骨髓间充质干细胞外泌体调控NLRP3炎性小体对糖尿病大鼠牙槽骨缺损的机制研究

Mechanism of bone marrow mesenchymal stem cell exosomes regulating NLRP3 inflammatory bodies on alveolar bone defect in diabetes rats

目的 探究骨髓间充质干细胞外泌体通过调控NLRP3炎性小体信号通路影响糖尿病大鼠牙槽骨缺损愈合相关分子机制。方法 选取50只SD大鼠纳入研究,随机选取10只大鼠作为对照组,其余大鼠构建糖尿病+双侧上颌牙槽骨缺损模型,设置模型组、骨髓间充质干细胞外泌体组(BMMSC-Exos组)、MCC950组(NLRP3抑制剂)、BMMSC-Exos+MCC950组,其中BMMSC-Exos组、MCC950组、BMMSC-Exos+MCC950组SD大鼠分别尾静脉注射BMMSC-Exos及MCC950试剂,模型组注射等量盐水。模型构建28d后取大鼠上颌骨组织。TEM检测BMMSC-Exos形态,Westernblot检测BMMSC-Exos标志性蛋白CD9、CD63、CD81表达。检测各组大鼠空腹血糖水平、HE染色分析各组大鼠牙槽骨炎性浸润及Lance-Sandhu评分;Western blot检测各组大鼠牙槽骨组织内NLRP3炎性小体信号通路关键蛋白(NLRP3、caspase-1、IL-1β)、骨代谢关键蛋白(OPG、ALP、Collal)表达。结果 BMMSC-Exos直径大小在100 nm左右,呈双凹圆盘状态,膜结构完整;与纯BMMSCs相比,BMMSC-Exos标志物蛋白CD9、CD63、CD81的含量表达明显提升(P <0.05)。糖尿病大鼠模型建立成功,BMMSC-Exos干预、NLRP3抑制剂(MCC950)下调大鼠血糖水平(P <0.05);两者联合可以进一步下调大鼠血糖水平(P <0.05);与对照组相比,糖尿病牙槽骨缺损模型建立可以下调骨再生Lane-Sandhu评分、OPG、ALP、Collal的表达(P <0.05),上调NLRP3、caspase-1、IL-1β表达(P <0.05);BMMSC-Exos干预可以再次上调Lane-Sandhu评分及OPG、ALP、Collal蛋白表达(P<0.05),下调NLRP3、caspase-1、IL-1β的表达(P <0.05)。与模型组相比,BMMSC-Exos及MCC950干预后大鼠骨再生Lane-Sandhu评分、骨代谢关键蛋白(OPG、ALP、Collal)的表达提升(P<0.05);两者联合可进一步上调大鼠骨再生Lane-Sandhu评分及骨代谢关键蛋白表达(P <0.05)。结论 骨髓间充质干细胞外泌体通过抑制NLRP3炎症小体活性来改善糖尿病牙槽骨缺损大鼠骨代谢,降低炎症反应,促进骨缺损的愈合。

Objective To explore the molecular mechanism of bone marrow mesenchymal stem cell exosomes on alveolar bone defect healing in diabetic rats.Methods A total of 50 SD rats were selected to be included in this study,and 10 rats were randomly selected as the control group.The remaining rats were used to construct the diabetes plus bilateral maxillary alveolar bone defect model.The model group,BMMSC-Exos group,MCC950 group(NLRP3 inhibitor),BMMSC-Exos and MCC950 group were set up.The BMMSC-Exos and MCC950 group of SD rats were injected with BMMSC-Exos and MCC950 reagents through the tail vein,while the model group was injected with an equal amount of saline;After 28 days of model construction,the maxilla tissue of rats was taken.TEM was used to detect the morphology of BMMSC-Exos,and Western blot was used to detect the expression of BMMSC-Exos marker proteins CD9,CD63,and CD81.Fasting blood glucose levels,HE staining analysis of alveolar bone inflammatory infiltration and Lance-Sandhu score in each group of rats were analyzed;The NLRP3 inflammasome signaling pathway key proteins(NLRP3,caspase-1,IL-1β)in alveolar bone tissue of rats in each group,and the expression of key proteins in bone metabolism(OPG,ALP,Collal)were analyzed by Western blot.Results The diameter of BMMSC-Exos is around 100 nm,presenting a double concave disc state.Compared with pure BMMSCs,the membrane structure is intact,and the expression of BMMSC-Exos marker proteins CD9,CD63,and CD81 was significantly increased(P<0.05).The model of diabetic rats was successfully established.BMMSC-Exos intervention and NLRP3 inhibitor(MCC950)reduced the blood glucose level of the rats(P<0.05);and combination of the two treatments further reduced the blood sugar level of the model rats(P<0.05).Compared with control group,the bone regeneration Lane-Sandhu score,OPG,ALP,Collal expression were down-regulated,while the NLRP3,caspase-1,IL-1βexpression levels were up-regulated,in the diabetic alveolar bone defect model(P<0.05).BMMSC-Exos intervention greatly up-regulated the Lane-Sandhu score and the expression of OPG,ALP,and Collal proteins(P<0.05),and down-regulated the NLRP3,caspase-1,and IL-1βexpression(P<0.05).Compared with the model group,BMMSC-Exos and MCC950 intervention increased the Lane-Sandhu score of bone regeneration and the expression of key bone metabolism proteins(OPG,ALP,Collal)in rats(P<0.05).Combination of the two treatments further up-regulated the Lane-Sandhu score and expression of key bone metabolism proteins in rat bone regeneration(P<0.05).Conclusion The exosomes of bone marrow mesenchymal stem cells can benefit the bone metabolism of diabetic rats with alveolar bone defect by inhibiting the activity of NLRP3 inflammasome,reducing the inflammatory reaction and promoting the healing of bone defect.