柚皮苷诱导自噬对骨关节炎模型大鼠炎性因子的影响

Effect of Naringin-induced autophagy on inflammatory factors in a rat model of osteoarthritis

目的探讨柚皮苷诱导自噬对骨关节炎模型大鼠血清和关节软骨中炎性因子表达的影响。方法在2022年11—12月,由温州市人民医院骨科将30只健康Sprague-Dawley大鼠通过随机数字表法分为对照组、模型组及低、中、高剂量实验组各6只。切除模型组及低、中、高剂量实验组大鼠右膝关节内侧半月板和前交叉韧带,构建骨关节炎模型;对照组施行假手术。低、中、高剂量实验组建模期间分别灌胃25 mg·kg^(-1)·d^(-1)、50 mg·kg^(-1)·d^(-1)、100 mg·kg^(-1)·d^(-1)柚皮苷,对照组与模型组则灌胃等量0.9%氯化钠注射液。造模成功后通过国际骨关节炎研究学会(OARSI)评分和滑膜评分评价骨关节炎严重程度;以酶联免疫吸附试验(ELISA)法检测大鼠血清和软骨细胞中白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)水平;反转录-聚合酶链式反应(RT-PCR)法检测软骨细胞中IL-1β、IL-6和TNF-αmRNA的表达;以蛋白质印迹(WB)法检测软骨细胞中自噬活性和AKT/mTOR信号通路变化。结果对照组、模型组及低、中、高剂量实验组大鼠OARSI评分分别为(0.80±0.75)分、(9.40±1.02)分、(8.20±1.33)分、(6.80±1.17)分和(4.60±1.50)分;滑膜评分分别为(0.40±0.49)分、(3.80±0.75)分、(3.40±0.49)分、(2.20±0.98)分和(1.60±0.80)分;大鼠血清中IL-1β水平分别为(186.48±50.31)ng/L、(817.43±66.99)ng/L、(533.42±45.67)ng/L、(462.90±21.43)ng/L和(396.64±24.66)ng/L;IL-6水平分别为(448.25±89.42)ng/L、(1762.49±171.95)ng/L、(1517.08±83.87)ng/L、(1019.78±103.32)ng/L和(819.42±169.37)ng/L;TNF-α水平分别为(419.67±60.99)ng/L、(1287.40±184.68)ng/L、(948.73±87.82)ng/L、(860.55±102.21)ng/L和(726.95±15.65)ng/L;模型组OARSI评分、滑膜评分、血清IL-1β、IL-6、TNF-α表达水平及软骨细胞磷酸化AKT(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)蛋白表达量比对照组高,高剂量实验组OARSI评分、滑膜评分、血清IL-1β、IL-6、TNF-α含量以及软骨细胞p62、p-AKT、p-mTOR蛋白表达量比模型组低,高剂量实验组软骨细胞LC3-Ⅱ蛋白表达量比模型组高,差异均有统计学意义(均P<0.05)。结论柚皮苷通过抑制AKT/mTOR信号通路,激活自噬,降低骨关节炎模型大鼠炎性因子分泌,最终抑制骨关节炎。

Objective To investigate the effect of Naringin-induced autophagy on the expression of inflammatory factors in serum and chondrocytes in a rat model of osteoarthritis.Methods From November 2022 to December 2022,30 healthy Sprague-Dawley rats were randomly divided into a control group,a model group,and low-dose,medium-dose,and high-dose experimental groups,with six rats in each group.The model group and the low-dose,medium-dose,and high-dose experimental groups underwent resection of the medial meniscus and anterior cruciate ligament of the right knee to establish a rat model of osteoarthritis.The control group underwent a sham operation.During the modeling period,the low-dose,medium-dose,and high-dose experimental groups were administered Naringin at 25,50,and 100 mg/kg per day,respectively,while the control group and model group received an equal volume of 0.9%sodium chloride injection.After successful modeling,the Osteoarthritis Research Society International(OARSI)score and synovial score were used to assess the severity of osteoarthritis.The levels of interleukin(IL)-1β,IL-6,and tumor necrosis factor(TNF)-αin serum and chondrocytes were determined using an enzyme-linked immunosorbent assay.The mRNA expressions of IL-1β,IL-6,and TNF-αin chondrocytes were detected by reverse transcription-polymerase chain reaction.The changes in autophagy activity and the AKT/mTOR signaling pathway in chondrocytes were detected by western blotting.Results The OARSI scores for the control,model,and low-dose,medium-dose,and high-dose experimental groups were(0.80±0.75)points,(9.40±1.02)points,(8.20±1.33)points,(6.80±1.17)points,and(4.60±1.50)points,respectively.The synovial scores for these groups were(0.40±0.49)points,(3.80±0.75)points,(3.40±0.49)points,(2.20±0.98)points,and(1.60±0.80)points,respectively.The serum levels of IL-1βin these groups were(186.48±50.31)ng/L,(817.43±66.99)ng/L,(533.42±45.67)ng/L,(462.90±21.43)ng/L,and(396.64±24.66)ng/L,respectively.IL-6 levels in these groups were(448.25±89.42)ng/L,(1762.49±171.95)ng/L,(1517.08±83.87)ng/L,(1019.78±103.32)ng/L,and(819.42±169.37)ng/L,respectively.The TNF-αlevels in these groups were(419.67±60.99)ng/L,(1287.40±184.68)ng/L,(948.73±87.82)ng/L,(860.55±102.21)ng/L,and(726.95±15.65)ng/L,respectively.The OARSI scores,synovial scores,serum levels of IL-1β,IL-6,and TNF-α,as well as the protein expressions of phosphorylated AKT(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)in chondrocytes,were significantly higher in the model group compared with the control group(all P<0.05).In contrast,the OARSI scores,synovial scores,serum levels of IL-1β,IL-6,and TNF-α,as well as the protein expressions of p62,p-Akt,and p-mTOR in chondrocytes,were significantly lower in the high-dose experimental group compared with the model group(all P<0.05).However,the protein expression of LC3-II in chondrocytes was significantly higher in the high-dose experimental group compared with the model group(P<0.05).Conclusion Naringin can inhibit osteoarthritis in a rat model of osteoarthritis by inhibiting the Akt/mTOR signaling pathway,activating autophagy,and reducing the secretion of inflammatory factors.