唑来膦酸促进去势大鼠牙槽骨的成骨作用

Zoledronic acid promotes alveolar bone formation in ovariectomized rats

背景:唑来膦酸主要作用于破骨细胞的形成,也能抑制成骨细胞的凋亡及成骨细胞增殖和分化,但唑来膦酸对去势大鼠牙槽骨促成骨作用及机制尚存在争议。目的:基于牙槽骨NLPR3/Caspase-1/IL-1β信号通路,探讨唑来膦酸对去势大鼠牙槽骨促成骨作用的机制。方法:选取36只SD雌性大鼠,随机分为对照组、模型组、唑来膦酸组,每组12只,后两组采用双侧卵巢切除术建立骨质疏松大鼠模型,唑来膦酸组于造模术后12周一次性皮下注射20μg/kg唑来膦酸,对照组及模型组注射同等剂量生理盐水。药物干预4周后麻醉大鼠,收集腹主动脉血清及取牙槽骨进行相关检测。ELISA检测大鼠血清中碱性磷酸酶、骨钙素及白细胞介素1β的表达水平;苏木精-伊红染色观察各组大鼠牙槽骨病理结构变化;TUNEL染色检测各组大鼠牙槽骨成骨细胞凋亡情况;钙黄绿素荧光标记检测牙槽骨骨形成情况;Western blot检测各组大鼠牙槽骨骨组织中NLPR3、Caspase-1、白细胞介素1β蛋白的表达水平及Runt相关转录因子2、碱性磷酸酶和骨钙素蛋白的表达情况。结果与结论:(1)与模型组相比,唑来膦酸组大鼠血清中碱性磷酸酶及白细胞介素1β表达水平显著降低(P<0.01),骨钙素表达水平显著升高(P<0.05);(2)与模型组相比,唑来膦酸组牙槽骨病理结构有所改善;(3)唑来膦酸组TUNEL阳性细胞数明显低于模型组(P<0.001);(4)唑来膦酸组骨小梁矿化沉积率和新骨形成率较模型组高(P<0.05);(5)Western blot结果显示,与对照组相比,模型组NLPR3、Caspase-1、白细胞介素1β蛋白表达均升高(P<0.05),Runt相关转录因子2、碱性磷酸酶、骨钙素蛋白表达均降低(P<0.05);而唑来膦酸组NLPR3、Caspase-1、白细胞介素1β蛋白表达水平均降低(P<0.05),Runt相关转录因子2、碱性磷酸酶、骨钙素蛋白表达水平均升高(P<0.05);(6)提示唑来膦酸可以影响血清中成骨相关因子及炎性因子的表达来改善骨代谢及炎性反应状态,增强成骨细胞分化功能,延缓骨质疏松形成;唑来膦酸可改善牙槽骨骨组织结构、减少牙槽骨成骨细胞凋亡及NLPR3、Caspase-1、白细胞介素1β蛋白表达,增加牙槽骨骨小梁矿化沉积率、新骨形成率及Runt相关转录因子2、碱性磷酸酶、骨钙素蛋白表达,其可能与抑制NLRP3/Casapse-1/IL-1β信号通路有关。

BACKGROUND:Zoledronic acid mainly acts on the formation of osteoclasts and also inhibits osteoblast apoptosis,proliferation,and differentiation.However,the effect and mechanism of zoledro nic acid on alveolar bone formation in ovariectomized rats are still controversial.OBJECTIVE:To explo re the osteogenic effect of zoledro nic acid on alveolar bone of ovariectomized rats via alveolar nucleotide-binding oligomerization domainlike receptor protein 3(NLRP3)/cysteinyl-aspartate protease-1(Caspase-1)/interleukin-1β pathway.METHODS:Totally 36 female Sprague-Dawley rats were randomly divided into control group,model group,and zoledronic acid group,with 12 rats in each group.Bilate ral oophorecto my was used to establish an animal model of osteoporosis in the latter two groups.The zoledronic acid group was subcutaneously given 20μg/kg zoledro nic acid at 12 weeks after modeling,while the control and model groups were injected with the same dose of normal saline.All animals were anesthetized after 4-week drug intervention to collect serum samples from the abdominal aorta and alveolar bone samples.Serum alkaline phosphatase,osteocalcin and inte rleukin-1β levels were detected by ELISA.Hematoxylin-eosin staining was used to observe the pathological changes of rat alveolar bone.TUNEL staining was used to detect the apoptosis of osteoblasts in rat alveolar bone.The formation of alveolar bone was detected by calcein fluorescent labeling.Western blot assay was used to detect the levels of NLPR3,Caspase-1 and inte rleukin-1β in alveolar bone tissue as well the protein expression of Runt related transcri ption factor 2,alkaline phosphatase,and osteocalcin.RESULTS AND CONCLUSION:Compared with the model group,the expression level of alkaline phosphatase in rat serum decreased in the zoledro nic acid group(P<0.01),while the expression level of osteocalcin increased significantly(P<0.05).Compared with the model group,the pathological structure of alveolar bone in the zoledronic acid group was improved.The number of TUNEL positive cells in the zoledro nic acid group was significantly lower than that in the model group(P<0.001).The mineralization deposition rate and new bone formation rate of trabecular bone in the zoledronic acid group were higher than those in the model group(P<0.05).(5)Western blot res ults showed that compared with the control group,NLRP3,Caspase-1,interleukin-1β proteins was highly expressed in the model group(P<0.05),while the expressions of Runt related transcription factor 2,alkaline phosphatase,and osteocalcin proteins were decreased(P<0.05).Howeve r,treatment with zoledronic acid decreased the protein expression of NLRP3,Caspase-1,and interleukin-1β(P<0.05),but increased the protein expression of Runt related transc ription factor 2,alkaline phosphatase,and osteocalcin(P<0.05).The above results show that zoledronic acid can affect the expression of osteogenic related fa ctors and inflammatory factors in serum to improve bone metabolism and inflammatory response,enhance the differentiation function of osteoblasts,and delay the formation of osteoporosis.Zoledronic acid can improve alveolar bone structure,reduce alveolar bone osteoblast apoptosis and the protein expression of NLRP3,Caspase-1,and interleukin-1β,increase the mineralization deposition rate and new bone formation rate of alveolar bone trabecula and the protein expression of Runt related transc ription factor 2,alkaline phosphatase,and osteocalcin,which may be related to the inhibition of NLRP3/Caspase-1/interleukin-1β signaling pathway.