石胆酸对于BMSCs成骨-成脂分化平衡的影响

Impact of lithocholic acid on the osteogenic and adipogenic differentiation balance of bone marrow mesenchymal stem cells

目的探讨石胆酸(lithocholic acid,LCA)对于BMSCs成骨-成脂分化平衡的影响。方法取10周龄SPF级C57BL/6J雌性小鼠12只,随机分为实验组(行双侧卵巢切除术)和对照组(仅移除卵巢周围相同体积脂肪组织),每组6只。术后每周测量体质量,4周取小鼠子宫称重;取股骨标本行micro-CT扫描和三维重建,分析骨量变化;取胫骨标本行HE染色并计算骨髓腔区域中骨髓脂肪空泡数量和面积百分比;取血清样本,采用ELISA法检测骨转换标志物表达;取肝脏样本行实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)检测胆汁酸代谢关键酶相关基因cyp7a1、cyp7b1、cyp8b1及cyp27a1表达。采用离心法分离2只10周龄C57BL/6J雌性小鼠BMSCs,取第3代细胞分别给予0、1、10、100μmol/L LCA干预并行细胞计数试剂盒8法检测细胞毒性,经成骨及成脂诱导分化7 d后分别行ALP染色和油红O染色,并行RT-qPCR检测成骨相关基因ALP、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)、骨钙素(osteocalcin,OCN)以及成脂细胞相关基因脂联素(Adiponectin,Adipoq)、脂肪酸结合蛋白(fatty acid binding protein 4,FABP4)、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)表达。结果与对照组比较,实验组小鼠体质量升高、子宫萎缩、骨量降低、骨髓脂肪扩张、骨代谢呈高骨转换状态;RT-qPCR检测示肝脏胆汁酸代谢关键酶相关基因cyp7a1、cyp8b1、cyp27a1表达降低(P<0.05),而cyp7b1表达差异无统计学意义(P>0.05)。LCA干预浓度为1、10、100μmol/L时,对BMSCs均无明显毒性作用,且LCA以剂量依赖性抑制BMSCs成骨相关基因ALP、Runx2、OCN表达,ALP染色阳性区域减少;同时,LCA促进成脂相关基因Adipoq、FABP4、PPARγ表达,油红O染色阳性区域增加。结论绝经后小鼠骨质疏松病理状态下胆汁酸代谢发生改变,次级胆汁酸LCA以干扰BMSCs成骨-成脂分化平衡方式影响骨重塑。

Objective To Investigate the effects of lithocholic acid(LCA)on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group(undergoing bilateral ovariectomy)and a control group(only removing the same volume of adipose tissue around the ovaries),with 6 mice in each group.The body mass was measured every week after operation.After 4 weeks post-surgery,the weight of mouse uterus was measured,femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass.Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area.ELISA was used to detect the expression of bone turnover markers in the serum.Liver samples were subjected to real-time fluorescence quantitative PCR(RT-qPCR)to detect the expression of key genes related to bile acid metabolism,including cyp7a1,cyp7b1,cyp8b1,and cyp27a1.BMSCs were isolated by centrifugation from 2 C57BL/6J female mice(10-week-old).The third-generation cells were exposed to 0,1,10,and 100μmol/L LCA,following which cell viability was evaluated using the cell counting kit 8 assay.Subsequently,alkaline phosphatase(ALP)staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction.RT-qPCR was employed to analyze the expressions of osteogenic-related genes,namely ALP,Runt-related transcription factor 2(Runx2),and osteocalcin(OCN),as well as adipogenic-related genes including Adiponectin(Adipoq),fatty acid binding protein 4(FABP4),and peroxisome proliferator-activated receptor γ(PPARγ).Results Compared with the control group,the body mass of the mice in the experimental group increased,the uterus atrophied,the bone mass decreased,the bone marrow fat expanded,and the bone metabolism showed a high bone turnover state.RT-qPCR showed that the expressions of cyp7a1,cyp8b1,and cyp27a1,which were related to the key enzymes of bile acid metabolism in the liver,decreased significantly(P<0.05),while the expression of cyp7b1 had no significant difference(P>0.05).Intervention with LCA at concentrations of 1,10,and 100μmol/L did not demonstrate any apparent toxic effects on BMSCs.Furthermore,LCA inhibited the expressions of osteogenic-related genes(ALP,Runx2,and OCN)in a dose-dependent manner,resulting in a reduction in ALP staining positive area.Concurrently,LCA promoted the expressions of adipogenic-related genes(Adipoq,FABP4,and PPARγ),and an increase in oil red O staining positive area.Conclusion After menopause,the metabolism of bile acids is altered,and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs,thereby affecting bone remodelling.