Clematichinenoside AR (AR), a major active ingredient extracted from traditional Chinese herb Clematis chinensis Osbeck, has been demonstrated to possess anti-inflammatory and immune-modulatory activities in the treatment of experimental rheumatoid arthritis (RA). The therapeutic potential of AR was supposed to be closely correlated to its ability against tumor necrosis factor-α (TNF-α). Therefore, we aimed to explore the protective effects of Clematichinenoside AR against inflammation and cytotoxicity induced by human TNF-α. AR treatment significantly decreased IL-6 and IL-8 secretion, and attenuated MMP-1 production in human RA-derived fibroblast-like synoviocyte MH7A cells stimulated by recombinant human TNF-α (rhTNF-α). AR might antagonize rhTNF-α-induced responses in MH7A cells through inhibiting p38 and ERK MAPKs signal activation. In TNF-α-sensitive murine fibroblast L929 cells, AR treatment attenuated the proliferation inhibition ratio induced by rhTNF-α/ActD and antagonized rhTNF-α-induced cytotoxicity. The cellular and nuclear morphological alterations in apoptotic characteristics induced by rhTNF-α/ActD in L929 cells were observed to be attenuated by the pretreatment with AR under a phase-contrast and fluorescence microscopy, respectively. The Annexin V-FITC/PI double-staining assay was performed to confirm that AR pretreatment obviously decreased the cell death. The antagonistic effects of AR against rhTNF-α-induced cytotoxicity might be potentially attributed to the degeneration of reactive oxygen species and the increasing of mitochondrial membrane potential, along with the suppression of durative phosphorylation of c-Jun N-terminal kinase (JNK). Collectively, our results indicated that AR antagonizes the inflammatory and cytotoxic activities induced by human TNF-α effectively in vitro, which provided further evidence for a novel mechanism underlying AR for treating RA correlating with excessive TNF-α production.

鸡油皂苷AR（AR），是从中草药铁线莲中提取的主要活性成分Osbeck已被证明在治疗类风湿性关节炎（RA）中具有抗炎和免疫调节活性。据认为，AR的治疗潜力与其抗肿​​瘤坏死因子-α（TNF-α）的能力密切相关。因此，我们的目的是探讨鸡油树皂苷AR对人TNF-α诱导的炎症和细胞毒性的保护作用。在重组人TNF-α（rhTNF-α）刺激下，AR治疗显着降低了IL-6和IL-8的分泌，并减弱了人RA衍生的成纤维样滑膜细胞MH7A细胞中MMP-1的产生。AR可能通过抑制p38和ERK MAPKs信号激活来拮抗rhTNF-α诱导的MH7A细胞应答。在TNF-α敏感的鼠成纤维细胞L929细胞中，AR处理减弱了rhTNF-α/ ActD诱导的增殖抑制率，拮抗rhTNF-α诱导的细胞毒性。在相差和荧光显微镜下，rhTNF-α/ ActD诱导的L929细胞凋亡特征的细胞和核形态学改变分别被AR预处理所减弱。进行膜联蛋白V-FITC / PI双重染色测定以确认AR预处理明显降低了细胞死亡。AR对rhTNF-α诱导的细胞毒性的拮抗作用可能归因于活性氧的变性和线粒体膜电位的增加，以及对c-Jun N端激酶（JNK）持续磷酸化的抑制。总的来说，在体外，这为AR治疗与过量TNF-α产生相关的RA的新机制提供了进一步的证据。