X. Wu, Y. Cai, K. Xu, L. Yang, P. Xu, Department of Joint Surgery, Xi'an Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, China S. Lu, Department of Genetics and Molecular Biology, Xi'an Jiaotong University Health Science Center, Xi'an, China X. Shi, Department of Oncology, Qianfoshan Hospital, Shandong University, Jinan, China Z. Huang, Department of Joint Surgery, Shiquan County Hospital of Traditional Chinese Medicine, Ankang, China.
Clin Orthop Relat Res. 2019 Dec;477(12):2785-2797. doi: 10.1097/CORR.0000000000001016.
Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Studies have found that enhancement of autophagy, an intracellular catabolic process, may limit the pathologic progression of OA. Chloramphenicol is a potent activator of autophagy; however, the effects of chloramphenicol on articular cartilage are unknown.
QUESTIONS/PURPOSES: Using human OA knee chondrocytes in vitro, we asked, does chloramphenicol (1) activate autophagy in chondrocytes; (2) protect chondrocytes from IL-1β-induced apoptosis; and (3) reduce the expression of matrix metallopeptidase (MMP)-13 and IL-6 (markers associated with articular cartilage degradation and joint inflammation). Using an in vivo rabbit model of OA, we asked, does an intra-articular injection of chloramphenicol in the knee (4) induce autophagy; (5) reduce OA severity; and (6) reduce MMP-13 expression?
Human chondrocytes were extracted from 10 men with OA undergoing TKA. After treatment with 25 μg/mL, 50 μg/mL, or 100μg/mL chloramphenicol, the autophagy of chondrocytes was detected with Western blotting, transmission electron microscopy, or an autophagy detection kit. There were four groups in our study: one group was untreated, one was treated with 100 μg/mL chloramphenicol, another was treated with 10 ng/mL of IL-1β, and the final group was treated with 10 ng/mL of IL-1β and 100 μg/mL of chloramphenicol. All groups were treated for 48 hours; cell apoptosis was detected with Western blotting and flow cytometry. Inflammation marker IL-6 in the cell culture supernatant was detected with an ELISA. Articular cartilage degradation-related enzyme MMP-13 was analyzed with Western blotting. A rabbit model of OA was induced by intra-articular injection of type II collagenase in 20 male 3-month-old New Zealand White rabbits' right hind leg knees; the left hind leg knees served as controls. Rabbits were treated by intra-articular injection of saline or chloramphenicol once a week for 8 weeks. Autophagy of the articular cartilage was detected with Western blotting and transmission electron microscopy. Degeneration of articular cartilage was analyzed with Safranin O-fast green staining and the semi-quantitative index Osteoarthritis Research Society International (OARSI) grading system. Degeneration of articular cartilage was evaluated using the OARSI grading system. The expression of MMP-13 in articular cartilage was detected with immunohistochemistry.
Chloramphenicol activated autophagy in vitro in the chondrocytes of humans with OA and in an in vivo rabbit model of OA. Chloramphenicol inhibited IL-1-induced apoptosis (flow cytometry results with chloramphenicol, 25.33 ± 3.51%, and without chloramphenicol, 44.00 ± 3.61%, mean difference, 18.67% [95% CI 10.60 to 26.73]; p = 0.003) and the production of proinflammatory cytokine IL-6 (ELISA results, with chloramphenicol, 720.00 ± 96.44 pg/mL, without chloramphenicol, 966.67 ± 85.05 pg/mL; mean difference 74.24 pg/mL [95% CI 39.28 to 454.06]; p = 0.029) in chondrocytes. After chloramphenicol treatment, the severity of cartilage degradation was reduced in the treatment group (OARSI 6.80 ± 2.71) compared with the control group (12.30 ± 2.77), (mean difference 5.50 [95% CI 1.50 to 9.50]; p = 0.013). Furthermore, chloramphenicol treatment also decreased the production of MMP-13 in vitro and in vivo.
Chloramphenicol reduced the severity of cartilage degradation in a type II collagen-induced rabbit model of OA, which may be related to induction of autophagy and inhibition of MMP-13 and IL-6.
Our study suggests that an intra-articular injection of chloramphenicol may reduce degeneration of articular cartilage and that induction of autophagy may be a method for treating OA. The animal model we used was type II collagen-induced OA, which was different from idiopathic OA and post-traumatic OA. Therefore, we need to use other types of OA models (idiopathic OA or a surgically induced OA model) to further verify its effect, and the side effects of chloramphenicol also need to be considered, such as myelosuppression.
骨关节炎（OA）的特征在于关节软骨的退化。研究发现，增强自噬（一种细胞内分解代谢过程）可能限制 OA 的病理进展。氯霉素是一种有效的自噬激活剂;然而，氯霉素对关节软骨的影响尚不清楚。
问题/目的：我们在体外使用人 OA 膝关节软骨细胞，提出以下问题：（1）氯霉素是否激活软骨细胞中的自噬；（2）氯霉素是否保护软骨细胞免受 IL-1β诱导的凋亡；（3）减少基质金属蛋白酶（MMP）-13 和白细胞介素（IL）-6 的表达（与关节软骨降解和关节炎症相关的标志物）。使用兔 OA 模型，我们提出，关节内注射氯霉素（4）是否诱导自噬；（5）减轻 OA 严重程度；（6）减少 MMP-13 表达？
从 10 名接受 TKA 的 OA 男性患者中提取人软骨细胞。用 25μg/mL、50μg/mL 或 100μg/mL 氯霉素处理后，通过 Western blot、透射电镜或自噬检测试剂盒检测软骨细胞的自噬。研究中有四组：一组未处理，一组用 100μg/mL 氯霉素处理，另一组用 10ng/mL IL-1β处理，最后一组用 10ng/mL IL-1β和 100μg/mL 氯霉素处理。所有组均处理 48 小时；用 Western blot 和流式细胞术检测细胞凋亡。细胞培养上清液中的炎症标志物 IL-6 用 ELISA 检测。用 Western blot 分析与关节软骨降解相关的酶 MMP-13。通过向 20 只 3 个月大的新西兰白兔右后腿膝关节内注射 II 型胶原酶诱导 OA 兔模型，左后腿膝关节作为对照。每周一次向膝关节内注射生理盐水或氯霉素 8 周。通过 Western blot 和透射电镜检测关节软骨的自噬。用 Safranin O-fast green 染色和骨关节炎研究协会国际（OARSI）分级系统分析关节软骨退变。用 OARSI 分级系统评估关节软骨退变。用免疫组织化学检测关节软骨中 MMP-13 的表达。
氯霉素在 OA 患者的软骨细胞和体内兔 OA 模型中均能激活自噬。氯霉素抑制 IL-1 诱导的凋亡（用氯霉素处理的流式细胞术结果为 25.33±3.51%，无氯霉素处理的为 44.00±3.61%，平均差异 18.67%[95%CI 10.60 至 26.73]；p=0.003）和促炎细胞因子 IL-6 的产生（ELISA 结果，用氯霉素处理的为 720.00±96.44pg/mL，无氯霉素处理的为 966.67±85.05pg/mL；平均差异 74.24pg/mL[95%CI 39.28 至 454.06]；p=0.029）。在用氯霉素处理后，治疗组的软骨降解严重程度较对照组（12.30±2.77）降低（OARSI 6.80±2.71）（平均差异 5.50[95%CI 1.50 至 9.50]；p=0.013）。此外，氯霉素处理还减少了 MMP-13 的体外和体内产生。
在 II 型胶原诱导的兔 OA 模型中，氯霉素降低了软骨降解的严重程度，这可能与诱导自噬和抑制 MMP-13 和 IL-6 有关。
我们的研究表明，关节内注射氯霉素可能会减轻关节软骨的退化，而诱导自噬可能是治疗 OA 的一种方法。我们使用的动物模型是 II 型胶原诱导的 OA，与特发性 OA 和创伤后 OA 不同。因此，我们需要使用其他类型的 OA 模型（特发性 OA 或手术诱导的 OA 模型）进一步验证其效果，并且还需要考虑氯霉素的副作用，如骨髓抑制。
