Adiponectin inhibits H2O2⁃induced apoptosis and extracellular matrix degradation in rat nucleus pulposus cells through AMPK/mTOR pathway
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摘要:
目的：探讨脂联素（adiponectin，APN）对氧化应激下大鼠髓核细胞凋亡和细胞外基质（extracellular matrix，ECM）退变的保护作用及相关机制。方法：原代培养SD大鼠髓核细胞，CCK-8法检测细胞活力，以确定APN的最适保护浓度。将细胞分为对照组、H2O2组、APN+H2O2组及Compound C+APN+H2O2组，流式细胞术检测凋亡率，Western blot检测Bcl-2、Bax、Cleaved Caspase-3、基质金属蛋白酶-13（matrix metalloproteinase-13，MMP-13）及带有血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶-5（a disintegrin and metalloproteinase with thrombospondin motifs-5，ADAMTS-5）等蛋白的表达，RT-PCR检测Ⅱ型胶原（collagen type Ⅱ alpha 1 chain，COL2A1）、蛋白聚糖（aggrecan，ACAN）的mRNA水平，Western blot评估5′-单磷酸腺苷活化蛋白激酶（adenosine 5′-monophosphate-activated protein kinase，AMPK）和哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）的磷酸化水平。结果：1 μg/mL APN对200 μmol/L H2O2所致的细胞毒性起最佳拮抗作用。APN预处理显著抑制H2O2诱导的髓核细胞凋亡。相应地，Bax/Bcl-2比值及Cleaved Caspase-3 表达的增加也被APN抑制。尽管对ADAMTS-5无明显抑制作用，APN降低了H2O2诱导的MMP-13的表达。此外，H2O2对COL2A1及ACAN转录的抑制作用也被APN显著缓解。APN还促进AMPK磷酸化并抑制mTOR磷酸化，而AMPK抑制剂Compound C显著减轻了APN对mTOR磷酸化的抑制作用。APN对凋亡、分解代谢的抑制作用及其对合成代谢的促进作用也均被Compound C逆转。结论：APN通过AMPK/mTOR通路抑制H2O2诱导的大鼠髓核细胞凋亡及ECM退变。
Abstract:
Objective：To investigate the protective effects of adiponectin（APN）on apoptosis and extracellular matrix（ECM）degradation in rat nucleus pulposus（NP） cells under oxidative stress. Methods：Cell viability was detected by cell counting kit-8（CCK-8）to determine the optimal protective concentration of APN. Cells were randomly divided into the control group，the H2O2 group，the APN+H2O2 group and the compound C+APN+H2O2 group. Apoptosis incidence was evaluated by flow cytometry. The expression of Bcl-2，Bax，cleaved caspase-3，matrix metalloproteinase-13（MMP-13） and a disintegrin and metalloproteinase with thrombospondin motifs-5（ADAMTS-5）was detected by Western blot. The mRNA levels of collagen type Ⅱ alpha 1 chain（COL2A1） and aggrecan（ACAN） were detected by RT-PCR. The phosphorylation levels of adenosine 5′-monophosphate-activated protein kinase（AMPK）and mammalian target of rapamycin（mTOR） were evaluated by Western blot. Results：One μg/mL APN conferred the optimal protection against the cytotoxicity of 200 μmol/L H2O2. APN pretreatment significantly suppressed H2O2-induced apoptosis in NP cells. Consistently，the increase in Bax/Bcl-2 ratio and the expression of cleaved caspase-3 was also inhibited by APN. Although no notable inhibitory effect on ADAMTS-5 was observed，APN reduced the expression of MMP-13 induced by H2O2. Besides，the inhibition of H2O2 on the transcription of COL2A1 and ACAN was also notably abated by APN. APN also induced the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. Inhibition of AMPK with compound C alleviated the suppression of APN on mTOR phosphorylation. Moreover，the inhibition of APN on apoptosis and catabolism，together with the promotion of APN on anabolism were also reversed by compound C. Conclusion：APN inhibits H2O2-induced apoptosis and ECM degradation in rat NP cells in an AMPK/mTOR dependent manner.
