Role Of RELM-β Regulatory Signal Pathway PLC-IP₃/Ca²⁺in The Proliferation Of Rat Pulmonary Artery Smooth Muscle Cells And Its Mechanism

Objective:To study the relationship between resistin like moleculeβ(RELM-β)and monocrotaline-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs),and to explore the mechanism of PLC-IP3/Ca2+ signaling pathways in this processMethod1.Observe whether monocrotaline promotes or inhibits the proliferation of rat PASMCs.Further regulate the expression level of RELM-β,and observe whether its effect on monocrotaline affects the proliferation of rat PASMCs through the PLC-IP3/Ca2+ signaling pathwayThe Groups of Experiments:control group,MCT group,no-load lentivirus saline group,no-load lentivirus MCT group,silent lentivirus expression RELM-β saline group,silent lentivirus expression RELM-β MCT group,overexpress lentivirus RELM-β saline group,overexpress lentivirus RELM-β MCT groupThe SD rat primary PASMCs were cultured in vitro and passed to 3-5 generations.After adding the MCT,lentivirus transfection was used to intervened the expression level of RELM-β in the cells.The proliferation of PASMCs was detcted by EDU kit.And the average fluorescence intensity of intracellular free calcium was detected by flow cytometry.The expression level of RELM-β、PLC、IP3R was detected by QT-PCR and Western bolt methods2.Explore whether RELM-β affects PASMCs proliferation through PLC-IP3/Ca2+signaling pathwayThe SD rat primary PASMCs were cultured in vitro and passed to 3-5 generations.After synchronization treatment,recombinant RELM-β protein and signaling pathway inhibitor were addedDivide into four groups:1.Control group 2.normal saline add recombinant mouse RELM-β protein group 3.PLC inhibitor U73122 add recombinant mouse RELM-β protein group 4.IP3R inhibitor Xestospongin C add recombinant mouse RELM-β protein groupThe proliferation of PASMCs was detcted by EDU kit.And the average fluorescence intensity of intracellular free calcium was detected by flow cytometry.The expression level of RELM-β、PLC、IP3R was detected by QT-PCR and Western bolt methods.Results:1.effects of monocrotaline and intervention of RELM-β expression level on PLC-IP3R/Ca2+singal pathway and the proliferation of rat PASMCsEDU detects the proliferation rate of PASMCs:MCT group were higher than control group(P<0.05);silent lentivirus expressionRELM-β MCT group were higher than no-load lentivirus MCT group(P<0.05);overexpress lentivirus RELM-β MCT group were higer than no-load Lentivirus MCT group(P<0.05)The results of QT-PCR、Western bolt detects the mRNA and protein expression level shows that:MCT group were higher than control group(P<0.05);silent lentivirus expression RELM-β MCT group were lower than no-load lentivirus MCT group(P<0.05);overexpress lentivirus RELM-βMCT group were higher than no-load lentivirus MCT group(P<0.05).silent lentivirus expression RELM-β saline group were lower than no-load lentivirus saline group(P<0.05);overexpress lentivirus RELM-β saline group were higher than no-load lentivirus saline group(P<0.05);2.the mechanism of RELM-β and PLC-IP3/Ca2+signaling pathway on the proliferation of rat PASMCsThe results of Edu detects the proliferation and the flow cymetory detects the average fluorescence intensity of intracellular free calcium showed that:normal saline add recombinant mouse RELM-β protein group were higher than control group(P<0.05),the average fluorescence intensity were increased(P<0.05);PLC inhibitor add recombinant mouse RELM-β protein group were lower than the normal saline add recombinant mouse RELM-β protein group(P<0.05),the average fluorescence intensity were decreased(P<0.05);IP3R inhibitor add recombinant mouse RELM-β protein group were lower than the normal saline add recombinant mouse RELM-β protein group(P<0.05),the average fluorescence intensity were decreased(P<0.05)The results of QT-PCR and Western bolt detects the expression level of PLC、IP3R mRNA and protein shows that:the expression level of the normal saline add recombinant mouse RELM-β protein group were higher than control group(P<0.05);the expression level of PLC inhibitor add the recombinant mouse RELM-β protein group were lower than the normal saline add recombinant mouse RELM-β protein group(P<0.05);the expression level of IP3R inhibitor add recombinant mouse RELM-β protein group were lower than the normal saline add recombinant mouse RELM-β protein group(P<0.05)Conclusions:1.Monocrotaline can promote the proliferation of rat PASMCs,and RELM-β can regulate the proliferation of rat PASMCs induced by monocrotaline2.RELM-β affects the proliferation of PASMCs in rats induced by monocrotaline by regulating the PLC-IP3/Ca2+signaling pathway.