miR-34a基于AKT/mTOR通路对骨关节炎大鼠的影响及作用机制研究

目的　探究微小核糖核酸-34a（microRNA-34A，miR-34a）基于蛋白激酶B（AKT）/哺乳动物雷帕霉素靶蛋白（mTOR）通路对骨关节炎大鼠的影响及作用机制。方法　研究时间为2020年2月至2021年5月，选取43只6～9周龄健康雄性SD大鼠作前瞻性研究，其中随机选取10只分为空白组，剩余33只建立骨关节炎模型。成功建模30只，随机分为模型组、上调miR-34a组、沉默miR-34a组，各10只。观察病理组织，实时荧光定量PCR（real-time PCR，RT-PCR）法检测miR-34a表达，酶联免疫吸附实验法检测白细胞介素（interleukin，IL）-1β、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、IL-4、IL-10，蛋白免疫印迹法检测磷酸化蛋白激酶B（phosphorylated protein kinase B，pAKT）、磷酸化哺乳动物雷帕霉素靶蛋白（phosphorylated mammalian target of rapamycin，pmTOR）表达。其中多组间比较行F检验，两组间比较行独立样本t检验。结果　空白组、模型组、上调miR-34a组、沉默miR-34a组的miR-34a表达分别为（0.94±0.08）、（2.39±0.26）、（2.12±0.23）、（1.54±0.17），Makin评分分别为（0.35±0.05）、（4.13±0.19）、（3.02±0.16）、（1.15±0.09）分，IL-1β分别为（3.68±0.37）、（7.51±1.23）、（5.72±0.95）、（4.63±0.72）ng/L，TNF-α分别为（37.45±4.85）、（62.05±7.49）、（56.26±6.25）、（43.85±5.10）ng/L，IL-4分别为（115.75±16.81）、（50.43±9.42）、（75.82±11.94）、（93.85±13.86）pg/ml，IL-10分别为（106.49±17.51）、（60.85±10.23）、（76.81±13.07）、（91.05±15.02）pg/ml，pAKT分别为（1.00±0.01）、（1.95±0.29）、（1.71±0.26）、（1.71±0.26），pmTOR分别为（1.00±0.01）、（1.89±0.24）、（1.56±0.25）、（1.25±0.15）。与空白组相比，模型组、上调miR-34a组、沉默miR-34a组的miR-34a表达、Makin评分、IL-1β、TNF-α、pAKT、pmTOR较高，IL-4、IL-10较低，差异均有统计学意义（F/P值分别为25.290/0.001、91.260/0.001、14.144/0.001、13.077/0.001、15.525/0.001、17.580/0.001、16.080/0.001、10.676/0.001）；与模型组相比，上调miR-34a组、沉默miR-34a组miR-34a表达、Makin评分、IL-1β、TNF-α、pAKT、pmTOR较低，IL-4、IL-10较高，差异均有统计学意义（均P<0.05）；沉默miR-34a组与上调miR-34a组相比，沉默miR-34a组miR-34a表达、Makin评分、IL-1β、TNF-α、pAKT、pmTOR较低，IL-4、IL-10较高，差异均有统计学意义（均P<0.05）。结论　沉默miR-34a对骨关节炎大鼠有干预作用，减少大鼠出现炎性反应，降低Makin评分，其作用机制与AKT/mTOR信号通路有关。

Effect of miR-34a based on AKT/mTOR pathway on osteoarthritis rats and its mechanism

Objective　To explore the effect of microrNA-34A (miR-34a) on osteoarthritis in rats based on the AKT/mTOR pathway and its mechanism. Methods　The study was form February 2020 to May 2021. In this study, 43 healthy male SD rats were selected for prospective study, and they were 6-9 weeks old. Among them, 10 rats were randomly selected as a blank group, and The remaining 33 rats were established as the osteoarthritis models. The 30 rats successfully modeled were randomly divided into a model group, an up-regulated miR-34a group, and a silenced miR-34a group, with 10 rats in each group. The expression of miR-34a was detected by real-time polymerase chain reaction (RT-PCR), and The levels of interleukin-1β (Il-1β), tumor necrosis factor-α, TNF-α, interleukin-4 (IL-4), and interleukin-10 (IL-10) was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of phosphorylated protein kinase B (pAKT) and phosphorylated mammalian target of rapamycin (pmTOR) were detected by Western blot. F test was used for the comparison between ≥3 groups, and independent-sample t test was used for the comparison between two groups. Results　The expressions of miR-34a in the blank group, the model group, the up-regulated miR-34a group, and the silenced miR-34a group were (0.94±0.08), (2.39±0.26), (2.12±0.23), and (1.54±0.17), rthe Makin scores (0.35±0.05), (4.13±0.19), (3.02±0.16), and (1.15±0.09), the IL-1β levels (3.68±0.37), (7.51±1.23), (5.72±0.95), and (4.63±0.72) ng/L, the TNF-α levels (37.45±4.85), (62.05±7.49), (56.26±6.25), and (43.85±5.10) ng/L, the IL-4 levels (115.75±16.81), (50.43±9.42), (75.82±11.94), and (93.85±13.86) pg/ml, the IL-10 levels (106.49±17.51), (60.85±10.23), (76.81±13.07), and (91.05±15.02) pg/ml, the pAKT (1.00±0.01), (1.95±0.29), (1.71±0.26), and (1.71±0.26), and the pmTOR (1.00±0.01), (1.89±0.24), (1.56±0.25), and (1.25±0.15). Compared with those in the blank group, the expression of miR-34a, Makin score, IL-1β, TNF-α, pAKT and pmTOR were higher in than those in the model group, the up-regulated miR-34a group, and the silenced miR-34a group, while IL-4 and IL-10 were lower those in the model group (F=25.290, P<0.001; F=91.260, P<0.001; F=14.144, P<0.001; F=13.077, P<0.001; F=15.525, P<0.001; F=17.580, P<0.001; F=16.080, P<0.001; and F=10.676, P<0.001). Compared with those in the model group, the expression of miR-34a, Makin score, IL-1β, TNF-α, pAKT, and pmTOR were lower than those in the up-regulated miR-34a group and the silenced miR-34a group, while IL-4 and IL-10 were higher, with statistical differences. Compared with those in the up-regulated miR-34a group, the expression of miR-34a, Makin score, IL-1β, TNF-α, pAKT, and pmTOR were lower than those in the miR-34a silencing group, while IL-4 and IL-10 were higher than those in the miR-34a silencing group, with statistical differences (all P<0.05). Conclusion　Silencing of miR-34a has an intervention effect in osteoarthritis rats, and reduces the inflammatory response and the Makin score, and the mechanism of action is related to the AKT / mTOR signaling pathway.