【摘要】 目的 探究地黄梓醇对RP5-894D12.5/miR-1972/JUN轴的调控作用及其对滑膜成纤维细胞炎症和铁死亡的影响。方法 （1）体内实验:将40只C57BL/6小鼠随机分为假手术组、模型组、地黄梓醇组和塞来昔布组,每组10只。除假手术组外,其余组小鼠通过内侧半月板失稳术建立滑膜炎模型。术后证实造模成功后,地黄梓醇组给予地黄梓醇200 mg/kg灌胃,塞来昔布组给予塞来昔布28.6 mg/kg灌胃,模型组和假手术组灌胃等量生理盐水,均1次/d。连续灌胃4周后,HE染色观察膝关节滑膜组织形态并计算Krenn’s评分,免疫组化法检测膝关节滑膜组织中JUN蛋白阳性表达情况,ELISA法检测血清白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、Fe²⁺、丙二醛（MDA）、还原型谷胱甘肽（GSH）水平。（2）体外实验:实验设空白组、诱导组、地黄梓醇低剂量组、地黄梓醇中剂量组、地黄梓醇高剂量组,除空白组外,其余组利用IL-1β诱导人滑膜成纤维细胞建立炎症模型。ELISA法检测细胞中IL-1β、TNF-α、Fe²⁺、MDA、GSH水平,Western blot法检测细胞中谷胱甘肽过氧化物酶4抗体（GPX4）、溶质载体家族7成员11（SLC7A11）、JUN蛋白表达情况,RT-qPCR法检测细胞中RP5-894D12.5、miR-1972、JUN mRNA表达情况。结果 体内实验中,模型组滑膜组织肥厚增生,大量炎性细胞浸润,血管翳形成,Krenn’s评分和血清IL-1β、TNF-α、Fe²⁺、MDA水平均明显高于假手术组（P均<0.05）,滑膜组织中JUN蛋白阳性表达平均光密度值和血清GSH水平均明显低于假手术组（P均<0.05）;地黄梓醇组和塞来昔布组滑膜组织损伤和滑膜增生明显减轻,少量炎性细胞浸润,Krenn’s评分和血清IL-1β、TNF-α、Fe²⁺、MDA水平均明显低于模型组（P均<0.05）,滑膜组织中JUN蛋白阳性表达平均光密度值和血清GSH水平均明显高于模型组（P均<0.05）。体外实验中,与空白组比较,诱导组细胞中IL-1β、TNF-α、Fe²⁺、MDA水平和miR-1972相对表达量均明显升高（P均<0.05）,GSH水平和GPX4、SLC7A11、JUN蛋白相对表达量及RP5-894D12.5、JUN mRNA相对表达量均明显降低（P均<0.05）;与诱导组比较,地黄梓醇各组细胞中IL-1β、TNF-α、Fe²⁺、MDA水平和miR-1972相对表达量均明显降低（P均<0.05）,GSH水平和GPX4、SLC7A11、JUN蛋白相对表达量及RP5-894D12.5、JUN mRNA相对表达量均明显升高（P均<0.05）;地黄梓醇高剂量组细胞中IL-1β、TNF-α、Fe²⁺、MDA水平和miR-1972相对表达量均明显低于地黄梓醇低、中剂量组（P均<0.05）,GSH水平和GPX4、SLC7A11、JUN蛋白相对表达量及RP5-894D12.5、JUN mRNA相对表达量均明显高于地黄梓醇低、中剂量组（P均<0.05）。结论 地黄梓醇可通过调控RP5-894D12.5/miR-1972/JUN轴而抑制滑膜成纤维细胞炎症及铁死亡,缓解滑膜炎疾病进展。更多还原

【Abstract】 Objective It is to investigate the regulatory effect of rehmannia catalpol（RC） on RP5-894D12. 5/miR-1972/JUN axis and its effect on inflammation and ferroptosis of synovial fibroblasts. Methods（1）In vivo experiments: Forty C57BL/6 mice were randomly divided into sham operation group,model group,RC group and celecoxib group,with 10 mice in each group. The mice of all groups except for the sham operation group were prepared by medial meniscus destabilization to establish models of synovitis. Following surgical confirmation of successful modeling,the RC group was given RC200 mg/kg by gavage,the celecoxib group was given celecoxib 28. 6 mg/kg by gavage,while the model and sham operation groups were given an equal volume of physiological saline,all administered once daily. After 4 weeks of continuous administration,the morphology of knee synovial tissue was observed by HE staining and Krenn’s score was calculated,the positive protein expression of JUN in knee synovial tissue was detected by immunohistochemistry,and the serum levels of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,Fe²⁺,malondialdehyde（MDA） and glutathione（GSH） were measured by ELISA.（2）In vitro experiments: A blank group,an induction group,low-dose,medium-dose and high-dose groups of RC were set up in the experiment. Inflammatory models were established by inducing human synovial fibroblasts with IL-1β in all groups except for the blank group. The levels of IL-1β,TNF-α,Fe²⁺,MDA,and GSH in the cells were measured by ELISA,the protein expressions of glutathione peroxidase 4（GPX4）, solute carrier family 7 member 11（SLC7A11） and JUN in the cells were detected by Western blot,and mRNA expressions of RP5-894D12. 5,miR-1972 and JUN in the cells were detected by RT-qPCR. Results In vivo experiments revealed that there were synovial tissue hypertrophy and hyperplasia,extensive inflammatory cell infiltration and pannus formation in the model group,the Krenn’s score and serum levels of IL-1β,TNF-α,Fe²⁺and MDA were significantly higher,while the mean optical density value of JUN protein expression in synovial tissue and serum level of GSH were significantly lower than those in the sham operation group（all P < 0. 05）; the synovial tissue damage and hyperplasia were alleviated with less inflammatory cell infiltration in the RC group and celecoxib group,the Krenn’s score and serum levels of IL-1β,TNF-α,Fe²⁺and MDA were significantly lower,while the mean optical density value of JUN protein expression in synovial tissue and serum level of GSH were significantly higher than those in the model group（all P < 0. 05）. In vitro experiments,compared with the blank group,the levels of IL-1β,TNF-α,Fe²⁺,MDA,and relative expression of miR-1972 were significantly increased,while the level of GSH and relative protein expressions of GPX4,SLC7A11,JUN,and relative mRNA expressions of RP5-894D12. 5 and JUN were significantly decreased in the induced cells（all P < 0. 05）. Compared with the induction group,the levels of IL-1β,TNF-α,Fe²⁺,MDA,and relative expression of miR-1972 were significantly decreased,while the level of GSH and relative protein expressions of GPX4,SLC7A11,JUN,and relative mRNA expressions of RP5-894D12. 5 and JUN were significantly increased in the cells of each RC group（all P < 0. 05）; the levels of IL-1β,TNF-α,Fe²⁺,MDA,and relative expression of miR-1972 were significantly lower,while the level of GSH and relative protein expressions of GPX4,SLC7A11,JUN,and relative mRNA expressions of RP5-894D12. 5 and JUN were significantly higher in the high dose groups of RC than those in the medium dose and low dose groups of RC（all P < 0. 05）. Conclusion Rehmannia catalpol can inhibit inflammation and ferroptosis in synovial fibroblasts to delay the development of synovitis via regulating the RP5-894D12. 5/miR-1972/JUN axis.更多还原