The Role And Mechanism Of TRPV4 Receptor In The Inflammatory Response Induced By Monocrotaline In Rats With Pulmonary Arterial Hypertension

BackgroundPulmonary artery hypertension（PAH）is defined as a hemodynamic and pathophysiological status which is characterized by the increase of pulmonary artery pressure and pulmonary circulatory resistance.PAH is a chronic pulmonary circulatory disease with poor diagnosis and high mortality,and serious PAH may lead to right heart failure.It is either an independent disease or complication of some other diseases.Rats with PAH induced by monocrotaline（MCT）is one of the classical and widely used PAH models,which is related to the injury of endothelial cells caused by the toxic products of liver metabolism of MCT,the release of a variety of inflammatory mediators,and the contraction of pulmonary arteries and pulmonary vascular remodeling.The pathogenesis of PAH mainly focuses on genetics,hypoxia,endothelial injury and inflammation,among which inflammatory response has attracted much attention of researchers.Inflammatory mediators can cause vasoconstriction and promote vascular remodeling.More inflammatory cytokines（e.g.IL-1β&IL-6）are released during PAH and inhibiting the production of inflammatory cytokines can effectively reverse MCT-induced pulmonary artery hypertension,suggesting that inflammatory response is one of the important pathological mechanisms underlying the pathogenesis of PAH.It has been reported that an increase of intracellular free calcium concentration([Ca²⁺]_i)in the cytoplasm of pulmonary arterial smooth muscle cells（PASMCs）,inflammatory response and the proliferation of PASMCs play a pivotal role in the pathogenesis of PAH.Transient receptor potential channels（TRP）are a kind of non-selective cation channels superfamily,consisting more than 50 members.Transient receptor potential vanilloid 4（TRPV4）is one of member of TRPV subfamily.TRPV4 is selectively permeable to Ca²⁺and widely expressed in various tissues and cells in mammals.TRPV4 is highly expressed in both vascular endothelial cells and smooth muscle cells.Activation of TRPV4 in the endothelial cells may promote the release of endothelium-derived relax factor through increasing[Ca²⁺]_i,while activation the receptor in vascular smooth cells may participate in the process of contraction of smooth muscle cells by increasing[Ca²⁺]_i.Therefore,TRPV4 plays an important role in regulating vasomotor activities.Previous studies have shown that activation of TRPV4 leads to inflammatory response in nociceptors.Activation of TRPV4 can promote the proliferation and relocalization of vascular endothelial cells and thus is involved in vascular remodeling.However,it is still unclear whether TRPV4 is involved in the pathogenesis of PAH.The present study first examined the expression of TPV4 in PAMS in MCT-induced PAH model rats and further explored the involvement of TRPV4-mediated inflammation in the pathogenesis of PAH.ObjectivesTo study the involvement of TRPV4 in the inflammatory response in PAH rats and its underlying mechanisms.Materials and MethodsPart Ⅰ:Preparation for MCT-induced PAH model rats and effects of TRPV4 blockage on the inflammatory response of PAMS in PAH model rats Methods1.Experimental animal grouping:40 male SD rats were randomly divided into 4groups as follows:control group:10 rats were intraperitoneally（ip.）injected with equal amount of 0.9%normal saline;MCT group:10 rats were injected（ip.）with MCT（60 mg/kg）for once;HC group:10 rats were injected（ip.）with TRPV4 blocker HC-067047（10mg/kg）once daily and injected for 28 consecutive days;MCT+HC group:10 rats were injected（ip.）with HC-067047 and 30 min later injected with MCT.After that,rats were injected with HC-067047 once daily and injected for 27consecutive days.2.Preparation for MCT-induced PAH model rats（MCT rats）:Rats were injected（ip.）with 2%MCT（60 mg/kg）for once.The parameters of PAH by right cardiac catheterization were monitored 28 days later:（1）m PAP（mean pulmonary arterial pressure）;（2）PASP（pulmonary arterial systolic pressure）;（3）RV RVHI（right ventricular hypertrophy index,RVHI=×100%）.Pathological LV（10）S sections of lung tissue and HE staining:pathological changes of lung tissue,pulmonary vessels and inflammatory response;the medium thickness and lumen diameter of pulmonary arterial wall;the calculation of pulmonary arterioles wall thickness（WT%）were measured.3.Western blot:Western blot was used to examine the changes of TRPV4protein level in the pulmonary vascular smooth muscle tissue.4.Immuno-histochemical staining:pulmonary vascular remodeling and changes in inflammatory response of pulmonary artery smooth muscle tissues were examined.Results1.The mPAP,PASP,and RVHI were significantly higher in MCT rats than those in control rats,which indicated the success of preparation for PAH model rats.2.In MCT rats,HE staining showed that the pulmonary artery wall was significantly thickened,the hyperplasia of smooth muscle layer was significantly thickened,the lumen was narrowed,and the percentage（WT%）of medium thickness of pulmonary arterioles was significantly increased.3.In MCT rats,there were significant neutrophil and monocyte infiltration in the lung tissue and pulmonary artery,and the expression of CD68 and MPO（inflammatory markers）in the pulmonary artery smooth muscle tissue was significantly increased.4.The protein level of TRPV4 in the pulmonary vascular smooth muscle tissue increased significantly in MCT rats.5.HC had no effect on the morphological structure and the expression of CD68and MPO in the lung tissue in control rats,but markedly attenuated the changes of morphological structure and the expression of CD68 and MPO in MCT rats.ConclusionsThe expression of TRPV4 is up-regulated in the pulmonary artery smooth muscle tissue of MCT-induced PAH model rats.Blocking TRPV4 can inhibit the inflammatory response of the pulmonary artery smooth muscle tissue in MCT-induced PAH model rats.Part Ⅱ:TRPV4-mediated inflammatory response in pulmonary artery smooth muscle in MCT-induced PAH model ratsMethods1.Experimental animal grouping:20 male SD rats were randomly divided into 2groups as follows:control group:10 rats were injected（ip.）with equal amount of0.9%normal saline;MCT group:10 rats were injected with MCT to prepare for the PAH model rats（the same as Part I）.2.The sample extraction of rats’pulmonary artery smooth muscle and PASMC culture:pulmonary artery smooth muscle tissue was isolated from lung tissue of neonatal SD rats after anesthesia.Digestion was terminated after digestive enzyme I（30 minutes）and digestive enzyme II（60 minutes）at 37℃,respectively.The cells were added 20%FBS DMEM and inoculated in 60 mm culture dish,which were cultured in a incubator containing 5%CO2 at 37℃.The cells could be passed for 5-6generations,each passage lasted for 3-4 days,the first three generations were taken for reserve.3.Drug treatment:control group:PASMCs were cultured with normal culture medium;GSK1016790A group:TRPV4 agonist GSK1016790A（50n M）was added in the culture medium and PASMCs were cultured for 24 hours;Ac-YVAD-cmk group:caspase-1 invertase inhibitor Ac-YVAD-cmk（10μM）was added in the culture medium and PASMCs were cultured for 24 hours;GSK+Ac group:50n M GSK1016790A and 10μM Ac-YVAD-cmk were added in the culture medium and PASMCs were cultured for 24 hours.4.Western blot:Western blot was used to examine the changes of NLRP3,ASC,caspase-1,IL-1β,IL-6 and TNF-αin the pulmonary artery smooth muscle tissue and PASMCs.Results1.The protein levels of NLRP3,ASC and caspase-1,as well as those of inflammatory cytokines IL-1β,IL-6 and TNF-αin the pulmonary artery smooth muscle tissue of MCT rats significantly increased.2.After treated with TRPV4 agonist GSK1016790A,the protein levels of NLRP3,ASC and caspase-1,as well as those of IL-1β,IL-6 and TNF-αin PASMCs significantly increased.3.Caspase-1 inhibitor Ac-YVAD-cmk had no effect on the protein levels of IL-1β,IL-6 and TNF-αin normal PASMCs,but markedly attenuated GSK1016790A-induced increase of IL-1β,IL-6 and TNF-αprotein levels in PASMCs.ConclusionsActivation of TRPV4 may promote the activation of NLRP3 inflammasome in pulmonary artery smooth muscle to increase the release of inflammatory cytokines and thus enhance the inflammatory response.Blocking TRPV4 may alleviate MCT-induced rats inflammatory response in pulmonary artery smooth muscle by inhibiting TRPV4-mediated inflammatory response.