基于磷脂酶Cγ的自噬/凋亡拮抗调节对骨性关节炎软骨基质稳态的调控

Osteoarthritis (OA) is a chronic degenerative joint disease. It is characterized by a loss of chondrocyte and degradation of cartilage matrix, resulting in severe pain and physical disabilities to millions of people worldwide. Elucidating the regulatory mechanisms of cartilage matrix synthesis and degradation is one of the effective strategies for preventing cartilage damage and promoting repair. The chondrocyte is the sole cell type in cartilage and is strongly involved in maintaining the dynamic equilibrium between synthesis and degradation of the extracellular matrix. Roach et al. proposed the term ‘‘Chondroptosis’’ to suggest the type of cell death present in articular cartilage, which includes some elements of classical apoptosis and autophagy. Afterwards, Almonte-Becerril et al. reported that cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of osteoarthritis within an experimental model. The study of Caramés et al. showed that autophagy activation by rapamycin reduces severity of experimental osteoarthritis. López de Figueroa et al. reported that autophagy activation protects from mitochondrial dysfunction in human chondrocytes. These studies indicate there is an antagonism between autophagy and apoptosis in OA progression, and promoting autophagy /inhibiting apoptosis is effective for OA prevention and therapy. Our previous results that PLCγ was involved in OA progression through triggering mTOR signal pathway (crucial signal in autophagy regulation) to promote matrix degradation, indicating the involvement of PLCγ in autophagy. Meanwhile, there is a relationship between PLCγ and BCL2 in EBSS-induced autophagy, indicating PLCγ is involved in apoptosis by BCL2. These results showed that PLCγ could be involved in the regulation of autophagy and apoptosis simultaneously, in which mTOR and BCL2 are the crucial joints. However, the regulatory mechanism of PLCγ on the antagonism between autophagy and apoptosis is not clear. Understanding the role of PLCγ in regulating the antagonism between chondrocyte autophagy and apoptosis, and the maintenance of matrix homeostasis is beneficial to treat OA. Therefore, we plan to investigate the regulatory mechanism of PLCγ in rat IL-1β-treated OA chondrocytes and human OA chondrocytes in order to confirm that PLCγ could block autophagy, promote apoptosis, and disturb the homeostasis of matrix, in which mTOR and BCL2 are crucial. Meanwhile, the above results were detected in rat OA model using ACLT+MMx, and effective drugs and preparations to prevent and treat OA were screened by targeting PLCγ. As a result, elucidating the regulatory mechanism of PLCγ in the antagonism between autophagy and apoptosis, and the matrix homeostasis maintenance is beneficial to treat OA and screen the drugs and preparations for OA prevention and therapy.

软骨细胞凋亡是传统意义的凋亡与自噬的结合，自噬/凋亡间的拮抗作用贯穿OA病理进程。阐明调节自噬与凋亡间拮抗作用的信号分子机制，促使信号通路向自噬倾斜、减弱凋亡、可维持软骨基质稳态、减缓OA软骨损伤，有效防治OA。前期结果显示PLCγ可能通过mTOR参入自噬调节；自噬诱导过程中PLCγ与BCL2存在关联，提示PLCγ具备成为自噬/凋亡共同调控信号分子的基础。推测：PLCγ通过mTOR、BCL2两节点调节自噬/凋亡间拮抗作用，进而干扰OA软骨基质稳态维持。拟培养大鼠模拟OA软骨细胞与人OA软骨细胞，探讨基于PLCγ的自噬/凋亡拮抗调节对基质稳态的调控作用、确证BCL2与mTOR是其关键节点；建立大鼠OA模型，以PLCγ为靶点筛选活性物质调节自噬/凋亡比率、观察软骨改变情况，验证上述推测。从而阐明PLCγ调节自噬/凋亡间的拮抗作用对OA软骨基质稳态维持的意义，并为以PLCγ为靶点筛选药物提供依据

软骨细胞凋亡是传统意义的凋亡与自噬的结合，自噬/凋亡间的拮抗作用贯穿OA病理进程。阐明调节自噬与凋亡间拮抗作用的信号分子机制，促使信号通路向自噬倾斜、减弱凋亡、可维持软骨基质稳态、减缓OA软骨损伤，有效防治OA。我们的前期结果提示PLCγ具备成为自噬/凋亡共同调控信号分子的基础。我们推测：PLCγ通过mTOR、BCL2两节点调节自噬/凋亡间拮抗作用，进而干扰OA软骨基质稳态维持。为此，开展了以下几方面的研究：（1）检测PLCγ是否通过调节细胞自噬/凋亡间拮抗作用参与OA软骨基质稳态的调控。（2）构建大鼠OA模型，验证体外细胞实验结果；以PLCγ为靶点筛选促进自噬、抑制凋亡，进而维持基质稳态的活性物质。（3）体外培养处于OA不同病理阶段的人OA软骨细胞，检测筛选的活动物质对软骨基质稳态维持的功效。获得的主要研究成果（1）PLCγ1 活性参与软骨细胞的基质代谢调控，其抑制作用能够促进IL-1β处理的人OA软骨细胞与大鼠软骨细胞的基质合成(95%CI, 0.27-1.46 or 0.07-0.73(Collagen II), 0.22-1.09 or 0.18-1.04(Aggrecan))。（2）PLCγ1的抑制作用通过增强IL-1β处理的人OA软骨细胞与大鼠软骨细胞内的自噬流而促进了细胞外基质合成(95%CI, 0.06-0.61 or 0.18-0.74 (LC3BII/I), 0.38-2.42 or 0.66-1.78(P62)。（3）在IL-1β处理的大鼠软骨细胞（模拟OA状态）中，PLCγ1抑制作用通过促进Beclin1 从Beclin1-IP3R-Bcl-2复合物上解离、阻断mTOR/ULK1 信号促进自噬流的增强。（4）PLCγ1, Erk,与Akt 参与PLCγ1抑制作用诱导的自噬活动。（5）PLCγ1抑制作用诱导自噬的同时也通过提高ROS导致细胞的死亡。细胞死亡方式中caspase-3依赖的细胞凋亡占较大比例，细胞坏死也占一定比例，同时存在着自噬细胞死亡（Autophagic cell death）。（6）PLCγ1抑制作用通过促进自噬提高减缓3D培养的软骨球内的基质合成。（7）Ad-shPLCγ1注射到关节腔抑制软骨细胞内PLCγ1，进而增强细胞自噬流，促进细胞外基质合成，延缓大鼠OA模型的软骨损伤。研究结果为以PLCγ1为靶点预防OA提供了依据。