Abstract：
BACKGROUND: Facet joint osteoarthritis (FJOA) is a common lumbar osteoarthritis characterized by degeneration of small joint cartilage. Bushen Huoxue decotion (BSHXD) has good therapeutic effects on OA. Our work aimed to further probe the pharmacological effects of BSHXD-containing serum (BSHXD-CS) on FJOA and define underlying the mechanisms invovled.
METHODS: To establish a FJOA cell model, primary rat chondrocytes were treated with LPS. The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. The secretion levels of pro-inflammatory cytokines were measured by ELISA. Cell viability was determined by CCK8 assay. The global m6A level was detected by the kit, and NLRP3 mRNA m6A level was determined by Me-RIP assay. The molecular interactions were analyzed by RIP and RNA pull-down assays.
RESULTS: BSHXD-CS treatment relieved LPS-induced cell injury, inflammation, NLRP3 inflammasome and pyroptosis in chondrocytes (all p < 0.05). LPS-induced NLRP3 upregulation in chondrocytes was related to its high m6A modification level (p < 0.05). It was also observed that BSHXD-CS reduced LPS-induced m6A modification in chondrocytes via repressing STAT3 (all p < 0.05), suggesting BSHXD-CS could repress NLRP3 expression via m6A-dependent manner. Moreover, DAA, a m6A specific inhibitor, was proved to strengthen the protectively roles of BSHXD-CS on LPS-challenged pytoptosis (all p < 0.05).
CONCLUSIONS: BSHXD-CS inhibited NLRP3 inflammasome activation and pyroptosis in chondrocytes to repress OA progression by reducing RNA m6A modification.
METHODS: To establish a FJOA cell model, primary rat chondrocytes were treated with LPS. The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. The secretion levels of pro-inflammatory cytokines were measured by ELISA. Cell viability was determined by CCK8 assay. The global m6A level was detected by the kit, and NLRP3 mRNA m6A level was determined by Me-RIP assay. The molecular interactions were analyzed by RIP and RNA pull-down assays.
RESULTS: BSHXD-CS treatment relieved LPS-induced cell injury, inflammation, NLRP3 inflammasome and pyroptosis in chondrocytes (all p < 0.05). LPS-induced NLRP3 upregulation in chondrocytes was related to its high m6A modification level (p < 0.05). It was also observed that BSHXD-CS reduced LPS-induced m6A modification in chondrocytes via repressing STAT3 (all p < 0.05), suggesting BSHXD-CS could repress NLRP3 expression via m6A-dependent manner. Moreover, DAA, a m6A specific inhibitor, was proved to strengthen the protectively roles of BSHXD-CS on LPS-challenged pytoptosis (all p < 0.05).
CONCLUSIONS: BSHXD-CS inhibited NLRP3 inflammasome activation and pyroptosis in chondrocytes to repress OA progression by reducing RNA m6A modification.
摘要：
背景：小关节骨关节炎（FJOA）是一种常见的腰椎骨关节炎，其特征是小关节软骨的退变。补肾活血汤(BSHXD)对OA有较好的治疗作用。我们的工作旨在进一步探讨含BSHXD的血清（BSHXD-CS）对FJOA的药理作用，并确定潜在的机制。
方法：为了建立FJOA细胞模型，用LPS处理原代大鼠软骨细胞。采用qRT-PCR和westernblot检测mRNA和蛋白的表达,分别。通过ELISA测量促炎细胞因子的分泌水平。通过CCK8测定确定细胞活力。试剂盒检测到全球m6A水平，和NLRP3mRNAm6A水平通过Me-RIP测定法测定。通过RIP和RNA下拉法分析分子相互作用。
结果：BSHXD-CS治疗减轻了LPS诱导的细胞损伤，炎症,NLRP3炎性小体和软骨细胞的焦亡(均p<0.05)。LPS诱导的NLRP3在软骨细胞中的上调与其高m6A修饰水平有关(p<0.05)。还观察到BSHXD-CS通过抑制STAT3减少了LPS诱导的软骨细胞中的m6A修饰（所有p<0.05），提示BSHXD-CS可以通过m6A依赖性方式抑制NLRP3表达。此外，DAA,一种M6A特异性抑制剂，被证明可以增强BSHXD-CS对LPS攻击的脓毒性的保护作用（均p<0.05）。
结论：BSHXD-CS通过减少RNAm6A修饰，抑制软骨细胞中NLRP3炎性体活化和焦亡，从而抑制OA进展。
方法：为了建立FJOA细胞模型，用LPS处理原代大鼠软骨细胞。采用qRT-PCR和westernblot检测mRNA和蛋白的表达,分别。通过ELISA测量促炎细胞因子的分泌水平。通过CCK8测定确定细胞活力。试剂盒检测到全球m6A水平，和NLRP3mRNAm6A水平通过Me-RIP测定法测定。通过RIP和RNA下拉法分析分子相互作用。
结果：BSHXD-CS治疗减轻了LPS诱导的细胞损伤，炎症,NLRP3炎性小体和软骨细胞的焦亡(均p<0.05)。LPS诱导的NLRP3在软骨细胞中的上调与其高m6A修饰水平有关(p<0.05)。还观察到BSHXD-CS通过抑制STAT3减少了LPS诱导的软骨细胞中的m6A修饰（所有p<0.05），提示BSHXD-CS可以通过m6A依赖性方式抑制NLRP3表达。此外，DAA,一种M6A特异性抑制剂，被证明可以增强BSHXD-CS对LPS攻击的脓毒性的保护作用（均p<0.05）。
结论：BSHXD-CS通过减少RNAm6A修饰，抑制软骨细胞中NLRP3炎性体活化和焦亡，从而抑制OA进展。
