人退变椎间盘髓核组织中miR-146a的表达及对NP细胞增殖、凋亡和炎性细胞因子的影响作用机制

Expression of miR-146a in the Nucleus Pulposus Tissue of Human Degenerative Intervertebral Disc and Its Effect Mechanism on the Proliferation,Apoptosis and Inflammatory Cytokines of NP Cells

目的检测人退变椎间盘髓核组织中miR-146a的表达变化,并探讨其可能的作用机制。方法实时荧光定量PCR(qRT-PCR)检测人退变腰椎间盘髓核组织中miR-146a表达。用脂多糖(LPS)刺激人髓核(nucleus pulposus,NP)细胞模拟椎间盘变性,qRT-PCR检测NP细胞中miR-146a的表达变化。将miR-146a mimics或miR-146a inhibitor转染至NP细胞,然后LPS刺激NP细胞,观察miR-146a对NP细胞增殖活性、凋亡、TLR4蛋白表达及IL-1β,TNF-α和IL-6等炎性因子水平的影响。结果miR-146a在椎间盘退变(intervertebral disc degeneration,IVDD组)椎间盘髓核组织中表达(0.65±0.12)明显低于对照组(1.01±0.10),miR-146a在LPS组NP细胞中表达(0.29±0.11)明显低于阴性对照组(1.06±0.07),差异均有统计学意义(t=11.856,10.229,均P<0.01)。与阴性对照组比较,LPS组上清液中的IL-1β,TNF-α和IL-6含量明显升高,差异均有统计学意义(t=15.572~23.354,均P<0.001)。上调NP细胞miR-146a表达,能够提高LPS刺激的NP细胞的增殖活性(t=4.436~7.995,均P<0.05),降低其凋亡率(t=9.255,P=0.001),降低TLR4蛋白(t=5.881,P=0.004)和IL-1β,TNF-α和IL-6等炎性细胞因子水平(t=8.854~10.772,均P<0.01);而下调NP细胞miR-146a表达则出现相反的结果(t=2.977~6.777,均P<0.05)。结论miR-146a在退变椎间盘髓核组织中表达降低,miR-146a可能能够通过靶向TLR4调控NP细胞的增殖、凋亡和炎症反应,为椎间盘退变的生物治疗提供了新的方向。

Objective To detect the expression of miR-146a in nucleus pulposus of human degenerative intervertebral disc and explore its possible mechanism.Methods qRT-PCR was used to detect the expression of miR-146a in human degenerative lumbar disc nucleus pulposus.Human nucleus pulposus(NP)cells were stimulated with lipopolysaccharide(LPS)to simulate intervertebral disc degeneration.The expression of miR-146a in NP cells was detected by qRT-PCR.miR-146a mimics or miR-146a inhibitor were transfected into NP cells,and then LPS stimulated NP cells.The effects of miR-146a on proliferation,apoptosis,TLR4 protein expression and expression of IL-1β,TNF-αand IL-6 were observed.Results The expression of miR-146a in the intervertebral disc nucleus pulposus tissue of the(intervertebral disc degeneration IVDD)group(0.65±0.12)was significantly lower than that of the control group(1.01±0.10),and the expression of miR-146a in the NP cells of the LPS group(0.29±0.11)was significantly lower than that of the negative control group(1.06±0.07),the difference was statistically significant(t=11.856,10.229,all P<0.01).Compared with the negative control group,the levels of IL-1β,TNF-αand IL-6 in the supernatant of the LPS group were significantly increased,and the differences were statistically significant(t=15.572~23.354,all P<0.001).Up regulation of miR-146a expression in NP cells could increase the proliferation activity(t=4.436~7.995,all P<0.05),reduce the apoptosis rate(t=9.255,P=0.001),and decreased the levels of TLR4 protein(t=5.881,P=0.004)and inflammatory cytokines such as IL-1β,TNF-αand IL-6(t=8.854~10.772,all P<0.01),while down regulating the expression of miR-146a in NP cells had the opposite effect(t=2.977~6.777,all P<0.05).Conclusion The expression of miR-146a in nucleus pulposus of degenerative intervertebral disc was decreased.MiR-146a may be able to regulate the proliferation,apoptosis and inflammatory reaction of NP cells by targeting TLR4,which provides a new direction for the biological treatment of intervertebral disc degeneration.