Suppressing phosphoinositide-specific phospholipases Cγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.
Suppressing phosphoinositide-specific phospholipases Cγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.
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DOI：
10.1016/j.bone.2021.116262
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发表时间：
2021-11
期刊：
影响因子：
4.1
通讯作者：
Yue Wang;Tongen Zhang;Yang Xu;R. Chen;Ning Qu;Bing Zhang;Chun Xia
中科院分区：
文献类型：
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作者：
Yue Wang;Tongen Zhang;Yang Xu;R. Chen;Ning Qu;Bing Zhang;Chun Xia
磷脂酰肌醇特异性磷脂酶C-γ1（PLC-γ1）信号通路在骨关节炎（OA）软骨细胞代谢中起重要作用。然而，PLC-γ1在OA成骨细胞中的作用尚不清楚。方法分离人和大鼠的软骨下骨和颅骨，原代培养人和大鼠的非骨关节炎和骨关节炎成骨细胞，以及小鼠前成骨细胞系MC 3 T3-E1细胞。采用前交叉韧带切断术（ACLT），用微型电钻从股骨外上髁表面凿出骨道，建立大鼠膝关节OA模型。分别采用CT、micro-CT和番红O/Fast绿色染色评价软骨下骨结构和关节软骨的形态学特征。茜素红染色法测定矿化程度。分别通过qPCR、蛋白质印迹和免疫组织化学测定评价成骨细胞表型和矿化相关基因的表达和产生。结果ACLT大鼠模型软骨下骨相对骨密度和骨厚度在OA早期降低，在OA晚期增加。在ACLT后2周大鼠模型分离的体外成骨细胞中观察到ALP和OCN水平降低以及ARS含量的吸光度值，以及IL-1β处理（用于维持和模拟炎症状态）的人OA和大鼠成骨细胞。在OA或IL-1β处理的成骨细胞中，Atg 7水平和LC 3BII/I比值降低以及P62水平升高伴随ALP和OCN mRNA水平降低以及ARS含量吸光度值降低。ShRNA或抑制剂（U 73122）特异性抑制PLC-γ1可提高OA成骨细胞ALP和OCN mRNA水平及ARS含量的吸光度值，同时增加Atg 7水平和LC 3BII/I比值，并降低P62水平。抑制PLC-γ1对ALP和OCN mRNA水平和ARS含量的促进作用可被内质网应激激活剂HA 15以及自噬抑制剂CQ和3 MA逆转。从股骨外上髁表面注射PLC-γ1抑制剂U 73122可减少ACLT大鼠软骨下骨的异常形成，减轻关节软骨退变。结论OA软骨下骨结构的异常改变伴随着成骨细胞表型和矿化的改变。骨关节炎早期自噬受损导致成骨细胞矿化减少。PLC-γ1抑制通过增加OA成骨细胞的自噬促进成骨细胞矿化，这部分归因于ER应激的抑制。针对软骨下骨成骨细胞中的PLC-γ1可能通过同时治疗骨和软骨而更有效地治疗OA。总之，我们假设抑制PLCγ1通过增加自噬促进骨关节炎软骨下骨成骨细胞的矿化，从而改善关节软骨退变。
IntroductionPhosphoinositide-specific phospholipases C-γ1 (PLC-γ1) signaling has been shown to modulate osteoarthritis (OA) chondrocyte metabolism. However, the role of PLC-γ1 in OA osteoblasts remains unclear. Herein, whether and how PLC-γ1 was involved in mineralization in OA subchondral bone osteoblasts were investigated.MethodsPrimary non-OA and OA osteoblasts of human and rat isolated from the subchondral bone or the calvaria were culturedin vitro, as well as mouse pre-osteoblastic cell line MC3T3-E1 cells. Rat knee OA model was induced by anterior cruciate ligament transection (ACLT), in which bone canal was carried out from the surface of lateral epicondyle of femur using micro-electric drill. Morphological characteristics of subchondral bone structure and articular cartilage were assessed using CT, micro-CT, and Safranin O/Fast green staining, respectively. Mineralization was measured by alizarin red staining. The expression and production of genes involved in osteoblastic phenotype and mineralization were evaluated by qPCR, western blotting, and immunohistochemistry assays, respectively. The inhibitions were performed using inhibitors and ShRNAs.ResultsThe decreased relative bone density and thickness in the early stage of OA and the increased one in the late stage of OA were observed in subchondral bone of ACLT-rat model. Decreased ALP and OCN levels and absorbance values of ARS content were observed inin vitroosteoblasts isolated from 2 w post-ACLT rat model, as well as IL-1β-treated (for maintaining and mimicking inflammatory status) human OA and rat osteoblasts. Decreased Atg7 level and LC3BII/I ratio in combination with an increase in the P62 level, was concomitant with decreased ALP and OCN mRNA levels and absorbance values of ARS content in OA or IL-1β-treated osteoblasts. Specific inhibition of PLC-γ1 by ShRNAs or inhibitor (U73122) elevated ALP and OCN mRNA levels and absorbance values of ARS content accompanied with increased Atg7 level and LC3BII/I ratio in combination with a decrease in the P62 level in OA osteoblasts. Furthermore, the promoting effect of PLC-γ1 inhibition on ALP and OCN mRNA levels and absorbance values of ARS content was reversed by endoplasmic reticulum (ER) stress activator HA15, as well as autophagic inhibitors CQ and 3MA. Injection with PLC-γ1 inhibitor U73122 from the surface of lateral epicondyle of femur reduced aberrant subchondral bone formation and attenuated articular cartilage degeneration in ACLT-rat.ConclusionAberrant changes of OA subchondral bone structure were concomitant with altered osteoblastic phenotype and mineralization. Impaired autophagy contributed to decreased osteoblastic mineralization in the early stage of OA. PLC-γ1 inhibition promoted osteoblastic mineralization through increasing autophagy in OA osteoblasts, which was partially attributed to suppression of ER stress. Targeting PLC-γ1 in subchondral bone osteoblasts could be more efficacious for OA therapy through treating the bone and cartilage at the same time. In summary, we hypothesize that suppressing PLCγ1 promotes mineralization of osteoarthritic subchondral bone osteoblastsviaincreasing autophagy, thereby ameliorating articular cartilage degeneration.