Regulatory Effects Of RELM-? And PLC-IP₃/Ca²⁺ Signaling Pathway On Proliferation Of Hypoxic Human Pulmonary Artery Smooth Muscle Cells And Its Mechanism

Objective: To explore the relationship between hypoxia and RELM-? and whether RELM-? promotes the proliferation of human pulmonary artery smooth muscle cells through the downstream signaling pathway PLC-IP3-Ca²⁺ under hypoxia.Methods: The experiment was divided into two partsOne,Effects of hypoxic conditions and promotion and inhibition of RELM-? on the expression of RELM-?,PLC,and IP3 R in human pulmonary artery smooth muscle cells and their effects on proliferation of smooth muscle cells: The experiment was divided into 8 groups:?1?.RELM-?-overexpression group?lentiviral transfection??2?.over-expression control group?no-load lentivirus??3?.normoxic group?4?.hypoxia group?5?.RELM-? silencing normoxic group?lentiviral transfection??6?.silent Normoxic control group?lentivirus empty??7?.RELM-? silenced hypoxic group?lentiviral transfection??8?.Silencing control hypoxic group?lentivirus empty?.After the logarithmic growth phase 3–5 passages of human pulmonary artery smooth muscle cells?h PASMCs?were cultured in DMEM/F12 medium without fetal bovine serum for 24 h to synchronize the cell cycle,the RELM-?,PLC,IP3 R expression levels was detected by QT-PCR and Western blot methods.using the EDU kit to detect the proliferation of human pulmonary artery smooth muscle cells.Second,the effect of recombinant RELM-? protein on the proliferation of human pulmonary artery smooth muscle cells and PLC/IP3/Ca²⁺ pathway: The experiment was divided into four groups:?1?.Control group?2?.After adding normal saline,recombinant human RELM-? protein group was added.?3?.The PLC inhibitor U73122 was added to the recombinant human RELM-? protein group.?4?.After adding the IP3 R inhibitor xestosponginc,the recombinant human RELM-? protein group was added.The expression levels of RELM-?,PLC and IP3 R were detected by QT-PCR and Western blot respectively.The proliferation of human pulmonary arterial smooth muscle cells was detected by EDU kit,and intracellular free calcium([Ca²⁺]i)was labeled by Fluo-2.,Confocal microscopy was used to detect [Ca²⁺]i concentration.result:Part ?: Effects of hypoxia and RELM-? expression on the expression of RELM-?,PLC,and IP3 R in human pulmonary artery smooth muscle cells and their effects on proliferation of smooth muscle cells:In the hypoxia group,compared with the normoxic group: Western blot results showed that the expression of RELM-?/PLC/IP3 R protein increased?0.511±0.015,0.250±0.051/0.567±0.086,0.415±0.076/0.544±0.127,0.269±0.072,respectively?.???P<0.05?.QT-PCR results showed that the expression of RELM-?/PLC/IP3 R m RNA increased?4.670±0.495,2.208±0.357/7.161±1.418,3.963±1.407/4.591±1.237,2.572±0.499??P<0.05?;Cell proliferation was enhanced?31±3.00,16±1.73??P<0.01?.Lentivirus overexpression group compared with empty control group: Western blot results showed that RELM-?/PLC/IP3 R showed increased protein expression?0.758±0.016,0.242±0.07/0.733±0.159,0.411±0.063/0.934±0.231,0.236± 0.049??P<0.05?QT-PCR results showed increased expression of RELM-?/PLC/IP3R?7.687±1.061,2.226±0.312/10.447±2.802,3.941±1.508/6.600±1.363,2.581±0.495??P < 0.05?.<0.05);EDU immunofluorescence showed increased cell proliferation?35.33 ± 0.58,27 ± 3.00??P <0.05?.Lentivirus silencing group and empty control group: Western blot results showed decreased expression of RELM-?/PLC/IP3 R protein?0.088±0.057,0.248±0.096/0.120±0.018,0.319±0.055/0.091±0.028,0.201±0.029??P<0.05?.QT-PCR results showed that the expression of RELM-?/PLC/IP3 R decreased?1.036±0.045,1.753±0.276/1.181±0.094,2.426±0.485/1.051±0.029,1.559±0.032??P<0.05?;EDU immunofluorescence results The cell proliferation was decreased?8±2.65,14.33±2.52??P<0.05?.After hypoxia in the lentiviral-silence group,the expression of RELM-?/PLC/IP3 R protein was significantly decreased after Western blot?0.107±0.070,0.511±0.135/0.137±0.017,0.603±0.077/0.080?.± 0.006,0.487 ± 0.036)?P < 0.05?.QT-PCR results showed that RELM-?/PLC/IP3 R m RNA expression was significantly reduced?1.066±0.035,4.593±0.487/1.229±0.047,7.029±1.569/1.203±0.122,4.646±1.213??P<0.05?;EDU immunofluorescence The results showed that cell proliferation was significantly reduced?8.67±2.08,18.33±3.06??P<0.05?.The results of Western blot showed no significant changes in the expression of RELM-?/PLC/IP3 R protein between the lentiviral group and the lentiviral group?0.088±0.057,0.107±0.070/0.120±0.018,0.137?.± 0.017/0.091 ± 0.028,0.080 ± 0.006)?P> 0.05?.QT-PCR results showed no significant changes in the expression of RELM-?/PLC/IP3 R m RNA,and the difference was not statistically significant?1.036±0.045,1.066±0.035/1.181±0.094,1.229±0.047/1.051±0.029,1.203±0.122?.P>0.05);EDU immunofluorescence showed no significant changes in cell proliferation,the difference was not statistically significant?8±2.65,8.67±2.08??P>0.05?.Part ?: Effect of recombinant RELM-? protein on the proliferation of human pulmonary artery smooth muscle cells and PLC/IP3/Ca²⁺ pathway:After the recombinant human RELM-? protein was added,the protein expression of PLC/IP3 R was significantly increased?0.645±0.817,0.155±0.372/0.907±0.096,0.191±0.041??P<0.05?.The expression of PLC/IP3 R m RNA was significantly increased?7.743±0.4651.022±0.013 / 7.581±0.653,1.045±0.045??P<0.05?;the intracellular calcium concentration was increased by Fluo-2 labeling intracellular free calcium?0.269±0.046,0.153±0.012??P<0.05?.EDU immunofluorescence showed that cell proliferation was enhanced?35±1.00,15.33±4.51??P<0.05?;Adding PLC inhibitor U73122 followed by recombinant human RELM-? protein and recombinant human RELM-? protein plus saline increased the protein expression of IP3 R significantly?0.597±0.113,0.907±0.096??P<0.05?.The expression of IP3 R m RNA was significantly decreased?2.772±0.334,7.581±0.653??P<0.05?.The intracellular calcium concentration of intracellular free calcium detected by Fluo-2 was decreased?0.122±0.023,0.269±0.046??P<0.05?.EDU immunofluorescence showed that cell proliferation was decreased?15.67±2.08,35±1.00??P<0.05?.After the addition of IP3 R inhibitor xestosponginc plus recombinant human RELM-? protein and recombinant human RELM-? protein plus saline,there was no significant change in the protein expression of PLC?0.499±0.235,0.645±0.817?.?P>0.05?.There was no significant difference in the expression of PLC m RNA?3.722±1.215,7.743±0.465??P>0.05?.The intracellular calcium concentration of intracellular free calcium detected by Fluo-2 labeling was decreased?0.101±0.014,0.269±0.046??P< 0.05?;EDU immunofluorescence showed decreased cell proliferation?14.67 ± 2.08,35 ± 1.00??P <0.05?.Conclusion: 1.Hypoxia can promote the expression of RELM-?,PLC and IP3,and promote the proliferation of human pulmonary artery smooth muscle cells.2.RELM-? may affect the proliferation of human pulmonary artery smooth muscle cells through the downstream signaling pathway PLC-IP3/Ca²⁺.