Cheng Peng, Li Huan, Chen Hai-Wei, Wang Zhi-Qiang, Li Pei-Wu, Zhang Hai-Hong
Department of Emergency Medicine, Lanzhou University Second Hospital, Lanzhou, Gansu, 730000, People's Republic of China.
Department of Orthopedics, Lanzhou University Second Hospital, Lanzhou, Gansu Province, 730030, People's Republic of China.
In Vitro Cell Dev Biol Anim. 2025 Mar;61(3):288-297. doi: 10.1007/s11626-025-01021-6. Epub 2025 Mar 18.
Low back pain (LBP) is a common symptom of intervertebral disc degeneration (IDD). However, the pathogenesis of IDD is not well understood. Several studies have shown that patients with IDD experience aberrant changes in DNA methylation. 5-Azacytidine (5Aza) is a nucleoside-based DNA methyltransferase inhibitor that inhibits DNA methylation. Therefore, this study investigated whether 5Aza can improve the apoptosis of nucleus pulposus (NP) cells and ER stress (ERS) induced by il-1β by inhibiting PPARγ methylation and its potential pathogenesis. NP cell viability was detected using Cell Counting Kit-8 (CCK-8). Methylation-specific PCR (MSP) was used to evaluate the DNA methylation level. TUNEL was used to evaluate the apoptosis of NP cells. Western blot determined the expression levels of DNMT1, DNMT3a, PPARγ proteins, and ERS-related indexes (C/EBP homology protein (CHOP), GRP78, ATF-6) and apoptosis-related indexes (Bcl-2, Bax, Caspase-3) protein expression levels. 5Aza can inhibit the expression of DNMT1 and DNMT3a and promote PPARγ by modifying the methylation of PPARγ promoter. Western blot (Bcl-2, Bax, Caspase-3, CHOP, GRP78, ATF-6), TUNEL, and CHOP immunofluorescence results showed that 5Aza attenuated IL-1β-induced apoptosis and ERS of NP cells. When pretreated with PPARγ inhibitor (T0070907), the protective effect of 5Aza on IL-1β-induced apoptosis and ERS in NP cells is weakened, suggesting that 5Aza inhibits IL-1β-induced NP cell apoptosis and ERS by promoting the expression of PPARγ. 5Aza preserves PPARγ by inhibiting the expression of DNMT1/DNMT3a, which can significantly reduce IL-1β damage in NP cells. Our findings suggest that preserving PPARγ through DNA demethylation may be an attractive strategy for preventing or treating IDD.
下腰痛（LBP）是椎间盘退变（IDD）的常见症状。然而，IDD的发病机制尚未完全明确。多项研究表明，IDD患者存在DNA甲基化异常变化。5-氮杂胞苷（5Aza）是一种基于核苷的DNA甲基转移酶抑制剂，可抑制DNA甲基化。因此，本研究探讨了5Aza是否能通过抑制PPARγ甲基化及其潜在发病机制来改善白细胞介素-1β（IL-1β）诱导的髓核（NP）细胞凋亡和内质网应激（ERS）。使用细胞计数试剂盒-8（CCK-8）检测NP细胞活力。采用甲基化特异性PCR（MSP）评估DNA甲基化水平。TUNEL法评估NP细胞凋亡情况。蛋白质免疫印迹法测定DNA甲基转移酶1（DNMT1）、DNA甲基转移酶3a（DNMT3a）、PPARγ蛋白以及ERS相关指标（C/EBP同源蛋白（CHOP）、葡萄糖调节蛋白78（GRP78）、活化转录因子6（ATF-6））和凋亡相关指标（Bcl-2、Bax、半胱天冬酶-3）的蛋白表达水平。5Aza可通过修饰PPARγ启动子的甲基化来抑制DNMT1和DNMT3a的表达并促进PPARγ表达。蛋白质免疫印迹法（Bcl-2、Bax、半胱天冬酶-3、CHOP、GRP78、ATF-6）、TUNEL法以及CHOP免疫荧光结果显示，5Aza可减轻IL-1β诱导的NP细胞凋亡和ERS。用PPARγ抑制剂（T0070907）预处理后，5Aza对IL-1β诱导的NP细胞凋亡和ERS的保护作用减弱，这表明5Aza通过促进PPARγ表达来抑制IL-1β诱导的NP细胞凋亡和ERS。5Aza通过抑制DNMT1/DNMT3a的表达来维持PPARγ水平，这可显著减轻IL-1β对NP细胞的损伤。我们的研究结果表明，通过DNA去甲基化来维持PPARγ水平可能是预防或治疗IDD的一种有吸引力的策略。
