Activation Of Cannabinoid Receptor Type Ⅱ-CB2 Ameliorates Cardiac Fibrosis After Myocardial Infarction Via Nrf2-mediated Inhibition Of TGFβ-1/Smad3 Pathway

【Background】:In recent decades,with the development of cardiac interventional techniques,for instance,percutaneous coronary intervention(PCI),Percutaneous transluminal coronary angioplasty(PTCA),the mortality of myocardial infarction has decreased significantly.However,the cardiac fibrosis which caused by the imbalance between the secretion and degradation of Extracellular Matrix(ECM)after myocardial infarction(MI)severely impaired the diastolic and systolic function of heart,leading to death as a result.Hence how to effectively curb the process of cardiac fibrosis after MI has become a heat issue nowadays.No available certain anti-cardiac fibrosis drugs or techniques.In clinical practice,the anti-remodeling drugs such as Angiotensin converting enzyme inhibitors(ACEIs),Angiotensin Ⅱ receptor antagonists(ARBs),and beta blockers have been used to prevent the remodeling process thereby ameliorated the cardiac fibrosis.Although these drugs have exerted certain effects on fibrosis,it faced with contraindications,adverse effects and limited targets.Therefore,we should find more effective targets and develop new agents urgently to reduce the cardiac fibrosis.The cannabinoid receptor agonists effects through binding with the cannabinoid receptor.According to receptor distribution in tissue,the cannabinoid receptor can be divided into 2 types,namely,cannabinoid receptor type I(CB1)and cannabinoid receptor type Ⅱ(CB2),CB1 and CB2 both are seven-transmembrane and G protein-coupled receptor,CB1 is abundant in the central nervous system.conversely,the CB2 receptor mainly restricted to periphery tissues and immune cells.In recent years,the cardiac protection of CB2 receptor has aroused wide concerns.Meanwhile,a multitude of reports pointed that activation of CB2 reduced hepatic collagen content in cirrhotic rats and could exert antifibrotic effects in experimental dermal fibrosis mice.In the present research,both in vivo and in vitro models models were conducted to imitate cardiac fibrosis post MI.We attempted to evaluate the impact of CB2 receptor agonist AM1241 on cardiac fibrosis post MI and investigate the underlying mechanisms with a focus on Nrf2 and TGF-β1/Smad3 signaling pathways.【Aims】:1.Observe the effects of AM1241 on cardiac fibrosis in vivo.2.Evaluate the impact of AM1241 on transformation and secretion function of CFs subjected to H/SD injury in vitro.3.Explore the relationship between Nrf2,ROS and TGF-β1/Smad3 pathway.【Methods】:1.Established The Myocardial infarction(MI)mice model.After operation,AM1241 was intraperitoneally injected for 7 consecutive days.Echocardiograph was conducted to assess cardiac function.Fibrosis markers such as type I and type ⅡI collagen,fibronectin,Plasminogen activator inhibitor(PAI)-1 and tissue inhibitor of metalloprotease(TIMP)-1 were examined by Western blot,while collagens were directly observed by Sirius-red staining.2.Primary cultured cardiac fibroblasts(CFs)were isolated from the left ventricular of neonatal C57BL/6 mice.After identity the purity of CFs,CFs were subjected to hypoxia/serum deprivation(H/SD)injury to simulate ischemic conditions in vivo.Immunofluorescent staining and Western blot were applied to explore the effects of AM1241 on transformation and secretion function of CFs subjected to H/SD injury.Nrf2 siRNA were applied to explore the role of Nrf2 and TGF-β1/ Smad3 pathway in this process.3.Flow cytometry analysis and assay kits were applied to explore the effects of AM1241 on oxidative stress levels in CFs subjected to H/SD injury.Western Blot and small interfering RNA targeted to Nrf2(Nrf2 siRNA)were used to explore the relationship between Nrf2,ROS and TGF-β1/Smad3 pathway.【Results】:1.Content of collagen reflected by picrosirius red staining were notably higher in remote region of MI group(18.37±1.12%)as compared to sham group,which was attenuated by AM1241 treatment(11.40±0.66%);Western blot observed significant decreased expressions of cardiac collagen I,collagen ⅡI,fibronectin,PAI-1 and TIMP-1 in MI+AM1241group as compared to MI group;The results of echocardiograph at 4 weeks after MI revealed that AM1241 significantly increased LVEF,LVFS in MI+AM1241 group compared with that of MI group(EF: 60.04±5.93% vs.42.87±3.16%,P < 0.05;FS: 26.17±1.88% vs.21.64±1.03%,P < 0.05).Meanwhile,AM1241 treatment also decreased LVEDV,LVESV as compared to MI group(LVEDV: 0.32±0.01 ml vs.0.38±0.02 ml,P < 0.05;LVESV: 0.12±0.01 ml vs.0.19±0.01 ml,P< 0.05).2.Western blot and Immunofluorescent staining confirmed the results that AM124-1 significantly suppressed the transformation and collagen production of CFs under H/SD condition via Nrf2 signaling,Nrf2 siRNA,However,partially abolished this trend.Western blot revealed that H/SD could induce a mild elevation of CB2 receptor,phosphorylationAkt(p-Akt),nucleus Nrf2,and HO-1in cardiac fibroblasts(P < 0.05 vs.Control),the effect of which was further augmented by AM1241 treatment(P < 0.05 vs.H/SD).However,treatment with a CB2 receptor selective antagonist AM630 reversed these effects of AM1241(P < 0.05 vs.H/SD+AM1241).These data suggested a pivotal role of CB2 receptor/Akt/Nrf2/HO-1 signaling pathway in the anti-fibrotic effects of AM1241.3.Flow cytometry analysis indicated that AM1241 significantly reduced oxidative stress levels in CFs subjected to H/SD injury.Furthermore,AM1241 significantly decreased H/SD-induced up-regulation of malondialdehyde(MDA)(2.45±0.29 vs.3.42±0.27 nmol/mg protein,P < 0.05).Moreover,AM1241 increased H/SD-reduced GSH level and SOD activity(GSH: 17.96±1.90 vs.9.62±1.84 nmol/mg protein,P < 0.05;SOD: 126.10±4.90 vs.77.85±2.61 U/mg protein,P < 0.05).In addition,Western blot assay revealed that the levels of TGF-β1,Smad3 phosphorylation significantly increased under H/SD conditions as compared to Control group(P < 0.05).However,these increases were inhibited by AM1241.In contrast,silencing the Nrf2 by siRNA partially abolished the AM1241’s effect.【Conclusion】:1.CB2 receptor agonist AM1241 mainly ameliorated cardiac fibrosis in remote region after MI in mice and partly prevented the loss of cardiac systolic and diastolic function.2.Activation of CB2 receptor by AM1241 inhibited H/SD-induced fibroblast-tomyoblast transformation and collagen production of CFs in an Nrf2 dependent manner.3.CB2 receptor agonist AM1241 alleviated myocardial interstitial fibrosis via Nrf2-mediated down-regulation of TGF-β1/ Smad3 pathway,which suggested that CB2 receptor activation might represent a promising target for retarding cardiac fibrosis after MI.