少阳膝痹方调节LXR-α/NF-κB信号通路对膝骨关节炎大鼠的干预作用及机制

作者	单位
张轩鸣;郭杨;潘娅岚;刘孟敏;涂鹏程;陆超然;马勇	1.南京中医药大学骨伤修复与重建新技术实验室（南京 210023）,2.南京中医药大学慢性病中医护理干预实验室（南京 210023）, 3.南京中医药大学中医学院中西医结合医学院（南京 210023）

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中文摘要:

目的观察少阳膝痹方对大鼠膝骨关节炎的干预效应，初步从LXR-α/NF-κB信号通路探讨其可能的作用机制。方法采用改良hulth法建立大鼠双侧膝关节膝骨关节炎模型，随机分为假手术组、模型组、美洛昔康组、低、中、高剂量中药复方组，模型制备成功后灌胃给药，给药4周后观察软骨表面大体形态学，HE染色观察软骨病理变化， ELISA法检测血清中基质金属蛋白抑制剂-1（TIMP-1）、前列腺素2E（PGE2）、基质金属蛋白13（MMP13）指标，Western Blot检测大鼠膝关节TNF-α、调节肝X受体α（LXR-α）、核因子κB（NF-κB） P65、NF-κB P-P65蛋白的表达；建立膝关节软骨细胞培养体系，通过LXR-α激动剂及抑制剂干预，分为空白对照组、模型组、中药复方组、中药复方+抑制剂组、中药复方+激动剂组，检测PGE2、MMP13及TNF-α的表达情况。结果术后4周，大体形态学观察显示中药复方各组软骨表面较模型组更完整，HE染色显示各中药复方组关节软骨略有损伤，潮线略有杂乱，骨皮质略有损伤，较模型组有所改善。与假手术组比较，模型组 MMP13浓度、PGE2、TNF-α蛋白表达、P-P65蛋白表达、P-P65/P65相对蛋白表达升高（P＜0.05），NF-κB P65蛋白表达下降（P＜0.05）。与模型组比较，美洛昔康组、各剂量中药复方组MMP13浓度、P-P65蛋白表达、P-P65/P65蛋白相对表达降低，TIMP-1浓度、TIMP-1/MMP13比值、LXR-α基因及蛋白表达升高（P＜0.05）；美洛昔康组、低、高剂量中药复方组PGE2、TNF-α蛋白降低，中剂量中药复方组TNF-α蛋白表达量亦降低（P＜0.05）。与空白对照组比较，模型组MMP13、PGE2蛋白表达升高（P＜0.05）；与模型组比较，中药复方组及中药复方+激动剂组 MMP13、PGE2蛋白表达量降低（P＜0.05），TNF-α基因表达升高（P＜0.05）；与中药复方+抑制剂组比较，中药复方组、中药复方+激动剂组MMP13、PGE2表达量降低（P＜0.05），TNF-α基因表达降低（P＜0.05）。结论少阳膝痹方可降低血清中的MMP13、PGE2改善炎性微环境，减轻软骨退变，降低软骨中炎性因子TNF-α表达，这可能与上调LXR-α表达、降低NF-κB P65、P-P65有关。

英文摘要:

Objective To evaluate the intervention effect of Shaoyang Xibi Decoction（SXD） on rat knee osteoarthritis and to initiate an exploration into its probable mechanism of action through the LXR-α/NF-κB signaling pathway. Methods A rat bilateral knee osteoarthritis model was established by the modified Hulth technique and was subsequently segregated into six groups： sham operation group，model group，meloxicam group，and low，medium，and high dose Chinese medicine compound group. Following the successful preparation of the model，the drug was administered via gavage. After 4 weeks of treatment，the gross morphology of the cartilage surface was examined，while HE staining was employed to observe pathological changes in cartilage. ELISA was used to assay serum matrix metalloproteinase inhibitor-1（TIMP-1），prostaglandin E2（PGE2），and matrix metalloproteinase 13（MMP13） levels. Western Blot analysis was utilized to detect rat knee joint TNF-α，liver X receptor α （LXR-α），nuclear factor κB （NF-κB） P65，and NF-κB P-P65 proteins expressions. A knee joint cartilage cell culture system was established and intervened with LXR-α agonist and inhibitor. The cells were divided into blank controlgroup，model group，Chinese medicine compound group，Chinese medicine compound+agonist group and Chinese medicine compound+inhibitor group. The expression of PGE2 and TIMP-1 proteins were measured. Results Four weeks after surgery，gross morphology observations indicated that the cartilage surface integrity in the Chinese medicine compound groups was superior to that of the model group. HE staining revealed mild damage in joint cartilage，slightly disordered tide lines，and minor damage to the bone cortex within each Chinese medicine compound group，showing an improvement in comparison to the model group. Compared with the sham operation group，the model group exhibited increased MMP13 concentration，PGE2，TNF-α protein expression，P-P65 protein expression，P-P65/P65 relative protein expression（P＜0.05），and a decrease in NF-κB P65 protein expression （P＜0.05）. Compared with the model group，meloxicam and the various doses of Chinese medicine compound groups demonstrated reductions in MMP13 concentration，P-P65 protein expression，P-P65/P65 relative protein expression，and increased in TIMP-1 concentration，TIMP-1/MMP13 ratio，LXR-α gene，and protein expression（P＜0.05）. The meloxicam，low，and high dose Chinese medicine compound groups exhibited decreased PGE2 and TNF-α protein expression（P＜0.05），while the medium dose group also showed reduced TNF-α protein expression （P＜0.05）. Compared with the blank control group，the model group had increased MMP13 and PGE2 protein expression （P＜0.05）. Compared with the model group，MMP13 and PGE2 protein expression decreased in the Chinese medicine compound group and Chinese medicine compound+agonist grou（P＜0.05），while TNF-α gene expression increased （P＜0.05）. In comparison to the Chinese medicine compound+inhibitor group，MMP13 and PGE2 expression decreased in the Chinese medicine compound group and Chinese medicine compound+agonist group（P＜0.05）， and TNF-α gene expression decreased （P＜0.05）. Conclusions SXD demonstrates potential efficacy in reducing serum MMP13 and PGE2 levels， ameliorating the inflammatory microenvironment，alleviating cartilage degeneration，and decreasing inflammatory factor TNF-α expression in cartilage. These effects may be associated with the upregulation of LXR-α expression and the downregulation of NF-κB P65 and P-P65.

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