Establishment Of TRPM_2.5-Induced Pulmonary Hypertension Rat Model And Its Potential Mechanism Research

【BACKGROUND】Pulmonary arterial hypertension（PAH）is a group of diseases characterized by gradual increase in pulmonary circulation resistance.Pathological changes include pulmonary vasoconstriction,pulmonary vascular remodeling such as proliferation and hypertrophy of endothelial cells,pulmonary artery smooth muscle cells（PASMCs）and pulmonary fibroblasts and extracellular matrix deposition,etc.,results in thickening of the pulmonary arterial vessel wall and narrowing of the lumen,which may ultimately lead to right heart failure and death.More and more research shows that particulate matter（PM）is closely related to the pathogenesis of PAH.Ambient air particles with an aerodynamic diameter of 2.5μm or less are called atmospheric fine particles(PM_2.5).PM_2.5 can reach the respiratory gas exchange area and enter the pulmonary circulation,which has a significant impact on human health.With the economic development of China,the number of vehicles has increased dramatically.The PM_2.5 generated by motor vehicles is considered to be the main source of PM_2.5 in the urban atmosphere.To investigate the influence of traffic-related PM_2.5 on pulmonary arterial hypertension and its potential mechanism can help us better understand and eliminate the effects of PM_2.5.Our previous study showed that Ca²⁺-sensing receptor（Ca SR）and canonical transient receptor potential protein（TRPC）are involved in traffic-related PM_2.5(TRPM_2.5)induced pulmonary artery smooth muscle cell proliferation under hypoxic conditions and participated in the pathogenesis of PAH.At present,there is still no research on whether TRPM_2.5 causes the occurrence of PAH animal model.The specific mechanism of whether Ca SR or TRPC is involved in TRPM_2.5-induced PAH is not yet well illucidated.【OBJECTS】Determine whether TRPM_2.5 can cause pulmonary hypertension and its possible mechanisms.【METHODS】1.Establishment of TRPM_2.5-induced PAH rat model.1.1 Thirty-six male Sprague-Dawley（SD）rats were randomly divided into the control group（Control）and the motor vehicle exhaust group（MVE）.The MVE group was exposed daily once for 2 hours,2 times per day,5 days per week,with exposures of 4weeks,12 weeks and 24 weeks,respectively.The control group was placed in the normal environment in the animal center.1.2 The PAH was assessed by measuring right ventricular systolic pressure（RVSP）,right ventricle hypertrophy index（RVHI）,and wall thickness（WT%）,wall area（WA%）of pulmonary arteries.1.3 Pulmonary arterial remodeling was observed by HE staining.Immunohistochemistry and immunofluorescence were used to detectα-SMA staining on pulmonary artery smooth muscle and factor VIII and v WF expression on endothelial cells.2.The mechanism of PAH induced by TRPM_2.5.2.1 Twenty-four male Sprague-Dawley（SD）rats were randomly divided into the control group（Control）,the control+NPS2143 group（Control+NPS2143）,the motor vehicle exhaust group（MVE）,and the motor vehicle exhaust+NPS2143 group（MVE+NPS2143）.After establishing long-term TRPM_2.5 exposure-induced rat PAH model,animals were harvested by intraperitoneal injection of Ca SR inhibitor（allosteric modulator）NPS2143 from 16 weeks up to 24 weeks after TRPM_2.5 exposure.The normal control group was intraperitoneally injected with DMSO as normal control.2.2 The change of PAH was assessed by measuring RVSP,RVHI,and WT%,WA%of pulmonary arteries.2.3 The expression of Ca SR and TRPC1 in lung tissue of different groups was detected by western-blot.【RESULTS】1.Establishment of TRPM_2.5-induced PAH rat model.1.1 After 4 weeks of TRPM_2.5 exposure,compared with the control group（RVSP:22.6±0.6 mm Hg;RVHI:20±0.0%,n﹦6）,MVE group（RVSP:22.5±0.6 mm Hg;RVHI:20±0.0%,n﹦6）had no significant changes,and there was no statistically significant difference（P>0.05）.After 24 weeks of TRPM_2.5 exposure,the RVSP of MVE group（RVSP:26.5±0.6 mm Hg;RVHI:18.5±0.0%,n﹦6）was significantly higher than that of the control group（RVSP:23.1±0.5 mm Hg;RVHI:17.4±0.0%,n﹦6）,and the difference was statistically significant（P<0.001）.No significant change in RVHI was observed（P>0.05）.1.2 TRPM_2.5 can cause pulmonary artery remodeling after 4 weeks,12 weeks and 24weeks exposure.In the MVE group with a pulmonary artery diameter in the range of0-50μm,after 4 weeks,12 weeks and 24 weeks of TRPM_2.5 exposure,the WT%were37.2±7.6,39.7±14.4,50.6±10.8 and the WA%were 63.3±10.1,64.2±17.3 and 76.4±10.0,respectively.WT%in normal group were 26.9±6.8,25.3±7.2,23.8±7.1 and the WA%were 48.4±11.0,47.0±11.6 and 44.2±11.0,respectively.All above were statistically different between the two groups（P<0.05）.In the MVE group with a pulmonary artery diameter in the range of 50-100μm,after4 weeks,12 weeks and 24 weeks of TRPM_2.5 exposure,the WT%were 20.6±3.8,23.7±6.5,30.9±9.0 and the WA%were 43.0±9.0,50.2±11.6 and 55.0±10.8,respectively.WT%in normal group were 16.1±3.3,14.7±3.4,15.7±4.0 and the WA%were 38.0±7.3,34.4±8.6 and 34.3±8.1,respectively.All above were statistically different between the two groups（P<0.05）.In the MVE group with a pulmonary artery diameter in the range of 100-150μm,after 4weeks,12 weeks and 24 weeks of TRPM_2.5 exposure,the WT%were 20.6±3.8,23.7±6.5,30.9±9.0 and the WA%were 43.0±9.0,50.2±11.6 and 55.0±10.8,respectively.WT%in normal group were 16.1±3.3,14.7±3.4,15.7±4.0 and the WA%were 38.0±7.3,34.4±8.6and 34.3±8.1,respectively.All above were statistically different between the two groups（P<0.05）.In the MVE group with a pulmonary artery diameter in the range of 100-150μm,after4 weeks,12 weeks,and 24 weeks of TRPM_2.5 exposure,the WT%were 17.8±4.1,19.3±4.2,24.2±10.2 and the WA%were 44.7±11.4 41.4±11.3 and 48.4±14.4,respectively.WT%in normal group were 14.8±3.6,14.5±2.9,12.0±4.4 and WA%were 41.4±10.5,37.7±9.4 and 29.8±8.3,respectively.There was a statistically significant difference between the two groups after 24 weeks of TRPM_2.5exposure（P<0.05）.After 4 weeks and 12 weeks of TRPM_2.5 exposure,pulmonary arterial remodeling occurred in the medium and small pulmonary arteries.After 24 weeks of TRPM_2.5exposure,pulmonary arterial remodeling all occured in large,medium,and small pulmonary arteries.1.3 Immunohistochemistry and immunofluorescence showed that the pulmonary vascular endothelial cells in rats were damaged and detached,and the endothelial cell integrity was impaired after 24 weeks of TRPM_2.5 exposure.2.The mechanism of PAH induced by TRPM_2.5.2.1 After 24 weeks exposure of TRPM_2.5,the RVSP of the control group,MVE group,and MVE+NPS2143 group were 23.1±0.5 mm Hg,26.5±0.6 mm Hg and 23.7±0.7 mm Hg（n﹦6）and the RVHI were 17.4±0.0%,18.5±0.0%and 17.4±0.0%（n﹦6）,respectively.The RVSP of the MVE group was significantly different from that of the control group（P<0.05）.There was no statistically significant difference in the RVHI（P>0.05）.The RVSP of MVE+NPS2143 group was significantly lower than that of MVE group,there was statistically significant difference（P<0.05）.There was no significant difference in RVHI between MVE+NPS2143 group and MVE group（P>0.05）.2.2 After 24 weeks of TRPM_2.5 exposure,pulmonary arteries were significantly thickened and the pulmonary arterial lumen become narrow in the MVE group（n﹦6）with pulmonary artery diameters of 0-50μm,50-100μm,and 100-150μm compared with the control group（n﹦6）.Compared with the MVE group（n﹦6）,the thickness of the pulmonary artery wall in the MVE+NPS2143 group（n﹦6）was significantly decrease.2.3 Western blot analysis showed that the protein expression levels of Ca SR and TRPC1 in the MVE group（Ca SR:1.16±0.32,TRPC1:1.40±0.31,n=7）were significantly higher than those in the control group（Ca SR:0.48±0.27,TRPC1:0.68±0.22,n=7）,the differences were statistically significant（P<0.01）.The protein expression levels of Ca SR and TRPC1 in the MVE+NPS2143 group（Ca SR:0.63±0.24,TRPC1:0.78±0.28,n=7）were significantly lower than those in the MVE group（Ca SR:1.16±0.32,TRPC1:1.40±0.31,n=7）,and the differences were statistically significant（p<0.01）.【CONCLUSIONS】1.Long-term exposure to TRPM_2.5can cause pulmonary hypertension in rats.2.TRPM_2.5 exposure causes pulmonary hypertension by upregulating Ca SR.