摘要
目的 探讨炎性微环境牙周膜成纤维细胞(periodontal ligament fibroblasts,PDLFs)整合素α5(integrinα5)对 NOD 样受体热蛋白结构域相关蛋白 3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)表达的影响.方法 获得单位实验动物伦理委员会的批准,以脂多糖(lipopolysaccharide,LPS)(0.5、5、50 μg/mL)预处理大鼠PDLFs 24 h后更换为无血清DMEM培养基,24h后收集上清液分别与含10％无外泌体血清的DMEM培养基按体积比1∶1混合获得条件培养基(conditioned medium,CM),记作0.5-CM、5-CM、50-CM,另将含10％无外泌体血清的DMEM培养基记作0-CM.CM组以上述各浓度的CM培养PDLFs;抑制剂组以含有不同浓度整合素 α5 抑制剂 ATN-161(0、0.025、0.25、2.5、25、250 μg/mL)的 0-CM 培养 PDLFs;CCK-8 法检测上述各组CM和整合素α5抑制剂ATN-161对细胞活性的影响.根据CCK-8结果,在进一步的抑制剂干预实验中,将 PDLFs 培养于 0-CM、不含/含 25 μg/mL ATN-161 的 5-CM 及含 25 μg/mL ATN-161 的 0-CM 中,依次为 0-CM组、5-CM组、ATN-161+5-CM组及ATN-161组.利用Western blot及qRT-PCR检测整合素α5和NLRP3表达变化.体内实验将24只大鼠随机分为4组(n=6),对照组为健康大鼠,不做任何处理,其余3组大鼠每3天于大鼠左上颌第一磨牙腭侧分别注射40 μL的含25 μg/mL ATN-161的0-CM或5-CM(不含/含25 μg/mL ATN-161),记作ATN-161组、5-CM组和ATN-161+5-CM组.第30天取大鼠左上颌骨组织行Micro-CT、HE染色及免疫组化检测.结果 CCK-8检测显示,12h、24h时各组CM和25 μg/mL及以下浓度的ATN-161对细胞活性抑制无显著差异(P＞0.05);48h时50-CM和25、250 μg/mL ATN-161均显著抑制细胞活性(P＜0.05).在体外实验,相较于0-CM组,5-CM组大鼠PDLFs中整合素α5和NLRP3在蛋白和mRNA水平表达均显著升高(P＜0.05);25 μg/mL ATN-161干预显著抑制5-CM培养条件下整合素α5和NLRP3的表达(P＜0.05).在体内实验,与对照组相比,5-CM组和ATN-161+5-CM组牙槽骨吸收明显,牙周膜炎性细胞浸润增多,整合素α5和NLRP3阳性表达显著增加(P＜0.01).但ATN-161+5-CM组较5-CM组牙槽骨吸收较轻,牙周膜炎性细胞浸润较少,整合素α5和NLRP3阳性表达明显降低(P＜0.01).结论 体内外实验表明整合素α5介导炎性微环境下PDLFs的NLRP3表达,ATN-161抑制整合素α5表达从而显著下调NLRP3表达,发挥抑制炎症作用.
Abstract
Objective To investigate the effect of integrin α5 on the expression of NOD-like receptor thermal pro-tein domain associated protein 3(NLRP3)in periodontal ligament fibroblasts(PDLFs)within an inflammatory microenvi-ronment.Methods This study was approved by the Ethics Committee of Laboratory animals.After rat PDLFs were treated with LPS(0.5,5,and 50 μg/mL)for 24 h,the primary medium was discarded and replaced with serum-free cul-ture medium.After 24 h,the supernatant was collected and mixed with DMEM medium containing 10％exosome-free se-rum at a volume ratio of 1∶1 to obtain conditioned medium(CM).The groups were labeled as the 0.5-CM,5-CM,and 50-CM groups.In addition,PDLFs cultured in DMEM medium containing 10％exosome-free serum were considered the 0-CM group.PDLFs were cultured with the above CM.In the inhibitor group,PDLFs were cultured in 0-CM containing differ-ent concentrations of integrin α5 inhibitor ATN-161(0,0.025,0.25,2.5,25,and 250 μg/mL).The effect of CM and inte-grin α5 inhibitor ATN-161 on cell viability was assessed using the CCK-8 assay.According to the CCK-8 results,in fur-ther inhibitor intervention experiments,PDLFs were cultured in 0-CM,5-CM(without/with 25 μg/mL ATN-161),and 0-CM containing 25 μg/mL ATN-161,which were labeled as the 0-CM,5-CM,ATN-161+5-CM,and ATN-161 groups,respec-tively.The expression changes of integrin α5 and NLRP3 were detected using Western blot and qRT-PCR techniques.For in vivo experiments,24 rats were randomly divided into four groups(n=6).The control group contained healthy rats that received no treatment.The rats in the other three groups were injected with 40 μL of 0-CM containing 25 μg/mL ATN-161 or 5-CM(without or with 25 μg/mL ATN-161)on the palatal side of the left maxillary first molar every three days;these groups were classified as the ATN-161,5-CM,and ATN-161+5-CM groups,respectively.On the 30th day,the left maxillary tissue of rats was used for Micro-CT,HE staining,and immunohistochemical detection.Results The CCK-8 assay showed that CM,25 μg/mL ATN-161,and ATN-161 concentrations below 25 μg/mL had no significant effect on cell viability at 12 h and 24 h(P＞0.05).50-CM and 25 μg/mL ATN-161 significantly inhibited cell viability at 48 h(P＜0.05).For in vitro experiments,compared to the 0-CM group,both the protein and mRNA levels of integrin α5 and NLRP3 were significantly increased in rat PDLFs in the 5-CM group(P＜0.05).Intervention with 25 μg/mL ATN-161 significantly attenuated the enhancement of 5-CM on the expression of integrin α5 and NLRP3(P＜0.05).For in vivo ex-periments,compared to the control group,alveolar bone resorption and periodontal inflammatory cell infiltration were sig-nificantly increased in the 5-CM and ATN-161+5-CM groups,and the expression of integrin α5 and NLRP3 was signifi-cantly increased(P＜0.01).However,compared to the 5-CM group,the ATN-161+5-CM group had less alveolar bone resorption and fewer periodontal inflammatory cells.Further,the expression of integrin α5 and NLRP3 was significantly reduced(P＜0.01).Conclusion In vitro and in vivo experiments showed that integrin α5 mediated NLRP3 expression in PDLFs under an inflammatory microenvironment.ATN-161 inhibited the expression of integrin α5,thus significantly downregulating the expression of NLRP3,which plays a role in inhibiting inflammation.
维普
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