目的 探讨帕罗西汀对大鼠骨髓间充质干细胞(BMSCS)成骨分化的影响及机制。方法 从大鼠后肢分离培养原代BMSCS，流式鉴定BMSCS表面标志物。细胞计数试剂盒(CCK-8)法测定不同浓度的帕罗西汀对BMSCS的细胞毒性，采用1。25、2。50、5。00 μmol/L的浓度处理BMSCS。细胞分为GM组(生长培养基培养)、OS组(成骨诱导培养基培养)、LP组(1。25 μmol/L帕罗西汀+成骨诱导培养基培养)、MP组(2。50 μmol/L帕罗西汀+成骨诱导培养基培养)、HP组(5。00 μmol/L帕罗西汀+成骨诱导培养基培养)。1周后进行碱性磷酸酶(ALP)染色，3周后茜素红染色检测成骨分化水平。3周后提取细胞全蛋白，通过蛋白质印迹法(Western blot)检测成骨标志物OPN、Runx2的表达水平及核因子-κB(NF-κB)、磷酸化核因子-κB(p-NF-κB)的表达水平。添加NF-κB抑制剂PDTC后再次分组为OS、OS+PDTC、LP+PDTC、MP+PDTC、HP+PDTC组，同样分别进行ALP染色、茜素红染色及Western blot检测成骨分化水平和NF-κB表达水平。两样本间对比采取t检验，多组间差异采用单因素方差分析。结果 流式细胞检测CD73、CD90、CD29、CD44表达阳性，CD34、CD45表达阴性。OS组BMSCS的ALP染色和茜素红染色颜色较深，LP、MP、HP组染色较浅。GM 组 p-NF-κB 表达量低于 OS、LP、MP、HP 组(0。458±0。012 比 0。567±0。008、0。749±0。022、0。802±0。011、0。972±0。014，t=13。137、13。443、29。318、43。010，P＜0。05);NF-κB 表达量 OS 组与GM、LP、MP、HP 组差异无统计学意义(1。006±0。025 比 1。007±0。013、0。945±0。074、0。909±0。101、0。982±0。019，t=0。024、1。363、1。627、1。335，P＞0。05);Runx2 表达量 OS 组高于 GM、LP、MP、HP 组(0。955±0。011 比 0。585±0。014、0。746±0。012、0。584±0。016、0。489±0。013，t=36。083，21。910、33。210、67。948，P＜0。05);OPN 表达量 OS 组高于 GM、LP、MP、HP 组(0。918±0。015 比0。669±0。037、0。666±0。028、0。538±0。043、0。421±0。028，t=10。834，13。859、14。370、27。067，P＜0。05)。OS、OS+PDTC、LP+PDTC、MP+PDTC、HP+PDTC 组 ALP 染色和茜素红染色程度基本相同。p-NF-κB 表达量 OS 组高于 OS+PDTC、LP+PDTC、MP+PDTC 组 HP+PDTC 组(0。790±0。044比 0。450±0。032、0。439±0。015、0。481±0。020、0。449±0。024，t=10。821，12。956、11。024、11。705，P＜0。05);NF-κB 表达量 OS 组与 OS+PDTC、LP+PDTC、MP+PDTC、HP+PDTC 组差异无统计学意义(0。866±0。019 比 0。843±0。038、0。818±0。028、0。845±0。045、0。859±0。068，t=0。930、2。404、0。722、0。178，P＞0。05);OPN 表达量 OS 组与 OS+PDTC、LP+PDTC、MP+PDTC、HP+PDTC 组差异无统计学意义(0。902±0。032 比 0。925±0。041、0。890±0。052、0。963±0。042、1。003±0。093，t=0。785、0。311、2。004、1。789，P＞0。05);Runx2 表达量 OS 组与 OS+PDTC、LP+PDTC、MP+PDTC、HP+PDTC 组差异无统计学意义(0。967±0。052 比 0。898±0。095、0。951±0。015、0。950±0。055、0。921±0。055，t=1。113、0。532、0。392、1。068，P＞0。05)。结论 帕罗西汀可以通过激活大鼠BMSCS的NF-κB抑制大鼠BMSCS的成骨分化。
Paroxetine inhibits osteogenic differentiation of bone marrow mesenchymal stem cells through activation of nuclear factor-κB
Objective To investigate the effect and mechanism of paroxetine on osteogenic differ-entiation of rat bone marrow mesenchymal stem cells(BMSCs).Methods Primary BMSCs were isolated and cultured from the hind limbs of rats(Purchased from Shenzhen Bay Laboratory),and surface markers of BMSCs were identified by flow cytometry.Cell Counting Kit-8(CCK-8)assay was used to determine the cytotoxicity of paroxetine at different concentrations on BMSCs,with concentrations of 1.25,2.50,5.00 μmol/L used for treatment.The cells were divided into the GM group(cultured in growth medium),the OS group(cultured in osteogenic induction medium),the LP group(cultured in 1.25 μmol/L paroxe-tine+osteogenic induction medium),MP group(cultured in 2.50 μmol/L paroxetine+osteogenic induction medium),HP group(cultured in 5.00 μmol/L paroxetine+osteogenic induction medium).Alkaline phosphatase(ALP)staining was performed after 1 week,and alizarin red staining was performed after 3 weeks to detect the osteogenic differentiation level of cells in each group.Total protein was extracted from cells of each group after 3 weeks,and the expression levels of osteogenic markers OPN and Runx2,as well as nuclear factor-KB(NF-κB)and phosphorylated nuclear factor-KB(p-NF-κB)were detected by Western blotting.After adding NF-κB inhibitor PDTC,cells were grouped again into OS group,OS+PDTC group,LP+PDTC group,MP+PDTC group,and HP+PDTC group.ALP staining,alizarin red staining,and Western blotting were performed to detect the osteogenic differentiation level and NF-κB ex-pression level in each group.The comparison between two samples was conducted using t-test,and the differences between multiple groups were analyzed using one-way ANOVA.Results Flow cytometry showed positive expression of CD73,CD90,CD29,and CD44,and negative expression of CD34 and CD45.The OS group showed deeper ALP staining and alizarin red staining than the LP,MP,and HP groups.The expression of p-NF-κB in the GM group was lower than that in the OS group,LP group,MP group,and HP group(0.458±0.012 vs.0.567±0.008,0.749±0.022,0.802±0.011,0.972±0.014,t=13.137,13.443,29.318,43.010,P＜0.05).There was no statistically significant difference in NF-κB expression between the OS group and the GM group,LP group,MP group,and HP group(1.006±0.025 vs.1.007±0.013,0.945±0.074,0.909±0.101,0.982±0.019,t=0.024,1.363,1.627,1.335,P＞0.05).The expression of Runx2 in the OS group was higher than that in the GM group,LP group,MP group,and HP group(0.955±0.011 vs.0.585±0.014,0.746±0.012,0.584±0.016,0.489±0.013,t=36.083,21.910,33.210,67.948,P＜0.05).The expression of OPN in the OS group was higher than that in the GM group,LP group,MP group,and HP group(0.918±0.015 vs.0.669±0.037,0.666±0.028,0.538±0.043,0.421±0.028,t=10.834,13.859,14.370,27.067,P＜0.05).ALP staining and aliza-rin red staining in the OS group,OS+PDTC group,LP+PDTC group,MP+PDTC group,and HP+PDTC group showed similar degrees.The expression of p-NF-κB in the OS group was higher than that in the OS+PDTC group,LP+PDTC group,MP+PDTC group,and HP+PDTC group(0.790±0.044 vs.0.450±0.032,0.439±0.015,0.481±0.020,0.449±0.024,t=10.821,12.956,11.024,11.705,P＜0.05).The expression of NF-κB in the OS group was not significantly different from that in the OS+PDTC group,LP+PDTC group,MP+PDTC group,and HP+PDTC group(0.866±0.019 vs.0.843±0.038,0.818±0.028,0.845±0.045,0.859±0.068,t=0.930,2.404,0.722,0.178,P＞0.05).There was no statisti-cally significant difference in OPN expression between OS group and OS+PDTC group,LP+PDTC group,MP+PDTC group,HP+PDTC group(0.902±0.032 vs.0.925±0.041,0.890±0.052,0.963±0.042,1.003±0.093,t=0.785,0.311,2.004,1.789,P＞0.05).There was no statistically significant difference in the Runx2 expression between the OS group and the OS+PDTC group,LP+PDTC group,MP+PDTC group,HP+PDTC group(0.967±0.052 vs.0.898±0.095,0.951±0.015,0.950±0.055,0.921±0.055,t=1.113,0.532,0.392,1.068,P＞0.05).Conclusion Paroxetine can inhibit osteogen-ic differentiation of rat BMSCs by activating NF-κB.
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