化瘀祛湿方对miR⁃21靶向Skp2介导早期膝骨关节炎软骨细胞自噬的影响

Effect of Huayu Qushi Decotion on miR⁃21⁃targeted Skp2⁃mediated autophagy of early knee osteoarthritis

目的:研究miR‑21‑5p介导Skp2对膝OA软骨细胞自噬影响,探讨化瘀祛湿方对膝OA软骨细胞的保护作用。方法:(1)取全膝关节置换术的人体膝OA软骨组织,采用FISH单染试验检测miR‑21‑5p表达和定位,免疫荧光检测Skp2表达,TUNEL染色试验检测膝OA细胞凋亡,Western blot法检测Bax、Bcl‑2、Beclin1、CARM1、LC3Ⅱ/Ⅰ和Skp2相对表达量;(2)取SD大鼠36只,按随机数字法分为正常对照组、假手术组、模型组、模型+空载组、模型+miR‑21‑5p干扰组、模型+化瘀祛湿方组6组,每组6只;采用石蜡切片阿利新蓝染色观察软骨形态结构,qPCR、Western blot法检测各组软骨组织Beclin1、CARM1、LC3Ⅱ/Ⅰ、Skp2相对表达量,免疫组化检测Skp2的IOD/Area值。结果:FISH检测显示miR‑21‑5p在人体膝OA软骨组织中高表达,免疫荧光检测表明Skp2在人体膝OA软骨组织中低表达;TUNEL检测发现,人体膝OA软骨组织凋亡显著增加;Western blot法检测显示,与正常对照组相比,人体膝OA软骨组织中Bax、CARM1相对表达量较正常组显著升高,而Bcl‑2、LC3Ⅱ/Ⅰ、Skp2相对表达量明显降低,Beclin1相对表达量稍降低(P<0.05)。动物实验显示,采用miR‑21‑5p干扰处理后,Beclin1、CARM1、LC3Ⅱ/Ⅰ表达显著下降,Skp2稍降低;免疫组化检测发现Skp2 IOD/Area值与正常对照组相比,模型组IOD/Area值显著降低(P<0.05);与模型组相比,模型+化瘀祛湿方组IOD/Area值显著升高(P<0.05);石蜡切片阿利新蓝染色显示与假手术照组相比,模型+空载组、模型+miR‑21‑5p干扰组蓝色变浅,但模型组蓝色变深,模型+化瘀祛湿组蓝色较为明显。结论:miR‑21‑5p可负向调控Skp2,介导膝OA软骨细胞自噬,加速OA发展;化瘀祛湿方可能通过抑制miR‑21‑5p,提高Skp2表达,起到延缓膝OA退变的作用。

Objective:To study the effect of miR‑21‑5p mediated Skp2 on autophagy of knee OA chondrocytes,and to ex‑plore the protective effect of Huayu Qushi Decotion on OA chondrocytes.Methods:(1)Human knee OA cartilage tissues from total knee arthroplasty were taken,and miR‑21‑5p expression and localization were detected by FISH single‑staining assay,Skp2 expression by immunofluorescence,apoptosis by TUNEL staining assay,and relative expressions of Bax,Bcl‑2,Beclin1,CARM1,LC3Ⅱ/Ⅰand Skp2 were determined by Western blot analysis;(2)A total of 36 SD rats were divided into 6 groups according to the randomized numerical method with 6 rats in each group,including the normal control group,the sham operation group,the model group,the model+airborne group,the model+miR‑21‑5p interference group,the model+Huayu Qushi Decotion group;the morphology and structure of cartilage were observed by paraffin section alisin blue staining,and the cartilage was detected by qPCR and Western blot method in each group.The relative expressions of Beclin1,CARM1,LC3Ⅱ/Ⅰand Skp2 were detected by qPCR and Western blot,and the IOD/Area value of Skp2 was detected by immunohistochemistry.Results:FISH assay showed that miR‑21‑5p was highly expressed in human knee OA cartilage tissues,and immunofluorescence assay indicated that Skp2 was lowly expressed in human knee OA cartilage tissues.TUNEL assay revealed a significant increase in apoptosis in human knee OA cartilage tissues.Western blot assay showed that compared with the normal control group,the relative expressions of Bax and CARM1 in human knee OA cartilage tissues were significantly higher compared with the normal group,while the relative expression of Bcl‑2,LC3Ⅱ/Ⅰ,Skp2 were significantly lower,and the relative expressions of Beclin1 were slightly lower(P<0.05).Animal experiments showed that the expressions of Beclin1,CARM1,LC3Ⅱ/Ⅰwere significantly decreased and Skp2 was slightly decreased after treatment with miR‑21‑5p interference.Immunohistochemical detection revealed that the IOD/Area value of Skp2 was significantly decreased in the model group compared with that of the normal control group(P<0.05);compared with the model group,the IOD/Area value of the model+Huayu Qushi Decotion IOD/Area value was significantly higher in the model+Huayu Qushi Decotion compared with the model group(P<0.05).Alisin blue staining of paraffin sections showed that the blue color became lighter in the model+airborne group and the model+miR‑21‑5p interference group compared with that in the sham operation group,but the blue color became darker in the model group,and the blue color was more pronounced in the model+Huayu Qushi Decotion.Conclusion:miR‑21‑5p can negatively regulate Skp2,mediate knee OA chondrocyte autophagy,and accelerate the development of OA.The Huayu Qushi Decotion may play a role in delaying the degeneration of knee OA by inhibiting miR‑21‑5p and increasing the expression of Skp2.