TLR9-JNK信号通路调控缺氧诱导大鼠肺动脉平滑肌细胞增殖

TLR9-JNK signaling pathway regulates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells

目的研究Toll样受体-9(toll-like receptor 9,TLR9)在缺氧诱导大鼠肺动脉平滑肌细胞(rat pulmonary artery smooth muscle cells,RPASMCs)增殖中的作用及机制。方法采用MTT、Western blot、PCR法检测RPASMCs的相对增殖水平、蛋白表达水平、mRNA转录水平。①3%O_(2),36 h处理RPASMCs,构建缺氧诱导RPASMCs增殖的细胞模型,检测细胞相对增殖水平、TLR9、磷酸化c-jun氨基末端激酶(phosphorylated c-jun N-terminal kinase,p-JNK)蛋白表达水平。②缺氧条件下分别使用小干扰RNA序列(small interfering RNA sequence,siRNA)或20μmol/L TLR9激动剂ODN 1826敲低或活化TLR9,检测RPASMCs的细胞增殖水平和c-jun氨基末端激酶(c-jun N-terminal kinase,JNK)磷酸化水平。③缺氧时使用5μmol/L JNK抑制剂SP600125处理RPASMCs,检测缺氧条件下细胞相对增殖水平。④缺氧时复合使用20μmol/L TLR9激动剂ODN 1826和5μmol/L JNK抑制剂SP600125处理RPASMCs,检测RPASMCs的JNK磷酸化水平和相对增殖水平。结果缺氧组RPASMCs的相对增殖水平[(1.30±0.07),P<0.01]以及TLR9蛋白水平[(1.70±0.22),P<0.05]和JNK磷酸化[(1.83±0.18),P<0.01]都显著高于常氧组。敲低TLR9显著抑制缺氧诱导的RPASMC增殖[(0.64±0.09),P<0.01]并减少JNK磷酸化[(0.49±0.01),P<0.001]。TLR9激动剂显著促进RPASMCs增殖[(2.01±0.08),P<0.001]且增加JNK的磷酸化[(2.11±0.33),P<0.01]。JNK抑制剂抑制RPASMCs增殖[(0.70±0.06),P<0.05]。与缺氧+TLR9激动剂组相比,缺氧+TLR9激动剂+JNK抑制剂组RPASMCs JNK磷酸化[(0.82±0.14)vs(1.59±0.30),P<0.001]和相对增殖水平[(1.08±0.05)vs(1.35±0.02),P<0.001]都显著降低。结论缺氧时TLR9通过激活JNK通路促进大鼠肺动脉平滑肌细胞增殖。

Objective To investigate the role and mechanism of Toll-like receptor 9(TLR9)in hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(RPASMCs).Methods MTT assay,Western blotting and PCR were adopted to measure cell proliferation level,protein and mRNA levels of following molecules in RPASMCs.①After RPASMCs were treated with 3%O_(2)for 36 h to establish a hypoxic model,the cell proliferation and expression levels of TLR9 and phosphorylated c-jun N-terminal kinase(p-JNK)were detected at the same time.②After TLR9 was knocked down using small interfering RNA sequence(siRNA)or activated by 20μmol/L TLR9 agonist,ODN 1826,under hypoxia,the proliferation and p-JNK level were examined.③RPASMCs were treated with 5μmol/L JNK inhibitor SP600125 under hypoxia,and the proliferation was assayed.④Under hypoxia,20μmol/L ODN 1826 combined with 5μmol/L SP600125 were administered to RPASMCs,and the proliferation and level of p-JNK were measured.Results The proliferation of RPASMCs(1.30±0.07,P<0.01)and protein levels of TLR9(1.70±0.22,P<0.05)and p-JNK(1.83±0.18,P<0.01)were significantly higher in the hypoxia group than the normoxia group.Knock-down of TLR9 significantly inhibited hypoxia-induced proliferation of RPASMC(0.64±0.09,P<0.01)and reduced the JNK phosphorylation(0.49±0.01,P<0.001).TLR9 agonist,ODN 1826,significantly promoted the proliferation of RPASMCs(2.01±0.08,P<0.001)and increased JNK phosphorylation(2.11±0.33,P<0.01).JNK inhibitor SP600125 inhibited the proliferation(0.70±0.06,P<0.05).Compared with hypoxia+TLR9 agonist group,the level of p-JNK(0.82±0.14 vs 1.59±0.30,P<0.001)and cell proliferation(1.08±0.05 vs 1.35±0.02,P<0.001)were both decreased significantly in hypoxia+TLR9 agonist+JNK inhibitor group.Conclusion TLR9 promotes the proliferation of RPASMCs by activating the JNK pathway under hypoxia.