Effect Of T-Type Calcium Channel Inhibitors On MCT-Induced Pulmonary Arterial Hypertension In Rats

Objective:Investigating the effect of T-type calcium channel inhibitors,ProTx-I,micromolar Ni²⁺and Mibefradil on monocrotaline（MCT）-induced pulmonary arterial hypertension（PAH）in rats,and revealing the role of the Raf/MEK/ERK pathway coulped by T-type calcium channel pathway in PAH.Methods:Part I and animal studies:Forty male Sprague-Dawley rats were randomly divided into 5 groups:normal control group,MCT group,ProTx-1 group,micromolar Ni²⁺group and Mibefradil group（8 cases in each group）.Day 28 after MCT-injection,the expression of T-type calcium channels were detectd by the method of qPCR and Western Blot.The right ventricular systolic pressure（RVSP）,the right ventricular hypertrophy index（RS/LV+S）and the index of pulmonary vascular remodeling（MA%）were measured.The expression level of proliferating cell nuclear antigen（PCNA）,Cleaved Caspase-3 were detected by Western blot.Part II and vitro study:hPASMCs were cultured under 1%O2,the expression of T-type calcium channels were detectd,and the effect of T-type calcium channel inhibitor on the proliferation and apoptosis of hPASMCs were detected in using of CCK8 and FACS,then WB technology was used to test the Raf/MEK/ERK pathway protein experssion.Results:In vivo animal research:（1）The expression of CaV3.1 and CaV3.2 were significantly up-regulated in PAH compared with the control group（P<0.05）,and CaV3.3 showed no significant change.（2）RVSP and RV/LV+S in MCT group were significantly increased compared to Control Group（P<0.01）,meantimes,RVSP and RV/LV+S in intervention groups were significantly decreased compared to MCT group（P<0.05）.（3）MA%in control group was lower than that in MCT group（P<0.01）;3 intervention groups showed no significant differences（P>0.05）,however,they were all significantly lower than that in MCT group（P<0.05）.（4）The expression of PCNA in MCT group was higher than that in control group,meanwhile,3 intervention groups were significantly lower than that in MCT group.The expression of Cleaved Caspase-3 in intervention groups showed no significant changes compared with MCT group,respectively.In vitro cell research:（1）The expression of CaV3.1and CaV3.2 were significantly up-regulated in hypoxia-cultured hPASMCs compared with the control group（P<0.05）,and CaV3.3 showed no significant change.（2）T-type calcium channel inhibitors significantly ameliorate intracellular free calcium of hPASMCs.（3）T-type calcium channel inhibitors(ProTx-1,micromolar Ni²⁺and mibefradil)significantly decreased the proliferation of hypoxia-cultured hPASMCs and resulted in G1/S delay,and the apoptosis of hPASMCs showed no statistical difference bewteen in hypoxia group and in normoxia group.（4）The phosphorylation of Raf,MEK and ERK were supressed by T-type calcium channel inhibitors.Conclusions:1.T type calcium channel blockers(ProTx-1,micromolar Ni²⁺and mibefradil)could effectively alleviate the degree of right ventricular hypertrophy,and inhibite the proliferation of PASMC,and effectively mitigate the pulmonary artery wall hypertrophy,and then improve pulmonary vascular remodeling in MCT-induced rats.2.Raf/MEK/ERK pathway coulped by T-type calcium channel played an important role in the pathogenesis of hypoxia-induced PAH.T type calcium channel blockers,which improved pulmonary vascular remodeling,inhibited the phosphorylation of Raf/MEK/ERK signaling pathway,and then influenced the expression of CCND1,resulted in cell cycle progression,eventually leaded to supressing the proliferation of cells.3.T type calcium blockers might provide a new theoretical basis for treating PAH.