线粒体靶向肽SS-31对炎症诱导髓核细胞衰老和焦亡的作用及机制研究

Research on the role and mechanism of mitochondrial targeting peptide SS-31 in inflammation-induced senescence and pyroptosis of nucleus pulposus cells

中文关键词:  SS-31  髓核细胞  炎症  衰老  焦亡

英文关键词:SS-31  Nucleus pulposus cells  Inflammation  Senescence  Pyroptosis

中文摘要:

  【摘要】 目的：探讨线粒体靶向肽SS-31对炎症诱导的髓核（nucleus pulposus，NP）细胞衰老、焦亡的作用及机制。方法：体外提取培养6周龄SD雄性大鼠的NP细胞，分为对照组、脂多糖（lipopolysaccharide，LPS）组和LPS+SS-31组，其中对照组不做处理，LPS组以浓度为100μg/mL的LPS干预NP细胞24h，LPS+SS-31组在LPS干预前30min利用SS-31进行预处理。采用衰老相关β半乳糖苷酶（senescence associated β-galactosidase，SA-β-Gal）染色和Western blot（WB）实验评估各组细胞衰老及焦亡水平；采用透射电镜和氧耗率检测分析各组NP细胞线粒体功能；应用WB和免疫荧光检测各组磷酸化核因子-κB-p65（phosphorylated nuclear factor-κB-p65，p-NF-κB-p65）、环磷酸鸟苷-腺苷酸合成酶（cyclic GMP-AMP synthase，cGAS）和干扰素刺激基因（interferon stimulating gene，STING）的蛋白表达；苏木精-伊红（hematoxylin-eosin，HE）染色、免疫组化（immunohistochemistry，IHC）染色和WB实验分析体内注射SS-31对椎间盘退变、细胞衰老和焦亡的影响。结果：与LPS组相比，LPS+SS-31组的NP细胞SA-β-Gal染色阳性率、衰老标志蛋白（p53、p21）及焦亡相关分子（NLRP3、Caspase-1 p20）的表达均明显降低（P<0.05）。通过透射电镜观察发现LPS组NP细胞线粒体肿胀呈圆形，基质透亮，线粒体嵴间腔扩张；而LPS+SS-31组的线粒体肿胀明显消退，线粒体形态趋于正常。同时，与LPS组相比，LPS+SS-31组的NP细胞最大呼吸水平明显增强（P<0.05）。WB和免疫荧光检测结果显示，LPS干预明显上调了p-NF-κB-p65、cGAS和STING的表达（P<0.05），而SS-31的预处理显著抑制了LPS诱导的p-NF-κB-p65、cGAS和STING的表达（P<0.05）。体内实验证实局部注射SS-31可降低NP细胞衰老、焦亡，减轻纤维环穿刺诱导的椎间盘退变。结论：SS-31减轻了炎症诱导的NP细胞衰老和焦亡，这与其对NF-κB信号通路、cGAS/STING信号通路的抑制有关。

英文摘要:

  【Abstract】 Objectives: To investigate the effect and mechanism of mitochondrial targeting peptide SS-31 on senescence and pyroptosis of nucleus pulposus(NP) cells induced by inflammation. Methods: NP cells were isolated and cultured in vitro from 6-week-old male SD rats, and were divided into control group, lipopolysaccharide(LPS) group, and LPS+SS-31 group; And the control group of cells received no treatment, LPS group of cells were treated with 100μg/mL LPS for 24h, and LPS+SS-31 group of cells were pre-treated with SS-31 30min before LPS intervention. The levels of cellular senescence and pyroptosis were assessed in each group using β-galactosidase(SA-β-Gal) staining and western blot(WB) assay; Transmission electron microscopy and oxygen consumption rate(OCR) assay were used to analyze the mitochondrial function of NP cells in each group; The expressions of phosphorylated nuclear factor-κB-p65(p-NF-κB-p65), cyclic GMP-AMP synthase(cGAS), and interferon stimulating gene(STING) in each group were detected by WB and immunofluorescence; Hematoxylin-eosin(HE) staining, immunohistochemistry(IHC) staining and WB assay were performed to analyze the effects of in vivo injection of SS-31 on intervertebral disc degeneration, senescence, and pyroptosis. Results: The positive SA-β-Gal staining of NP cells, expression of senescence marker proteins(p53, p21), and pyroptosis-related molecules(NLRP3, Caspase-1 p20) were significantly lower in the LPS+SS-31 group compared with the LPS group(P<0.05); Transmission electron microscopy(TEM) showed that the mitochondrial swelling of NP cells was round, the matrix was translucent, and the interstitial lumen of the mitochondrial cristae was dilated in the LPS group, while the mitochondrial swelling was subsided, and the morphology of mitochondria tended to be normalized in the LPS+SS-31 group. Meanwhile, the maximum respiration level of NP cells was significantly enhanced in the LPS+SS-31 group compared with the LPS group(P<0.05). The results of WB and immunofluorescence showed that LPS significantly up-regulated the expression of p-NF-κB-p65, cGAS, and STING(P<0.05), while SS-31 pretreatment significantly inhibited the expression of p-NF-κB-p65, cGAS and STING in NP cells induced by LPS(P<0.05). Finally, in vivo experiments confirmed that local injection of SS-31 reduced the senescence and pyroptosis of NP cells, and alleviated the degeneration of intervertebral disc induced by annulus fibropuncture. Conclusions: SS-31 alleviates inflammation-induced senescence and pyroptosis of NP cells, which is related to the inhibition of NF-κB signaling pathway and cGAS/STING signaling pathway.