人参皂苷Rb1通过激活AMPK减轻大鼠创伤性骨关节炎

Ginsenoside Rb1 attenuates post-traumatic osteoarthritis by activating AMPK in rats

目的探讨人参皂苷Rb1(G-Rb1)治疗大鼠创伤性骨关节炎(PTOA)的分子机制。方法将大鼠分为假手术组、模型组、低、中、高剂量组和抑制剂组,每组12只。假手术组大鼠切开髌骨外侧皮肤后立即缝合伤口,其他组大鼠采用前十字韧带切断加半月板部分切除法建立PTOA动物模型。建模2 h后,假手术组和模型组大鼠灌胃2 mL生理盐水。低、中和高剂量组大鼠分别灌胃2 mL浓度为10,20,40 mg/(kg·d)的G-Rb1溶液。抑制剂组大鼠灌胃2 mL浓度为40 mg/(kg·d)的G-Rb1溶液,同时腹腔注射20 mg/(kg·d)AMPK抑制剂Compound C。各组大鼠均处理4周。采用苏木素伊红(HE)染色评价大鼠关节软骨形态。采用番红O/固绿染色法评价大鼠关节软骨退变,然后进行国际骨关节炎研究学会(OARSI)评分。采用TUNEL染色检测软骨细胞凋亡情况。通过酶联免疫吸附法(ELISA)测定大鼠血清中白细胞介素-1β(IL-1β)、IL-6和肿瘤坏死因子-α(TNF-α)水平。通过RT-qPCR测定大鼠软骨中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、CollagenⅡ、基质金属蛋白酶-13(MMP-13)、Runt相关转录因子2(Runx2)和骨形态发生蛋白-2(BMP-2)的mRNA水平。通过Western blot测定大鼠软骨中AMP活化蛋白激酶α(AMPKα)的磷酸化水平。结果与模型组比较,低、中和高剂量组大鼠的软骨损伤和退变明显减轻,OARSI评分和软骨细胞TUNEL阳性率均剂量依赖性降低(P<0.05),血清中IL-1β、IL-6和TNF-α水平剂量依赖性降低(P<0.05),Bax和MMP-13的mRNA相对表达量均剂量依赖性降低(P<0.05),Bcl-2、CollagenⅡ、Runx2和BMP-2的mRNA相对表达量均剂量依赖性升高(P<0.05),AMPKα相对磷酸化水平剂量依赖性升高(P<0.05)。Compound C减弱了高剂量G-Rb1对PTOA大鼠的治疗效果(P<0.05)。结论G-Rb1通过激活AMPK减轻大鼠PTOA进程。

Objective To explore the molecular mechanism of ginsenoside Rb1(G-Rb1)in the treatment of post-traumatic osteoarthritis(PTOA)in rats.Methods Rats were divided into sham group,model group,low,medium,high dose group and inhibitor group,with 12 rats in each group.Rats in sham group were sutured immediately after incision of the outer skin of the patella,and the rats underwent anterior cruciate ligament transection and partial meniscectomy to establish PTOA model in the other groups.At 2 h after modeling,rats in sham group and model group were given 2 mL saline.Rats were given 2 mL G-Rb1 solution at the doses of 10,20 and 40 mg/(kg·d)in low,medium and high dose groups,respectively.Rats in inhibitor group were intragastrically given 2 mL G-Rb1 solution at a dose of 40 mg/(kg·d),and intraperitoneally injected with AMPK inhibitor Compound C at a dose of 20 mg/(kg·d).Rats in all groups were treated for 4 weeks.The morphology of articular cartilage was evaluated by hematoxylin eosin(HE)staining.Articular cartilage degeneration was evaluated by Safranin O/fast green staining,and then OARSI score was performed.Apoptosis of chondrocytes was detected by TUNEL staining.The levels of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)in serum of rats were measured by enzyme-linked immunosorbent assay(ELISA).The mRNA levels of B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2-associated X protein(Bax),CollagenⅡ,matrix metalloproteinase-13(MMP-13),Runt-associated transcription factor 2(Runx2)and bone morphogenetic protein-2(BMP-2)in the cartilage were measured by RT-qPCR.The phosphorylation level of AMP-activated protein kinaseα(AMPKα)in the cartilage was measured by Western blot.Results Compared with model group,cartilage injury and degeneration were dose-dependently alleviated in low,medium and high dose groups,OARSI score and chondrocyte TUNEL positive rate were decreased in a dose-dependent manner(P<0.05),the levels of IL-1β,IL-6 and TNF-α in serum were decreased in a dose-dependent manner(P<0.05).The mRNA relative expression levels of Bax and MMP-13 were decreased in a dose-dependent manner(P<0.05),the mRNA relative expression levels of Bcl-2,CollagenⅡ,Runx2 and BMP-2 were increased in a dose-dependent manner(P<0.05),and the relative phosphorylation level of AMPKα was increased in a dose-dependent manner(P<0.05).Compound C attenuated the therapeutic effect of high dose G-Rb1 on PTOA rats(P<0.05).Conclusion G-Rb1 attenuates PTOA in rats by activating AMPK.