南蛇藤素调节HMGB1-RAGE信号通路对牙周炎大鼠牙周组织损伤的影响

Effects of Celastrol on Periodontal Tissue Injury in Periodontal Disease Rats by Regulating HMGB1-RAGE Signaling Pathway

目的:探究南蛇藤素(Celastrol,Cel)调节高迁移率组框1(high mobility group box 1,HMGB1)-晚期糖基化终产物(receptor for advanced glycation end products,RAGE)信号通路对牙周炎大鼠牙周组织损伤的影响及其机制。方法:建立牙周炎大鼠模型。将大鼠分为对照组(Control组)、模型组(Model组)、南蛇藤素低、中、高剂量组[Cel-L组,1 mg/(kg·d)Cel,Cel-M组,2 mg/(kg·d)Cel和Cel-H组,4 mg/(kg·d)Cel]和Cel-H+HMGB1重组蛋白[4 mg/(kg·d)Cel-H+R-HMGB1]。评定牙周组织炎症情况;微计算机断层扫描观察牙槽骨吸收情况;苏木精-伊红染色和抗酒石酸酸性磷酸酶染色分别观察牙周组织病理变化和破骨细胞数变化;酶联免疫吸附试验检测白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和γ-干扰素(interferon-gamma,IFN-γ)超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(Malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)、活性氧(reactive oxygen species,ROS)、一氧化氮(nitric oxide,NO)和前列腺素E2(prostaglandin E2,PGE2)水平;Westernblot检测HMGB1、RAGE蛋白水平。结果:与Control组相比,Model组大鼠牙龈乳头部分缺失和牙龈上皮侵蚀或溃疡,牙龈上皮和固有层发现大量炎性细胞浸润,牙周胶原束排列不规则,部分胶原纤维溶解变性,牙槽骨吸收明显;大鼠牙龈指数、牙龈出血指数、釉牙骨质界到牙槽嵴顶的距离值、牙周组织破骨细胞数、血清IL-6、TNF-α和IFN-γ水平、牙周组织MDA、ROS、NO和PGE2水平及HMGB1和RAGE蛋白表达显著增加(P<0.05),牙周组织SOD和CAT水平显著降低(P<0.05);与Model组相比,Cel-L组、Cel-M组和Cel-H组大鼠牙槽骨损伤和炎症侵蚀减轻,大鼠牙龈指数、牙龈出血指数、CEJ-ABC值、牙周组织破骨细胞数、血清IL-6、TNF-α和IFN-γ水平、牙周组织MDA、ROS、NO和PGE2水平及HMGB1和RAGE蛋白表达显著降低(P<0.05),牙周组织SOD和CAT水平显著增加(P<0.05),且呈剂量依赖性;HMGB1重组蛋白可逆转南蛇藤素对牙周炎大鼠牙周组织损伤的抑制作用(P<0.05)。结论:南蛇藤素通过抑制HMGB1-RAGE信号通路从而抑制牙周炎大鼠牙周组织损伤。

Objective:To investigate the effect of celastrol(Cel)on periodontal tissue injury in periodontal disease(PE)rats by regulating high mobility group protein 1(HMGB1)-advanced glycation end products(RAGE)signaling pathway and its mechanism.Methods:The periodontitisrat model was established rat model was established.The rats were separated into Control group,Model group,low,Celastrol groups[Cel-L group:1 mg/(kg·d)Cel;Cel-M group:2 mg/(kg·d)Cel;and Cel-H group:4 mg/(kg·d)Cel],and Cel-H+HMGB1 recombinant protein group group(4 mg/(kg·d)Cel+R-HMGB1).The inflammation of periodontal tissue was evaluated.Microcomputedtomography asappliedtoobservethealveolarboneresorption.He hematoxylin and eosin staining and Tartrate-resistant acid phosphatase staining were applied to observe the pathological changes of periodontal tissue and the number of osteoclast.Enzyme-linked immunosorbent assay(ELISA)detected interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interferon-gamma.IFN-γsuperoxide dismutase(SOD),Malondialdehyde(MDA),catalase CAT),reactive oxygen species(ROS),nitric oxide(NO)and prostaglandin E2(PGE2)levels.Western blot was applied to detect the levels of HMGB1 and RAGE proteins.Results:Compared with the control group,partial loss of gingival papilla and erosion or ulcer of gingival epithelium were found in the model group,a large number of inflammatory cells were found in the gingival epithelium and lamina propria,periodontal collagen bundles were arranged irregularly,some collagen fibers were dissolved and degenerated,and alveolar bone resorption was obvious.The gingival index,gingival bleeding index,cement-to-enamel junction-alveolar bone crest(CEJ-ABC)value,the number of osteoclast in periodontal tissue,the levels of IL-6,TNF-α,and IFN-γin serum,the levels of MDA,ROS,NO,and PGE2 in periodontal tissue,and the expressions of HMGB1 and RAGE proteins in periodontal tissue were obviously increased in rats(P<0.05).The levels of SOD and CAT in periodontal tissue were obviously reduced(P<0.05).Compared with the Model group,the alveolar bone damage and inflammatory erosion in the Cel-L,Cel-M,and Cel-H groups were reduced,the gingival index,gingival bleeding index,CEJ-ABC value,the number of osteoclast in periodontal tissue,the levels of IL-6,TNF-α,and IFN-γin serum,the levels of MDA,reactive oxygen species(ROS),NO,and PGE2 in periodontal tissue,and the expressions of HMGB1 and RAGE proteins in periodontal tissue of rats were obviously reduced(P<0.05).The levels of SOD and CAT in periodontal tissue were obviously increased(P<0.05)and dose-dependent.HMGB1 recombinant protein could reverse the inhibitory effect of celastrol on periodontal tissue injury in PE rats(P<0.05).Conclusion:Celastrol can inhibit the periodontal tissue injury in periodontal disease rats by inhibiting HMGB1-RAGE signaling pathway.