DING Ping,ZHANG Daiyang,LI Hewei,et al.Impacts and Mechanism Study of Asiaticoside on Cartilage Damage and OPG/RANK/RANKL Signaling Pathway in Rats with Knee Osteoarthritis[J].Chinese Journal of Traditional Medical Traumatology & Orthopedics,2022,30(09):7-13+19.





Impacts and Mechanism Study of Asiaticoside on Cartilage Damage and OPG/RANK/RANKL Signaling Pathway in Rats with Knee Osteoarthritis
1恩施土家族苗族自治州中心医院脊柱外科(湖北 恩施,445000)
DING Ping1ZHANG Daiyang1△LI Hewei2WU Yanru2ZHANG Guangheng3
1Department of Spinal Surgery,Central Hospital of Enshi Tujia and Miao Autonomous Prefecture,Enshi 445000,Hubei China; 2Liyuan Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China; 3Central Laboratory of Liyuan Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China.
积雪草苷 膝骨关节炎 骨保护素 核因子κB受体活化因子 核因子κB受体活化因子配体
asiaticoside knee osteoarthritis osteoprotegerin receptor activator of nuclear factor κB receptor activator of nuclear factor κB ligand
目的:探讨积雪草苷(ASS)对膝骨关节炎(KOA)大鼠软骨损伤和骨保护素(OPG)/核因子κB受体活化因子(RANK)/RANK配体(RANKL)信号通路的影响。方法:采用前交叉韧带切断术(ACLT)加内侧半月板切除术(MMx)诱导建立膝骨关节炎大鼠模型,建模后分为KOA组、ASS低剂量组(ASS-L组,16 mg/kg)、ASS高剂量组(ASS-H组,32 mg/kg)、ASS+si-NC组(32 mg/kg ASS+100 ng LV-si-NC载体)、ASS+si-OPG组(32 mg/kg ASS+100 ng LV-si-OPG载体),另设假手术(Sham)组,每组12只。给予相应的药物干预2周后,通过机械缩爪阈值(PWT)和热缩爪潜伏期(PWL)评估大鼠疼痛行为,酶联免疫吸附(ELISA)法检测血清炎性细胞因子白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)水平,苏木精-伊红(HE)和番红O/固绿染色观察关节软骨组织病理学变化,实时荧光定量PCR(RT-qPCR)检测软骨组织基质金属蛋白酶(MMP-13)、Ⅱ型胶原蛋白(COL2A1)、聚集蛋白聚糖(Aggrecan)mRNA表达,蛋白质印迹(Western Blot)检测软骨组织OPG、RANK、RANKL蛋白表达。结果:与Sham组相比,KOA组大鼠的PWT和PWL值、COL2A1 mRNA、Aggrecan mRNA和OPG蛋白水平显著降低,血清IL-1β、IL-6和TNF-α水平、软骨组织Mankin评分和OARSI评分、MMP-13 mRNA和RANK、RANKL蛋白水平显著升高,差异有统计学意义(P<0.05); 与KOA组相比,ASS-L组和ASS-H组大鼠的PWT和PWL值、COL2A1 mRNA、Aggrecan mRNA和OPG蛋白水平显著升高,血清IL-1β、IL-6和TNF-α水平、Mankin评分和OARSI评分、MMP-13 mRNA和RANK、RANKL蛋白水平显著降低,差异有统计学意义(P<0.05); 抑制OPG可明显逆转积雪草苷对膝骨关节炎大鼠软骨损伤的保护作用。结论:积雪草苷可减少促炎细胞因子的释放,抑制MMP-13表达和增加Ⅱ型胶原沉积,抑制关节软骨退变,并可能通过调节OPG/RANK/RANKL信号通路,发挥抗膝骨关节炎作用。
Objective:To investigate the impacts of asiaticoside(ASS)on cartilage damage and osteoprotegerin(OPG)/receptor activator of nuclear factor κB(RANK)/RANK ligand(RANKL)signaling pathway in knee osteoarthritis(KOA)rats.Methods:KOA rat model was established by induction of anterior cruciate ligament amputation(ACLT)and medial meniscectomy(MMx).After modeling,they were grouped into KOA group,ASS low-dose group(ASS-L group,16 mg/kg),ASS high-dose group(ASS-H group,32 mg/kg),ASS+si-NC group(32 mg/kg ASS+100 ng LV-si-NC carrier)and ASS+si-OPG group(32 mg/kg ASS+100 ng LV-si-OPG carrier),and another sham operation(Sham)group was set,with 12 animals in each group.After 2 weeks of corresponding drug intervention,the pain behavior of rats was evaluated by mechanical paw withdrawal threshold(PWT)and thermal paw withdrawal latency(PWL).The levels of serum inflammatory cytokines interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA).The histopathological changes of articular cartilage were observed by hematoxylin-eosin(HE)and safranin O/fast green staining.The mRNA expression of matrix metalloproteinase 13(MMP-13),collagen type Ⅱ(COL2A1)and aggrecan(Aggrecan)in cartilage tissue was measured by real-time quantitative PCR(RT-qPCR),and the protein expression of OPG,RANK and RANKL in cartilage tissue was measured by Western Blot.Results:Compared with the Sham group,the PWT and PWL values,COL2A1 mRNA,Aggrecan mRNA and OPG protein levels were significantly decreased in the KOA group; the serum IL-1β,IL-6 and TNF-α levels,cartilage tissue Mankin scores and OARSI scores,MMP-13 mRNA and RANK,RANKL protein levels were significantly increased(P<0.05).Compared with the KOA group,the PWT and PWL values,COL2A1 mRNA,Aggrecan mRNA and OPG protein levels of the ASS-L group and ASS-H group were significantly increased; the serum IL-1β,IL-6 and TNF-α levels,Mankin scores and OARSI scores,MMP-13 mRNA and RANK,RANKL protein levels were significantly decreased(P<0.05).Inhibition of OPG was able to significantly reverse the protective efficacy of ASS on cartilage injury in KOA rats(P<0.05).Conclusion:ASS can reduce the release of pro-inflammatory cytokines,inhibit the expression of MMP-13,increase the deposition of type Ⅱ collagen and inhibit the degeneration of articular cartilage,which may play an anti-KOA effect by regulating the OPG/RANK/RANKL signaling pathway.


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更新日期/Last Update: 2022-09-10