基于RANKL/RANK/TRAF6信号通路和肠道菌群探讨买麻藤醇对骨质疏松性骨缺损小鼠的影响  

Effect of gnetol on osteoporotic bone defect mice based on RANKL/RANK/TRAF6 and gut microbiota

作　　者：韦宇杭 曾高峰 WEI Yuhang;ZENG Gaofeng(School of Pharmacy,Guangxi Medical University,Nanning 530021,China;School of Public Health,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学药学院,南宁530021 [2]广西医科大学公共卫生学院,南宁530021

出　　处：《广西医科大学学报》2025年第2期174-184,共11页Journal of Guangxi Medical University

基　　金：国家自然科学基金(No.82260437);广西科技基地和人才专项资助项目(No.桂科AD23026324)。

摘　　要：目的:探索买麻藤醇治疗骨质疏松性骨缺损的潜在机制和对肠道菌群的影响。方法:将30只C57BL/6J小鼠随机分为假手术组、模型组、阿仑膦酸钠组、低剂量买麻藤醇组以及高剂量买麻藤醇组。通过双侧卵巢摘除术构建骨质疏松模型,后在此模型上使用脂多糖(LPS)构建颅骨缺损模型。使用Micro-CT和苏木精—伊红染色(HE)观察小鼠颅骨骨密度和骨微结构,使用酶联免疫吸附试验(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)和Ⅰ型胶原交联羧基端肽(CTX-I)的表达,实时荧光定量PCR(RT-qPCR)检测组织蛋白酶K(CTSK)的表达,蛋白质免疫印迹法检测NFATc1、c-fos、TNF-α、TRAP、CTSK、RANK、TRAF6、RANKL、p-p65、p-IκBα的表达,16sRNA测序检测假手术组、模型组和高剂量买麻藤醇组的肠道菌群丰度。结果:与假手术组相比,模型组小鼠颅骨BMD和骨微结构破坏明显,TRAP、CTSK、CTX-Ⅰ、TNF-α、c-Fos、NFATc1、破骨细胞分化通路(RANK、RANKL、TRAF6)和炎症通路(p-P65、p-IκBα)蛋白表达量提高,骨吸收标志物CTSK基因表达提高,肠道丰富度提高,正常肠道菌群群落结构破坏。买麻藤醇改善了骨质疏松状态下LPS导致的骨微结构受损和骨丢失现象,抑制对破骨细胞分化起促进作用的TNF-α、c-Fos和NFATc1、破骨细胞分化通路(RANK、RANKL、TRAF6)和炎症通路(p-P65、p-IκBα)的蛋白表达,降低CTSK基因表达;降低对骨组织起到负面作用的毛螺菌科、另枝菌属等有害菌的丰度,并提高丹毒丝菌科以及双歧杆菌科(包括双歧杆菌属)等有益菌属的丰度。结论:买麻藤醇有效改善了骨质疏松状态下LPS导致的骨微结构破坏和机体过强的骨吸收,下调RANKL/RANK/TRAF6信号通路的表达和抑制NF-κB信号通路的激活,并调节有益菌和有害菌的丰度。Objective:To explore the potential mechanism of gnetol in the treatment of osteoporotic bone defects and its effect on the gut microbiota.Methods:A total of 30 C57BL/6J mice were randomly divided into sham group,model group,alendronate sodium group,low-dose gnetol group,and high-dose gnetol group.An osteoporosis model was constructed by bilateral ovarian enucleation,and then a skull defect model was constructed using lipopolysaccharide(LPS)on this model.Micro-CT and hematoxylin-eosin(HE)staining were used to observe the bone mineral density and bone microstructure of the skull of mice,the expression of tumor necrosis factor-α(TNF-α)and C-terminal telopeptide of typeⅠcollagen(CTX-I)in the serum was detected by enzyme-linked immunosorbent assay(ELISA),the expression of cathepsin K(CTSK)was detected by reverse transcriptionquantitative polymerase chain reaction(RT-qPCR),and the expression of NFATc1,c-fos,TNF-α,TRAP,CTSK,RANK,TRAF6,RANKL,p-p65 and p-IκBαwas detected by western blotting.The abundance of gut microbiota in the sham,model and high-dose groups was detected by 16sRNA sequencing.Results:Compared with the sham group,the BMD and bone microstructure of the skull in the model group were significantly damaged.The protein expression levels of TRAP,CTSK,CTX-I,TNF-α,c-Fos,NFATc1,as well as those related to the osteoclast differentiation pathway(RANK,RANKL,TRAF6),inflammatory pathway(p-P65 and p-IκBα)were increased,and the expression of bone resorption marker CTSK gene was also increased.Moreover,the gut microbiota richness was increased,while the structure of the normal gut microbiota community was disrupted.Gnetol ameliorated impaired bone microarchitecture and bone loss caused by lipopolysaccharide(LPS)in osteoporotic states,inhibited the protein expression of TNF-α,c-Fos and NFATc1 which promote osteoclast differentiation,as well as the protein expression of the osteoclast differentiation pathways(RANK,RANKL,TRAF6)and inflammatory pathways(p-P65,p-IκBα),and reduced the gene expression of CTSK.It red

关 键 词：买麻藤醇 破骨细胞 骨质疏松性骨缺损 RANK/RANKL/TRAF6信号通路 肠道菌群 

分 类 号：R580[医药卫生—内分泌]