| Objective:To study the effect of resistin-like molecule-bata(RELM-β)on hypoxic pulmonary hypertension(HPH)and the regulation of pulmo nary artery smooth muscle cell(PASMCs)proliferation through PLC/IP3R/Ca2+ signal pathway.Methods:this experiment is divided into two parts:1.Animal experiment:to detect the hemodynamics,pulmonary vascular morphology and the expression of RELM-β,PLC and IP3R in lung tissue of SD rats.Male SD rats with RELM-β gene knockout(RELM-β-/-)and wild type(Wild type,WT)were interfered with normoxic(Normoxia,N)and chronic hypoxia(Chronic hypoxia,CH)respectively.Normoxic condition:21%O2 concentration environment.Chronic hypoxia:the rats were continuously exposed to(10±0.5)%O2 for 8 hours every day and repeated continuously for 21 days.The experiment was divided into four groups:N+WT,Null RELM-β-/-,CH+WT,CH+RELM-β-/-.The mean pulmonary artery pressure(mPAP)was measured by right cardiac catheterization.Measurement of right Ventricular hypertrophy Index(RVHI))by weighing method.The ratio of pulmonary artery wall thickness to extravascular diameter(WT%)is calculated to indicate the degree of pulmonary artery remodeling.The relative protein expressions of RELM-β,PLC and IP3R in lung tissue were detected by Western blot(WB).The relative mRNA expression of RELM-β and IP3R in lung tissue was detected by Quantitative Real-time PCR(qPCR).2.Cell experiment:to detect the expression of RELM-β,PLC,IP3R and Ca2+in PASMCs and the relationship between upstream and downstream,and the regulation of RELM-β on PASMCs proliferation through PLC/IP3R/Ca2+signal pathway.The PASMCs of RELM-β-/-and WT rats were isolated and cultured.The PASMCs was treated with hypoxia(cells were placed in three gas incubators of 1%02,94%N2,5%CO2 for 24 hours)and normoxic(cells were cultured in 21%O2,74,N2,5%CO2 cell incubator),respectively.Cell experiment groups:N+WT,Null RELM-β-/-,CH+WT,CH+RELM-β-/-.The "CH+WT group" was selected to detect the signal pathway and regulate the proliferation of PASMCs,which was verified by the detection of downstream signal molecules and PASMCs proliferation after the addition of PLC inhibitor U73122 and IP3R inhibitor Xestospongin C,respectively.The relative protein expression of RELM-β,PLC and IP3R in PASMCs was detected by WB,the relative mRNA expression of RELMβ and IP3R in PASMCs was detected by qPCR,the concentration of Ca2+in PASMCs was detected by flow cytometry(flow cytometry,FCM),and the proliferation of PASMCs was detected by EdU.Results:1.Animal experiment:chronic hypoxia treatment can aggravate mPAP,RVHI,WT%and up-regulate the expression of RELM-β,PLC and IP3R in lung tissue of SD rats,and knockout RELM-β can inhibit the above indexes.(1)comparison between hypoxia group and normoxia group:mPAP,RVHI and WT%in hypoxic group were higher than those in normoxic group(P<0.05).The expressions of RELM-β,PLC and IP3R in lung tissue of rats in hypoxia group were higher than those in normoxia group(P<0.05).(2)comparison between RELM-β-/-group and WT group:1)under normoxic condition,there was no significant difference in mPAP,RVHI,WT%between RELM-β-/-group and WT group(P>0.05).The expressions of RELM-β,PLC and IP3R in lung tissue of rats in RELM-β-/-group were lower than those in WT group(P<0.05);2)under hypoxia,mPAP,RVHI and WT%in RELM-β-/-group were lower than those in WT group(P<0.05).The expression of RELM-β,PLC and IP3R in RELM-β-/-group was lower than that in WT group(P<0.05).2.Cell experiment:PASMCs can up-regulate the expression of RELM-β,PLC,IP3R,the concentration of Ca2+and the proliferation of PASMCs after hypoxia treatment.RELM-β can promote the proliferation of PASMCs by regulating PLC/IP3R/Ca2+signal pathway.(1)comparison between hypoxia group and normoxia group:The expression of RELM-β,PLC,IP3R,the concentration of Ca2+and the rate of cell proliferation in PASMCs of hypoxia group were higher than those of normoxic group(P<0.05).(2)comparison between RELM-β-/-group and WT group:1)under normoxic condition,there was no significant difference in the concentration of Ca2+in PASMCs and the proliferation rate of PASMCs between RELM-β-/-group and WT group(P>0.05).The expression of RELM-β,PLC and IP3R in PASMCs of RELM-β-/-group was lower than that of WT group(P<0.05);2)under hypoxia condition,the concentration of Ca2+ in PASMCs and the proliferation rate of PASMCs in RELM-β-/group were lower than those in WT group(P<0.05),and the expression of RELM-β,PLC and IP3R in PASMCs in RELM-β-/-group was lower than that in WT group(P<0.05).(3)RELM-β regulates PASMCs proliferation through PLC/IP3R/Ca2+signal pathway.In PASMCs,knockout of RELM-β gene decreased the expression of PLC,IP3R,the concentration of Ca2+and the proliferation of PASMCs(P<0.05).The addition of U73122 could down-regulate the expression of IP3R,the concentration of Ca2+and the proliferation rate of PASMCs(P<0.05).The addition of Xestospongin C could down-regulate the concentration of Ca2+and the proliferation rate of PASMCs(P<0.05).Conclusions:Chronic hypoxia upregulates RELM-β.RELM-β regulates the proliferation of PASMCs through PLC/IP3R/Ca2+signaling pathway,leading to the occurrence and development of HPH. |
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