褪黑素对卵巢摘除大鼠体内炎症状态及BMSCs成骨能力影响的研究

Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats

目的探讨褪黑素(melatonin,MT)对卵巢摘除(ovariectomy,OVX)大鼠骨量、血清炎症因子水平和对脂多糖刺激下BMSCs培养液中炎症因子水平和成骨能力的影响。方法将15只12周龄SD大鼠随机分为3组,假手术组大鼠仅接受双侧侧腹部切开缝合,OVX组行双侧OVX,OVX+MT组在双侧OVX术后行100 mg/(kg·d)MT口服干预。8周后采用ELISA法检测大鼠血液样本中炎症因子IL-1β、IL-6和TNF-α表达水平;用Micro-CT检测股骨远端,观察骨量及骨微结构变化并对骨体积分数、骨小梁厚度和骨小梁数量进行定量检测。取3只3周龄SD大鼠,采用全骨髓培养法提取股骨骨髓中的BMSCs并传代培养。经鉴定后取第3~5代细胞,用不同浓度(0、1、10、100、1000μmol/L)MT干预并行细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞活力,筛选最佳浓度MT用于后续实验。将细胞分为成骨诱导组(A组)及成骨诱导+1/5/10μg/mL脂多糖组(B~D组),进行相应干预后用ELISA法检测细胞培养液中IL-1β、IL-6和TNF-α表达水平;根据CCK-8法和ELISA检测结果,用刺激炎症效果最显著浓度的脂多糖以及最佳药物浓度的MT加成骨诱导培养基对细胞进行干预,并设为E组,同样采用ELISA法检测各炎症因子表达水平;之后分别对A、D、E组行ALP染色和茜素红染色,并用实时荧光定量PCR(real time fluorescence quantitative PCR,RT-qPCR)法检测成骨相关基因Ⅰ型胶原α1链(collagen typeⅠalpha 1 chain,Col1a1)、RUNX家族转录因子2(RUNX family transcription factor 2,Runx2)表达水平。结果ELISA和Micro-CT检测示,与假手术组比较,OVX组大鼠骨量减少,血清炎症因子IL-1β、IL-6、TNF-α表达水平升高(P<0.05);经MT治疗后,OVX+MT组上述指标均得到改善(P<0.05)。成功提取大鼠BMSCs,CCK-8法检测示100μmol/L是不影响细胞活性的最大MT浓度,将其用于后续实验。ELISA检测示,加入脂多糖刺激后,B~D组细胞培养液中的炎症因子IL-1β、IL-6和TNF-α表达水平明显升高,与A组比较差异均有统计学意义(P<0.05);并且呈浓度依赖性,D组各炎症因子表达水平明显高于B、C组(P<0.05);E组加入MT干预后,各炎症因子表达水平较D组显著降低(P<0.05)。ALP染色、茜素红染色及RT-qPCR检测示,与A组比较,D组ALP和茜素红阳性面积百分比以及Col1a1、Runx2 mRNA相对表达量均显著降低,经MT干预后E组上述指标均显著提升,差异均有统计学意义(P<0.05)。结论MT可能通过降低大鼠体内炎症对绝经后骨质疏松症的骨量产生影响;MT能降低脂多糖刺激引起的BMSCs炎症,并减弱其对BMSCs成骨分化的抑制。

Objective To investigate the effects of melatonin(MT)on bone mass and serum inflammatory factors in rats received ovariectomy(OVX)and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells(BMSCs)stimulated by lipopolysaccharide.Methods Fifteen 12-week-old Sprague Dawley(SD)rats were randomly divided into 3 groups.The rats in Sham group only received bilateral lateral abdominal incision and suture,the rats in OVX group received bilateralOVX,and the rats in OVX+MT group received 100 mg/(kg·d)MT oral intervention after bilateral OVX.After 8 weeks,the levels of serum inflammatory factors[interleukin-1β(IL-1β),IL-6,and tumor necrosis factorα(TNF-α)]were detected using ELISA assay.Besides,the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure,and quantitatively measured bone volume fraction,trabecular thickness,and trabecular number.The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged.The 3rd-5th passage BMSCs were cultured with different concentrations of MT(0,1,10,100,1000μmol/L),and the cell viability was then detected using cell counting kit 8(CCK-8)to select the optimal concentration of MT for subsequent experiments.Cells were devided into osteogenic induction group(group A)and osteogenic induction+1/5/10μg/mL lipopolysaccharide group(group B-D).The levels of inflammatory factors(IL-1β,IL-6 and TNF-α)in cell culture medium were detected using ELISA assay after corresponding intervention.According to the results of CCK-8 method and ELISA detection,the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction,defining as group E,and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay.After that,alkaline phosphatase(ALP)staining and alizarin red staining were performed respectively in groups A,D,and E,and the expression levels of osteogenic related genes[collagen typeⅠalpha 1 chain(Col1a1)and RUNX family transcription factor 2(Runx2)]were also detected by real time fluorescence quantitative PCR(RT-qPCR).Results ELISA and Micro-CT assays showed that compared with Sham group,the bone mass of the rats in the OVX group significantly decreased,and the expression levels of serum inflammatory factors(IL-1β,IL-6,and TNF-α)in OVX group significantly increased(P<0.05).Significantly,the above indicators in OVX+MT group were all improved(P<0.05).Rat BMSCs were successfully extracted,and CCK-8 assay showed that 100μmol/L was the maximum concentration of MT that did not cause a decrease in cell viability,and it was used in subsequent experiments.ELISA assays showed that compared with group A,the expression levels of inflammatory factors(IL-1β,IL-6,and TNF-α)in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation(P<0.05),and in a concentration-dependent manner.Moreover,the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C(P<0.05).After MT intervention,the expression levels of inflammatory factors in group E were significantly lower than those in group D(P<0.05).ALP staining,alizarin red staining,and RT-qPCR assays showed that compared with group A,the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased,while the above indicators in group E significantly improved after MT intervention(P<0.05).Conclusion MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats;MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.