柚皮苷通过cAMP/PKA/CREB通路促进人脂肪间充质干细胞成骨分化  

Naringin Promotes Osteogenic Differentiation of Human Adipose Mesenchymal Stem Cells via the cAMP/PKA/CREB Pathway

作　　者：张亮亮 赵程锦[1] 周煜虎[1] 曹博[1] 段明明[1] 冯阳阳[1] ZHANG Liangliang;ZHAO Chengjin;ZHOU Yuhu(Yan'an University Affiliated Hospital,Shaanxi Yan'an 716000,China)

机构地区：[1]延安大学附属医院创伤骨科,陕西延安716000

出　　处：《河北医学》2022年第10期1597-1603,共7页Hebei Medicine

基　　金：国家自然科学基金资助项目,(编号:82160431)。

摘　　要：目的:探讨柚皮苷通过环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/cAMP反应元件结合蛋白(CREB)通路促进人脂肪间充质干细胞成骨分化的作用。方法:取人脂肪间充质干细胞培养传代,将细胞分为对照组、柚皮苷低、中、高剂量组、cAMP抑制剂(SQ22536)组、成骨分化诱导组(诱导组),柚皮苷低、中、高剂量组分别加12.5μmoL/L、25μmoL/L、50μmoL/L柚皮苷,SQ22536组加100μmoL/L SQ22536,诱导组加10~8moL/L地塞米松、10mmoL/Lβ-甘油磷酸钠和50mg/L抗坏血酸;对照组仅加等体积培养液,每组设置6个复孔。培养48h后,碱性磷酸酶(ALP)染色,磷酸对硝基苯酯(PNPP)法检测各组细胞ALP活性;茜素红S(ARS)染色观察各组细胞钙沉积情况;酶联免疫吸附试验(ELISA)检测各组细胞cAMP水平;实时定量聚合酶链反应(RT-qPCR)检测各组细胞PKA、CREB、矮小相关转录因子2(RUNX2)、骨钙素蛋白(OCN)、骨桥蛋白(OPN)信使核糖核酸(mRNA)表达;蛋白质免疫印迹法(WB)检测各组细胞PKA、CREB、RUNX2、OCN、OPN蛋白表达及p-PKA、p-CREB水平。结果:ALP染色后显示对照组细胞可见少量蓝色沉淀,SQ22536组蓝色沉淀最少,诱导组和柚皮苷3剂量组蓝色沉淀逐渐增多;与对照组比,SQ22536组ALP活性显著下降(P<0.01);与对照组和SQ22536组比,诱导组和柚皮苷3剂量组ALP活性显著上升(P<0.01);对照组存在少量钙沉积,SQ22536组细胞钙沉积显著减少,诱导组和柚皮苷3剂量组钙沉积逐渐增多;与对照组比,SQ22536组细胞cAMP水平、PKA、CREB、RUNX2、OCN、OPN mRNA和蛋白表达及p-PKA、p-CREB水平显著下降(P<0.01);与对照组和SQ22536组比,诱导组和柚皮苷3剂量组cAMP水平、PKA、CREB、RUNX2、OCN、OPN mRNA和蛋白表达及p-PKA、p-CREB水平上升,差异有统计学意义(P<0.05或P<0.01);诱导组和柚皮苷低剂量组上述指标差异无统计学意义(P>0.05);上述指标柚皮苷呈剂量依赖性。结论:柚皮苷可促进人脂肪间充质干细胞成骨分化,可能Objective:To investigate the role of naringin in promoting osteogenic differentiation of human adipose derived mesenchymal stem cells through the cyclic adenosine phosphate(cAMP)/protein kinase A(PKA)/cAMP response element binding protein(CREB)pathway.Methods:The cells were divided into control group,naringin low,medium and high dose group,cAMP inhibitor(SQ22536)group and osteogenic differentiation induction group(induction group).12.5μmoL/L,25μmoL/L and 50μmoL/L naringin were added to the naringin low,medium and high dose groups,respectively,and 100μmoL/L SQ22536 was added to the SQ22536 group.L SQ22536,10~8 moL/L dexamethasone,10 mmoL/L sodiumβ-glycerophosphate and 50mg/L ascorbic acid were added to the induction group;only equal volumes of culture solution were added to the control group,and six replicate wells were set up for each group.After 48h of incubation,ALP activity was measured by alkaline phosphatase(ALP)staining and p-nitrophenyl phosphate(PNPP);calcium deposition was observed by Alizarin Red S(ARS)staining;cAMP level was measured by ELISA;PKA,CREB,RUNX2,Osteocalcin(OCN),OPN messenger ribonucleic acid(mRNA)expression was measured by RT-qPCR.The expression of PKA,CREB,RUNX2,osteocalcin(OCN)and osteopontin(OPN)messenger ribonucleic acid(mRNA)was measured by RT-qPCR,and the expression of PKA,CREB,RUNX2,OCN,OPN and p-PKA and p-CREB were measured by WB.Results:ALP staining showed a small amount of blue precipitation in the control group,with the least amount of blue precipitation in the SQ22536 group and gradually increasing in the induction and naringin 3 kinds of dose groups.ALP activity was significantly decreased in the SQ22536 group compared to the control group(P<0.01);ALP activity was significantly increased in the induction and naringin 3 dose groups compared to the control and SQ22536 groups(P<0.01).A small amount of calcium deposition was present in the control group,significantly reduced in the SQ22536 group and gradually increased in the induction and naringin 3 dose groups;cellular cAMP

关 键 词：脂肪间充质干细胞 柚皮苷 环磷酸腺苷 蛋白激酶A CAMP反应元件结合蛋白 成骨分化 

分 类 号：R285[医药卫生—中药学]