Regulatory Effects Of RELM-β 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-β and whether RELM-β 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-β on the expression of RELM-β,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-Β-overexpression group（lentiviral transfection）（2）.over-expression control group（no-load lentivirus）（3）.normoxic group（4）.hypoxia group（5）.RELM-β silencing normoxic group（lentiviral transfection）（6）.silent Normoxic control group（lentivirus empty）（7）.RELM-β 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-β,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-β 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-β protein group was added.（3）.The PLC inhibitor U73122 was added to the recombinant human RELM-β protein group.（4）.After adding the IP3 R inhibitor xestosponginc,the recombinant human RELM-β protein group was added.The expression levels of RELM-β,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 Ⅰ: Effects of hypoxia and RELM-β expression on the expression of RELM-β,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-β/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-β/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-β/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-β/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-β/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-β/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-β/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-β/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-β/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-β/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 Ⅱ: Effect of recombinant RELM-β protein on the proliferation of human pulmonary artery smooth muscle cells and PLC/IP3/Ca²⁺ pathway:After the recombinant human RELM-β 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-β protein and recombinant human RELM-β 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-β protein and recombinant human RELM-β 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-β,PLC and IP3,and promote the proliferation of human pulmonary artery smooth muscle cells.2.RELM-β may affect the proliferation of human pulmonary artery smooth muscle cells through the downstream signaling pathway PLC-IP3/Ca²⁺.