression of RhoE protein in RhoE transgenic mice was significantly increased.The overexpression of RhoE significantly decreased the expression of F4/80 and Ly-6G in myocardium,and the mRNA levels of IL-1β,TNF-α,MMP2 and MMP9 in myocardium post MI.The myocardial infarction area was minified,the cardiac EF value was increased,and the survival rate of the mice 1 week after MI was similar to that of the WT group.Impression: RhoE has endogenous cardioprotective effects.RhoE overexpression can reduce myocardial damage,reduce inflammation,reduce infarct size,protect cardiac function,and improve short-term survival of mice after MI.Chapter 4: RhoE negatively regulates NF-κB activation and exerts myocardial protection in myocardial infarction Objective: To investigate the effect of RhoE on NF-κB signal activation in vitro and in vivo.Methods: MI model was established in RhoE deficient and overexpressed mice.The expression of p65 and p50 in nucleus was detected by Western blotting on day 3 post MI.Myocardial cells were isolated from RhoE deficient and overexpressed mice.TNF-α was used to stimulate cultured myocardial cells.Western blotting was used to detect the expression of nuclear p65 and p50.Results: The expression of nuclear p65 and p50 were significantly increased in the MI model of RhoE-deficient mice,while after RhoE overexpression,the expression of nuclear p65 and p50 were significantly decreased.In myocardial cells stimulated with or without TNF-α,RhoE deficiency promoted the significant increase of nuclear p65 and p50 expression,while RhoE overexpression significantly reduced nuclear p65 and p50 expression.Impression: RhoE can directly inhibit the nuclear translocation of p50 and p65 proteins in NF-κB signaling,negatively regulate the activation of NF-κB signaling,inhibit inflammation and exert myocardial protection.Chapter 5: ConclusionConclusion: RhoE has endogenous cardioprotective effect in myocardial infarction injury,which can improve the survival rate of mice after MI.RhoE may directly negatively regulate the NF-κB signal activation,reduce the nuclear translocation of NF-κB protein,inhibit the inflammation and reduce the infarct size,enhance cardiac function and reduce MI damage.