新mTOR抑制剂洋椿苦素对骨关节炎的保护作用及机制研究

Effect and mechanism of novel mTOR inhibitor cedrelone for osteoarthritis treatment

目的:探讨洋椿苦素对骨关节炎的治疗效果及其作用机制。方法:使用软骨细胞系C28/I2作为体外研究对象,采用MTT法检测洋椿苦素的细胞毒性,使用Western blot和免疫荧光检测洋椿苦素对哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)的抑制作用和对自噬的激活效果;选用C57BL/6J小鼠(n=40)建立右膝骨关节炎模型,同只小鼠的左膝作为正常对照,经腹腔分别给以洋椿苦素(n=20)和生理盐水(n=20)治疗后,利用Western blot、免疫组化、免疫荧光、番红O染色、HE染色、软骨损伤半定量评分系统及炎症分级系统,检测洋椿苦素对mTOR的抑制效果、自噬的激活情况、对软骨的保护和滑膜炎症抑制的效果。结果:(1)洋椿苦素可抑制C28/I2细胞mTOR的Ser-2448磷酸化位点,同时抑制核糖体S6蛋白激酶磷酸化(ribosomal protein S6,rpS6)的Thr-389和Ser-371位点,其最佳剂量为40μmol/L(Ser-2448:t=13.42,P=0.000;Thr-389:t=19.06,P=0.000;Ser-371:t=9.52,P=0.000),最佳作用时间为24 h(Ser-2448:t=34.82,P=0.000;Thr-389:t=3.14,P=0.007;Ser-371:t=19.32,P=0.000);(2)洋椿苦素可促进C28/I2细胞LC3II/LC3I的比值(t=8.15,P=0.002),并增加自噬小体阳性细胞的数量(t=9.71,P=0.000);(3)在骨关节炎中,洋椿苦素可抑制mTOR的磷酸化水平(t=19.03,P=0.005)及rpS6表达阳性的细胞数量(t=27.97,P=0.000),并增加LC3II/LC3I表达水平(t=24.65,P=0.000)和LC3表达阳性的细胞数量(t=33.89,P=0.000);(4)洋椿苦素可降低骨关节炎模型中关节软骨的损伤程度(t=8.32,P=0.009),增加软骨细胞的密度(F=3.59,P=0.013),抑制具有I型血小板结合蛋白基序的解聚蛋白样金属蛋白酶5(A disintegrin and metalloproteinase with thrombospondin type I motifs 5,ADAMTS-5)的表达(F=1.82,P=0.002);(5)在骨关节炎模型的滑膜组织中,洋椿苦素可通过对白介素-1β(Interleukine-1 beta,IL-1β)的抑制(F=3.20,P=0.000)从而抑制滑膜组织中的炎症反应(F=5.44,P=0.029)。结论:洋椿苦素作为一种潜在的m TOR抑制剂,在小鼠模型中可通过激活自噬降低关节软骨的损伤并保护软骨细胞,还可通过对ADAMTS-5和IL-1β的抑制实现对细胞外基质的保护和炎症的抑制,但对骨关节炎中软骨的损伤的保护效果及机制还需进一步研究。

Objective：To investigate the effect and mechanism of cedrelone for osteoarthritis treatment. Methods：C28/I2 cells were used to establish the cell model. MTT was used to detect the toxicity of cedrelone. Western blot and immunofluorescence were used to detect the effect of cedrelone on mTOR inhibition and autophagy activation. The right knees of C57BL/6J mice（n=40） were used to establish the osteoarthritis model,and the left knees of the same mice were used as the control;the mice were given cedrelone（n=20） and normal saline（n=20） treatment through intraperitoneal injection. The effects of cedrelone on mTOR inhibition and synovial in-flammation,activation of autophagy and protection of cartilage were detected through Western blot,safranin O stain,immunohisto-chemistry,immunofluorescence,semi-quantitative scoring sys-tem and inflammation grading system. Results：①For the C28/I2 cell,cedrelone could inhibit the mTOR phosphorylation in the site of Ser-2448,and suppress the phosphorylation of ribo-somal protein S6（rpS6） in the site of Thr-389 and Ser-371. The optimum dose was 40 μmol/L（Ser-2448：t=13.42,P=0.000;Thr-389：t=19.06,P=0.000;Ser-371：t=9.52,P=0.000）,and the best time was 24 h（Ser-2448：t=34.82,P=0.000;Thr-389：t=3.14,P=0.007;Ser-371：t=19.32,P=0.000）. ②The value of LC3II/LC3I（t=8.15,P=0.002） and the number of autophagosome positive cells（t=9.71,P=0.000） were significantly increased by cedrelone treatment in C28/I2 cells. ③In the osteoarthritis model,cedrelone could inhibit the phosphorylation level of mTOR（t=19.03,P=0.005） and the number of rpS6 positive cells（t=27.97,P=0.000）,as well as enhance the level of LC3II/LC3I（t=24.65,P=0.000） and the number of LC3 positive cells（t=33.89,P=0.000）. ④Cedrelone also could reduce the degradation of extracellular matrix through ADAMTS-5 in-hibition（F=1.82,P=0.002）,and then reduce the severity of osteoarthritis（t=8.32,P=0.009）,increase the number of chondrocytes（F=3.59,P=0.013）. ⑤In addition,in the synovial tissue,synovial inflammation was suppressed（F=5.44,P=0.029） by cedrelone through IL-1β inhibition（F=3.20,P=0.000）. Conclusion：Cedrelone acts as a potential mTOR inhibitor,not only reducing the severity of osteoarthritis and protection chondrocytes through autophagy activation,but also inhibiting the degradation of extracellular matrix and synovial inflammation by ADAMTS-5 and IL-1β inhibition in mice,however,the protective effect and mechanism of cedrelone for cartilage injury in osteoarthritis need to be further studied.