環境汙染物誘發軟骨細胞衰老且增強關節軟骨老化:以三氧化二砷和三丁基錫為例

Abstract

隨著醫療文化的提高，全球老年人口逐漸增加。其中亞洲老齡化社會正在快速增長。年齡的增長與骨關節炎的患病率之間存在關聯性。先前的流行病學研究表明女性相比於男性更容易患上更嚴重的膝關節關節炎。雖然關節軟骨會隨著年齡的增長而退化，但環境污染物(如金屬)接觸也可能是影響因素之一。一些研究表明接觸金屬會增加骨關節炎的風險，這可能是由於細胞衰老的介入。其中，砷(Arsenic, As)暴露會促進人類間充質乾細胞細胞衰老，並且在關節炎患者血清中發現含有砷的存在。在關節炎發展中，三丁基錫(Tributyltin, TBT)暴露會促進免疫衰老微環境，並且可以在人類的血液和組織中找到TBT。然而，As和TBT暴露對軟骨細胞的毒理作用和調控機制仍有待釐清。在本研究中，我們研究衰老和衰老相關分泌因子的影響，並分別研究了As (1-5 μM)和TBT (0.01-0.5 μM)暴露於人類關節軟骨細胞。As和TBT暴露軟骨細胞24小時顯著增加與衰老相關的beta-半乳糖苷酶活性和衰老標誌物p16、p53和p21的蛋白表達。As誘導p38 mitogen-activated protein kinases (p38)和c-Jun N-terminal kinase (JNK)蛋白磷酸化，並通過p38和JNK抑製劑逆轉軟骨細胞衰老。TBT誘導ataxia telangiectasia mutated kinase (ATM)蛋白磷酸化，並通過ATM抑製劑抑制γH2AX、p-p38、p-JNK、p16、p53和p21蛋白表達。再進一步在軟骨細胞中As和TBT顯著刺激衰老相關分泌表型(senescence-associated secretory phenotype, SASP)相關的mRNA基因表達，包括IL-1α/β、TGF-β、TNF-α、ICAM-1、CCL2、PAI-1和MMP-13，以及NF-κB-p65的磷酸化和GATA4的蛋白表達。同樣地，我們還觀察到在動物模式中給予含As (0.05和0.5 ppm)飲用水9個月或TBT (5和25 μg/kg)管餵處理28天，皆會導致關節軟骨老化和磨損。並且衰老相關蛋白和SASP相關蛋白在老鼠軟骨中的表達增強。總而言之，這些結果皆表明As和TBT的暴露可以在體外模式下引起人類關節軟骨細胞衰老，並且在體內模式下增強小鼠關節軟骨的老化和磨損。

With the improvement in medical culture, the global elderly population has gradually increased. Among them, Asia's aging society is growing rapidly. There is a conjunction between the increasing age and the prevalence of osteoarthritis (OA). The previous epidemiological study have displayed that women are more prone to develop more severe knee osteoarthritis compared to men. Although the articular cartilage degeneration with age, contact with environmental pollutants (such as metals) may also be one of the effects. Some studies have indicated that exposure to metals assisted OA risk, and that was possibly mediated by senescence. Arsenic exposure has been proposed to advance senescence in human mesenchymal stem cells and arsenic (As) levels in the serum of patients with arthritis have been found. Tributyltin (TBT) exposure has promoted the immunosenescence microenvironment in arthritis development and can be found in the blood and tissues of the mankind. However, the toxicological effects and mechanisms of As and TBT exposure on chondrocytes still remain to be clarified. In this study, we researched the effects of senescence and senescence-associated secretory factors and cultured human chondrocytes-articular cells exposed to As (1-5 μM) and TBT (0.01-0.5 μM), individually. As and TBT for 24 hours significantly increased the senescence-related beta-galactosidase activity and the protein expression levels of senescence markers p16, p53, and p21 in chondrocytes. As increased the phosphorylation of p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) proteins, and reversed chondrocyte senescence by p38 and JNK inhibitors. TBT induced the phosphorylation of ataxia telangiectasia mutated kinase (ATM) proteins and inhibited the protein expression levels of γH2AX, p-p38, p-JNK, p16, p53, and p21 by ATM inhibitor. Next, As and TBT significantly stimulated the mRNA expression of senescence-related secretory phenotype (SASP)-related factors, including IL-1α/β, TNF-α, TGF-β, ICAM-1, PAI-1, CCL2, and MMP-13, and the protein expression of phosphorylated NF-κB-p65 and GATA4 in chondrocytes. Similarly, we also observed that administration of As (0.05 and 0.5 ppm) drinking water for 9 months or TBT (5 and 25 μg/kg) gavage for 28 days resulted in aging and wear of articular cartilage in mouse models. The expression of senescence-associated protein and SASP-related protein were strengthened in the mouse cartilages. Taken together, these results suggest that As and TBT exposure can trigger human articular chondrocytes senescence in vitro and enhance mouse articular cartilage abrasion and aging in vivo.