Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (5): 1026-1035.doi: 10.12307/2025.208
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Wang Yuru1, Li Siyuan2, Xu Ye2, Zhang Yumeng2, Liu Yang2, Hao Huiqin1, 2
Received:
2023-12-13
Accepted:
2024-01-31
Online:
2025-02-18
Published:
2024-06-04
Contact:
Hao Huiqin, PhD, Professor, PhD supervisor, Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan 030032, Shanxi Province, China; Basic Laboratory of Integrated Chinese and Western Medicine, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China
About author:
Wang Yuru, Master candidate, Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan 030032, Shanxi Province, China
Supported by:
CLC Number:
Wang Yuru, Li Siyuan, Xu Ye, Zhang Yumeng, Liu Yang, Hao Huiqin. Effects of wogonin on joint inflammation in collagen-induced arthritis rats via the endoplasmic reticulum stress pathway[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(5): 1026-1035.
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2.1 实验动物数量分析 18只大鼠实验过程中无死亡脱失,全部进入结果分析。 2.2 汉黄芩素对CIA大鼠体质量、关节肿胀度、关节炎指数评分的影响 随着饲养周期的延长,各组大鼠体质量呈现平缓增长趋势,未表现出明显差异,见图2A。足爪肿胀度和关节炎评分被认为是反映药物对CIA大鼠缓解作用最直观的指标。如图2B所示,治疗0 d时,与健康对照组相比,其余两组大鼠的关节炎指数评分均显著增高(P < 0.01),表明造模成功;到给药第21天,汉黄芩素治疗组大鼠的关节炎指数评分(10.3±0.3)较CIA模型组(12.9±0.5)明显降低(P < 0.05);治疗第28天,与CIA模型组关节炎指数评分(12.6±0.5)比较,汉黄芩素治疗组的大鼠关节炎评分(9.3±0.4)显著下降(P < 0.01),且其踝关节左右径长(7.1±0.2) mm较CIA模型组(7.8±0.1) mm显著减小(P < 0.05),见图2C。 2.3 汉黄芩素对CIA大鼠踝关节病理损伤的影响 如图3所示,给药28 d后大鼠足外观结果:CIA模型组大鼠踝关节有明显肿胀,汉黄芩素治疗组较CIA模型组肿胀程度明显减轻。病理学苏木精-伊红染色结果显示,健康对照组大鼠踝关节组织结构清晰可见,关节面光滑平整;CIA模型组大鼠踝关节结构被破坏,关节面粗糙,软骨破坏和骨质侵蚀严重,关节面及骨质内大量炎症细胞浸润;汉黄芩素治疗组病理损伤较CIA模型组明显改善,炎症细胞浸润减少,软骨破坏和骨质侵蚀出现不同程度减轻,关节结构基本清晰完整。 番红O-固绿染色结果可见健康对照组大鼠软骨完整连续,表面平整,番红O着色较深;CIA模型组几乎不可见番红O着色,关节面粗糙糜烂,可见溃疡缺损;汉黄芩素治疗组可见软骨番红O着色,关节软骨表面轻微磨损、大致光整。滑膜组织中含有较多胶原纤维,Masson染色结果示健康对照组大鼠滑膜形态正常,与关节面保持一定间距;CIA模型组关节腔内胶原染色加重,滑膜细胞增生排列紊乱并侵入关节面,关节完整结构破坏;汉黄芩素治疗组关节腔隙内有胶原增生侵袭关节面,但骨和软骨的破坏表现较CIA组有所减轻。 2.4 汉黄芩素对CIA大鼠关节GRP78、CHOP mRNA和蛋白表达的影响 qRT-PCR结果显示,与健康对照组比较,CIA模型组大鼠关节组织GRP78、CHOP mRNA相对表达量均显著升高(P < 0.01);与CIA模型组比较,汉黄芩素治疗组GRP78、CHOP mRNA相对表达量明显降低(P < 0.05,P < 0.01),见图4A。蛋白印迹检测结果示与健康对照组比较,CIA模型组大鼠关节组织GRP78、CHOP蛋白表达显著升高(均P < 0.01);与CIA模型组比较,汉黄芩素治疗组GRP78、CHOP蛋白表达明显降低(P < 0.01,P < 0.05),见图4B,C。 2.5 汉黄芩素对CIA大鼠关节GRP78、CHOP 蛋白表达的免疫组化分析 免疫组化染色结果显示,健康对照组大鼠关节中GRP78、CHOP表达很少,几乎不可见,见图5A。与健康对照组相比,CIA模型组大鼠关节中表达显著增高(P < 0.01),主要定位于侵袭的滑膜组织和被侵蚀的关节面周围;汉黄芩素治疗组相比于CIA模型组,侵入关节的滑膜组织中和关节面周围的GRP78、CHOP表达显著降低(P < 0.01),见图5B。 2.6 汉黄芩素对FLS细胞毒性的影响 不同浓度汉黄芩素分别作用FLS细胞6,12,24 h后,CCK-8实验结果见表3,DMSO组与空白对照组相比差异无显著性意义;与DMSO组相比,汉黄芩素浓度在50 μmol/L及以下时,细胞存活率未有明显下降;在100 μmol/L时,细胞存活率略有下降,但只有作用时间为24 h时差异有显著性意义(P < 0.05);在200 μmol/L时,细胞存活率均明显下降 "
(P < 0.01)。因此,认为汉黄芩素在100 μmol/L以下时对细胞无明显的细胞毒性作用,选择25,50,100 μmol/L为后续实验汉黄芩素所采用的浓度。 2.7 各组FLS细胞上清炎症因子水平检测结果 与空白对照组相比,毒胡萝卜素组细胞上清肿瘤坏死因子α、白细胞介素1β水平显著增高(P < 0.01);与毒胡萝卜素组相比,毒胡萝卜素+25 μmol/L汉黄芩素组细胞上清肿瘤坏死因子α、白细胞介素1β无显著变化,毒胡萝卜素+50 μmol/L汉黄芩素组和毒胡萝卜素+100 μmol/L汉黄芩素组细胞上清肿瘤坏死因子α、白细胞介素1β水平均显著降低(P < 0.05,P < 0.01)。见图6。 2.8 各组FLS细胞状态变化和活性氧含量的检测结果 显微镜下FLS细胞形态显示,空白对照组细胞生长状态良好,轮廓显著为长梭形,呈典型纤维样细胞结构,贴壁牢靠,群体细胞相互连接成网;毒胡萝卜素组大部分细胞改变原有形态,呈多角形,有些轮廓不可见,出现模糊圆团样,群体细胞聚集成堆;随着汉黄芩素给药浓度的增加,长梭形细胞逐渐增多,多角形细胞减少,见图7A。图7B可见,与空白对照组相比,毒胡萝卜素组细胞中平均荧光强度明显增强,表明毒胡萝卜素使细胞内活性氧水平显著提高(P < 0.01)。与毒胡萝卜素组相比,毒胡萝卜素+25 μmol/L汉黄芩素组细胞平均荧光强度几乎无明显变化,提示25 μmol/L汉黄芩素对毒胡萝卜素作用的FLS细胞中活性氧水平无明显影响;毒胡萝卜素+100 μmol/L汉黄芩素组平均荧光强度明显降低(P < 0.01),提示100 μmol/L汉黄芩素降低了毒胡萝卜素作用的FLS细胞中的活性氧水平。荧光显微镜和流式细胞仪检测展现了相同的结果,见图7C,D。 2.9 各组FLS细胞中GRP78、IRE1α、XBP1s、CHOP mRNA表达 与空白对照组相比,毒胡萝卜素组FLS细胞中GRP78、IRE1α、XBP1s、CHOP mRNA表达显著增加(P < 0.05,P < 0.01)。与毒胡萝卜素组相比,毒胡萝卜素+25 μmol/L汉黄芩素组细胞4个mRNA表达水平均无显著变化;毒胡萝卜素+50 μmol/L汉黄芩素组GRP78、XBP1s mRNA表达水平明显降低(P < 0.05),IRE1α、CHOP mRNA表达虽有降低,但差异无显著性意义;毒胡萝卜素+100 μmol/L汉黄芩素组GRP78、IRE1α、XBP1s、CHOP mRNA表达水平均明显降低(P < 0.05,P < 0.01),见图8。以上结果显示汉黄芩素能下调毒胡萝卜素作用FLS细胞中GRP78、IRE1α、XBP1s、CHOP mRNA的相对表达量,且呈现出一定的剂量依赖性。 2.10 各组FLS细胞中GRP78、IRE1α、XBP1s、CHOP蛋白的表达水平 与空白对照组相比,毒胡萝卜素显著上调了FLS细胞中GRP78、IRE1α、XBP1s、CHOP蛋白水平(P < 0.01)。与毒胡萝卜素组相比,毒胡萝卜素+25 μmol/L汉黄芩素组细胞4个蛋白几乎无明显变化;毒胡萝卜素+50 μmol/L汉黄芩素组明显降低了GRP78、IRE1α、CHOP蛋白表达(P < 0.01),XBP1s表达虽也有降低,但差异无显著性意义;毒胡萝卜素+100 μmol/L汉黄芩素组GRP78、IRE1α、XBP1s、CHOP蛋白表达均明显降低(P < 0.05,P < 0.01),见图9。以上结果显示汉黄芩素能下调毒胡萝卜素作用FLS细胞中GRP78、IRE1α、XBP1s、CHOP蛋白的相对表达量,且呈剂量依赖性关系。 "
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