大黄素上调miR-218表达对慢性牙周炎大鼠骨密度和炎症反应的影响

Influences of emodin on bone mineral density and inflammatory response in rats with chronic periodontitis by up-regulating miR-218 expression

目的:探讨大黄素上调miR-218表达对慢性牙周炎大鼠骨密度(BMD)和炎症反应的影响。方法:构建大鼠慢性牙周炎模型,并随机分成模型组、大黄素低剂量组(20 mg·kg^(-1))、大黄素高剂量组(40 mg·kg^(-1))、NC组(miR-218 NC)和miR-218过表达组(miR-218 agomir)和大黄素+miR-218抑制组(大黄素40 mg·kg-1+miR-218 antagonist),每组10只,另选10只正常饲养大鼠作为正常组。qRT-PCR检测大鼠牙周组织中miR-218表达;Micro-CT扫描检测大鼠BMD及骨微结构变化;苏木素-伊红(HE)染色检测牙周组织病理变化;ELISA法检测血清中肿瘤坏死因子-α(TNF-α)、白介素(IL)-1β、IL-6水平变化;Western Blot检测牙周组织MMP-9、COLⅠ、COLⅣ及DSP蛋白表达。结果:与正常组比较,模型组大鼠出现炎症细胞浸润、较深牙周袋和明显骨吸收等现象,且上颌骨骨小梁数量(Tb·N)、骨小梁分离距离(Tb·Sp),血清中TNF-α、IL-1β、IL-6水平,牙周组织中MMP-9蛋白表达显著上升(P<0.05);BMD、骨体积分数(BV/TV)、骨小梁厚度(Tb·Th)及牙周组织中miR-218表达和COLⅠ、COLⅣ、DSP蛋白表达显著下降(P<0.05)。大黄素低、高剂量组牙周炎大鼠炎症细胞浸润和骨吸收等现象均获得改善,且上颌骨Tb·N、Tb·Sp,血清中TNF-α、IL-1β、IL-6水平,牙周组织中MMP-9蛋白表达显著下降(P<0.05);BMD、BV/TV、Tb·Th及牙周组织中miR-218表达和COLⅠ、COLⅣ及、DSP蛋白表达显著上升(P<0.05),miR-218过表达对牙周炎大鼠各项指标的影响趋势与大黄素低、高剂量组一致。与大黄素高剂量组相比,大黄素+miR-218抑制组上述各项指标均得以逆转(P<0.05)。结论:大黄素可改善慢性牙周炎大鼠BMD和炎症反应,其作用机制可能与上调miR-218表达有关。

Objective:To investigate the influences of emodin up-regulation of miR-218 expression on bone mineral density(BMD)and inflammatory response in chronic periodontitis rats.Methods:A rat model of chronic periodontitis was constructed and randomly grouped into model group,emodin low-dose group(20 mg·kg^(-1)),emodin high-dose group(40 mg·kg^(-1)),NC group(miR-218 NC),miR-218 overexpression group(miR-218 agomir),and emodin+miR-218 inhibitor group(emodin 40 mg·kg^(-1)+miR-218 antagonist group),with 10 rats per group.Another 10 normal-raised rats were gathered as the normal group.qRT-PCR was used to detect the expression of miR-218 in rat periodontal tissue;Micro-CT scanning was performed to detect the changes of BMD and bone microstructure in rats;hematoxylin-eosin(HE)staining was used to measure the pathological changes of periodontal tissue;ELISA was used to measure the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 in serum;Western Blot was used to measure the expression of MMP-9,COLⅠ,COLⅣand DSP proteins in periodontal tissue.Results:Compared with the normal group,the rats in the model group had inflammatory cell infiltration,deeper periodontal pockets and obvious bone resorption,the number of maxillary trabecular bone(Tb·N),separation distance of trabecular bone(Tb·Sp),the serum levels of TNF-α,IL-1β,IL-6,and the expression of periodontal tissue MMP-9 protein increased significantly(P<0.05);the BMD,bone volume to tissue volume(BV/TV),thickness of trabecular bone(Tb·Th),miR-218 expression and the expressions of COLⅠ,COLⅣand DSP in periodontal tissue decreased significantly(P<0.05).In emodin low,high-dose group,the inflammatory cell infiltration and bone resorption in periodontitis rats were improved,the maxillary Tb·N,Tb·Sp,the serum levels of TNF-α,IL-1β,IL-6,and the expression of MMP-9 protein in periodontal tissue decreased significantly(P<0.05);the BMD,BV/TV,Tb·Th,miR-218 expression and the expression of COLⅠ,COLⅣand DSP proteins in periodontal tissues increased significantly,the influence trend of miR-218 overexpression on periodontitis rats was consistent with that of emodin low-dose and high-dose groups(P<0.05).Compared with the emodin high-dose group,the above indexes were reversed in the emodin+miR-218 inhibition group(P<0.05).Conclusion:Emodin can improve BMD and inflammatory response in rats with chronic periodontitis,and its mechanism may be related to up-regulation of miR-218 expression.