基于JNK、p38/Smads信号级联研究左归丸含药血清对MC3T3-E1细胞的调控机制
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摘要
目的：基于“肾主骨”理论，探讨补肾滋阴代表方剂左归丸促进骨形成作用，及JNK、p38/Smads信号级联对其影响，以探讨左归丸潜在促进骨形成的作用机制。
材料与方法：采用血清药理学方法，将SPF级SD雌性大鼠60只（180~220g）按体质量随机分为空白对照组、左归丸组和倍美力组，每组各20只，依据人与大鼠等剂量换算公式折算大鼠左归丸等临床剂量为1.6g·Kg-1，大鼠倍美力结合雌激素片的等临床剂量为56.25μg·kg-1，使用前用蒸馏水将药物制成混悬液，空白对照组给予蒸馏水，灌服10ml·Kg-1体质量，2次·d-1，连续灌胃7d，末次给药2h后，麻醉大鼠，腹主动脉取血，室温静置2h，2500r·min-1，4℃离心20min，收集上清，同组血清混匀，56℃水浴灭活30min，制备大鼠含药血清。采用MC3T3-E1成骨细胞系体外研究模型，用含10%FBS的α-MEM培养液（100U·ml-1青霉素和100μg·ml-1链霉素）培养。细胞实验分为空白对照组（control）、SP600125组（SP）、SB203580组（SB）、左归丸组（ZG）、左归丸+SP600125组（ZG+SP）、左归丸+SB203580组（ZG+SB）、倍美力组（BML）、倍美力+SP600125组（BML+SP）、倍美力+SB203580组（BML+SB）。细胞接种24h后，换用含0.2%FBS的α-MEM培养液（100U·ml-1青霉素和100μg·ml-1链霉素），饥饿24h，吸弃旧培养液，加入含或不含JNK、p38MAPK特异抑制剂SP600125、SB203580(10μmol·L-1，溶于DMSO，体积分数<0.1%)的α-MEM培养液（100U·ml-1青霉素和100μg·ml-1链霉素），预处理60min后，各组加入相应血清（阴性对照孔、control组、SP组、SB组加入空白对照大鼠血清，ZG组、ZG+SP组、ZG+SB组加入左归丸大鼠含药血清，BML组、BML+SP组、BML+SB组加入倍美力大鼠含药血清），体积分数为15%，孵育48h。采用蛋白质印迹Western blot测定JNK和p38MAPK磷酸化蛋白表达水平，采用免疫荧光染色法进一步验证JNK和p38MAPK磷酸化蛋白表达水平。采用MTT法检测MC3T3-E1细胞增殖；采用流式细胞术分析细胞周期。采用对硝基苯磷酸盐(PNPP)法和改良钙钴染色法检测MC3T3-E1细胞碱性磷酸酶（ALP）活性；采用茜素红染色法检测MC3T3-E1细胞钙化结节形成。采用Western blot分析OB分泌蛋白情况，检测核结合因子（Runx2）、ALP、Ⅰ型胶原（ColI）、Smad2/3、 p-Smad2和p-Smad3蛋白表达水平。采用Q-PCR技术测定相关基因mRNA表达水平，检测MC3T3-E1细胞转化生长因子β1（TGF-β1）、Smad4、Runx2和ColI mRNA表达水平。
结果：
1.JNK信号通路参与左归丸含药血清诱导MC3T3-E1细胞分化的调控
1.1左归丸含药血清能够诱导MC3T3-E1细胞JNK信号通路活化
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞JNK蛋白磷酸化；ZG显著优于BML；加入SP600125后，显著抑制ZG和BML对JNK蛋白磷酸化的诱导作用。
1.2JNK信号通路主要在分化晚期参与左归丸含药血清对MC3T3-E1细胞ALP活性的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞48h和7d的ALP活性（P＜0.01）；对于48h的ALP活性的影响，ZG组显著优于BML组（P＜0.01）；加入SP600125后，显著抑制BML对48h和7d的ALP活性的刺激作用（P＜0.01），对ZG诱导48h的ALP活性的影响没有统计学意义（P>0.05），但显著抑制了ZG诱导7d的ALP活性。
1.3JNK信号通路参与左归丸含药血清促进MC3T3-E1细胞矿化作用的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞14d的矿化作用；加入SP600125后，显著抑制ZG和BML对矿化作用的诱导。
1.4JNK信号通路对左归丸含药血清干预Runx2和ALP蛋白表达的影响
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞48h的Runx2和ALP蛋白的表达；加入SP600125后，未能对抗ZG诱导48h的Runx2和ALP蛋白表达，也未能对抗BML诱导的Runx2蛋白的表达，但显著抑制了BML诱导的ALP蛋白表达。
2.p38MAPK信号通路参与左归丸含药血清诱导MC3T3-E1细胞分化的调控
2.1左归丸含药血清能够诱导MC3T3-E1细胞p38MAPK信号通路活化
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞p38磷酸化蛋白的表达；加入SB203580后，显著抑制ZG和BML对p38蛋白磷酸化的诱导作用。
2.2p38MAPK信号通路参与左归丸含药血清对MC3T3-E1细胞分化不同阶段ALP活性的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞48h和7d的ALP活性（P＜0.01）；加入SB203580后，显著抑制ZG和BML对48h和7d的ALP活性的刺激作用（P＜0.01）。
2.3p38MAPK信号通路参与左归丸含药血清促进MC3T3-E1细胞14d矿化作用的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞14d矿化作用；加入SB203580后，显著抑制ZG和BML对细胞矿化的诱导作用。
2.4左归丸含药血清通过p38MAPK信号通路的活化上调Runx2蛋白的表达
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞Runx2蛋白的表达；加入SB203580后，显著抑制ZG和BML对Runx2蛋白表达的诱导。
3.JNK、p38MAPK/Smads信号级联参与左归丸含药血清对MC3T3-E1细胞功能的调控
3.1左归丸含药血清上调MC3T3-E1细胞TGFβ1mRNA表达
结果显示，与control组比较，ZG组和BML均组能显著上调MC3T3-E1细胞TGFβ1mRNA的表达（P＜0.01）。
3.2左归丸含药血清诱导MC3T3-E1细胞磷酸化JNK和磷酸化p38蛋白表达
结果显示，与control组比较，ZG组和BML组均能显著诱导MC3T3-E1细胞磷酸化JNK和p38蛋白的表达；ZG组明显优于BML组；加入SP600125或SB203580后，显著抑制ZG和BML对磷酸化JNK或p38蛋白的诱导作用。
3.3JNK和p38MAPK信号通路参与左归丸含药血清干预MC3T3-E1细胞增殖的调控
结果显示，与control组比较，ZG和BML均能显著促进MC3T3-E1细胞增殖（P＜0.01）；ZG组显著优于BML组（P＜0.01）；加入SP600125或SB203580后，抑制ZG组和BML组对MC3T3-E1细胞增殖的诱导作用（P＜0.05或P＜0.01）。
3.4JNK和p38MAPK信号通路参与左归丸含药血清干预MC3T3-E1细胞周期分布的调控
细胞周期分析结果显示，与control组比较，ZG组G1期的细胞百分率显著下降（P＜0.01），S期的细胞百分率变化不显著（P>0.05），G2/M期的细胞百分率显著增加（P＜0.01）；与ZG组比较，ZG+SP组和ZG+SB组显著抵抗了ZG对细胞周期的调控作用（P＜0.01）。
3.5左归丸含药血清活化JNK和p38MAPK信号通路有助于磷酸化Smad2/3和上调ColI蛋白表达
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞Smad2/3蛋白磷酸化和上调ColI蛋白表达水平（P＜0.01）；对p-Smad2的诱导作用，ZG组显著优于BML组（P＜0.01），对ColI蛋白表达的上调作用，ZG组优于BML组（P＜0.05），对p-Smad3的诱导作用，两组比较无统计学意义（P>0.05）；加入SP600125或SB203580后，显著抑制ZG和BML对MC3T3-E1细胞Smad2/3蛋白磷酸化的诱导作用（P＜0.01），显著抑制ZG对MC3T3-E1细胞ColI蛋白表达的上调作用（P＜0.01），加入SP600125后显著抑制BML诱导的ColI蛋白表达（P＜0.01），加入SB203580后抑制BML诱导的ColI蛋白的表达（P＜0.05）。
3.6左归丸含药血清干预Smad4、Runx2和ColI mRNA表达作用及JNK和p38MAPK通路对其影响
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞Smad4、Runx2和ColI mRNA的表达（P＜0.01）；对Smad4和Runx2mRNA表达诱导作用ZG组与BML组比较无统计学意义（P>0.05），对ColI mRNA诱导作用ZG组显著优于BML组（P＜0.01）；加入SP600125后，对ZG诱导Smad4和Runx2mRNA表达的抑制作用没有统计学意义（P>0.05），可以显著抑制ZG诱导的ColI mRNA表达（P＜0.01），对BML诱导的ColI mRNA表达的抑制作用没有统计学意义（P>0.05），可以抑制BML诱导的Smad4和Runx2mRNA的表达（P＜0.05或P＜0.01）；加入SB203580后，对ZG诱导Smad4mRNA表达的抑制作用没有统计学意义（P>0.05），可以显著抑制ZG诱导的Runx2和ColI mRNA表达（P＜0.01），对BML诱导的Smad4mRNA表达的抑制作用没有统计学意义（P>0.05），可以抑制BML诱导的Runx2和ColI mRNA的表达（P＜0.05或P＜0.01）。
结论：
1.左归丸含药血清具有诱导MC3T3-E1细胞增殖、分化和矿化作用。
2.JNK、p38MAPK/Smads信号级联参与MC3T3-E1细胞的增殖与分化。
3.左归丸含药血清可能部分通过活化JNK和p38MAPK通路调控MC3T3-E1细胞功能。
4.左归丸含药血清可能部分通过对JNK、p38MAPK/Smads信号级联的调节而达到促骨形成作用。
Purpose：To explore the mechanisms of ZuoGui pill to promote bone formation and themediation of JNK/p38MAPK/Smads signaling cascade on it.
Material and method：Sixty special pathogen free female Sprague-Dawley rats (180~220g) were randomly distributed into three groups. Rats were orally administratedphysiological saline (negative group), ZGP (1.6g/kg) and Premarin suspension (56.25μg/kg,positive group) twice a day for7days. The serum was collected from femoral artery at thetime of2h after last orally administration, then the serum was inactivated at56°C for30min.Murine MC3T3-E1subclone14preosteoblast cell lines (MC3T3cells) were cultured inα-Minimum Essential Medium (α-MEM) supplemented with10%fetal bovine serum (FBS)containing100U/mL penicillin and100μg/mL streptomycin. Cells were assigned to ninegroups, including control, SP600125, SB203580, Zuogui Pills, Zuogui Pills plus SP600125,Zuogui Pills plus SB203580, premarin, premarin plus SP600125and premarin plus SB203580group. MC3T3cells growth arrested for24h in α-MEM containing0.2%FBS andpenicillin-streptomycin, then cells were preincubated in the absence or presence of a specificinhibitors of JNK or p38MAPK (SP600125or SB203580) for60min, then cultured inmedium containing15%control serum, ZGP and Premarin pharmacological serum in theabsence or presence of10μmol/L SP600125or SB203580for48h. Then the effects of ZGPpharmacological serum on cells were observed. phosphorylated level of JNK and p38wereassessed by western blot analysis and immunofluorescence. Cell viability was assayed byMTT method after the cell cultured with ZGP pharmacological serum for48h. Cell cycle wasanalyzed by flow cytometry after propidium iodide staining. The test for alkaline phosphatase(ALP) activity was carried out with PNPP for48h and ALP staining method on the seventhday. Alizarin red staining method was applied to the detection of the mineralizationdeposition. Runx2, ALP, type I collagen (ColI), Smad2/3, p-Smad2and p-Smad3proteinexpression were assayed by western blot analysis. TGFβ1, Smad4, Runx2and ColI mRNAexpression were evaluated by the method of real-time quantitative reverse transcriptase polymerase chain reaction (Q-PCR).
Results：
1.JNK signaling mediates ZGP pharmacological serum dependent increase of celldifferentiation
1.1phosphorylated level of JNK could be increased by ZGP pharmacological serumsignificantly
The results showed that phosphorylated level of JNK could be promoted by ZGP andpremarin pharmacological serum compared with controls significantly. The effect induced byZGP was better than premarin. The phosphorylated level of JNK induced by ZGPpharmacological serum and premarin was inhibited by SP600125.
1.2JNK signaling mediates ZGP pharmacological serum dependent increase of ALP activityin telophase
As compared with the control group, ZGP and premarin group markedly promoted ALPactivity in48h and7days incubation of MC3T3-E1cells (P＜0.01). ALP activity inducedin48h incubation by ZGP pharmacological serum was better than premarin pharmacologicalserum (P＜0.01). ALP activity induced by ZGP pharmacological serum in48h incubationwas unaffected by SP600125(P>0.05), but ALP activity induced by ZGP pharmacologicalserum in the seventh day incubation was opposed by SP600125.
1.3JNK signaling mediates ZGP pharmacological serum dependent increase of mineralizeddeposition
As compared with the control group, ZGP and premarin group significantly promotedmineralized deposition in14days incubation of MC3T3-E1cells. Mineralized depositioninduced by ZGP and premarin pharmacological serum in the fourteenth day incubation wasopposed by SP600125.
1.4Effects of ZGP pharmacological serum on Runx2and ALP protein expression ofMC3T3-E1cells
As compared with the control group, ZGP and premarin group markedly promoted Runx2andALP protein expression in48h incubation of MC3T3-E1cells. Runx2and ALP proteinexpression induced by ZGP pharmacological serum and Runx2protein expression induced by premarin pharmacological serum in48h incubation was unaffected by SP600125, but ALPprotein expression induced by premarin pharmacological serum in the seventh day incubationwas opposed by SP600125.
2.p38MAPK signaling mediates ZGP pharmacological serum dependent increase of celldifferentiation
2.1phosphorylated level of p38could be increased by ZGP pharmacological serumsignificantly
The results showed that ZGP and premarin pharmacological serum significantly promotedphosphorylated level of p38compared with controls. SB203580inhibited the phosphorylatedlevel of p38induced by ZGP pharmacological serum and premarin via blocking the p38.
2.2p38MAPK signaling mediates ZGP pharmacological serum dependent increase of ALPactivity
As compared with the control group, ZGP and premarin group markedly promoted ALPactivity in48h and7days incubation of MC3T3-E1cells (P＜0.01). ALP activity induced in48h incubation by ZGP pharmacological serum was better than premarin pharmacologicalserum (P＜0.01). ALP activity induced by ZGP and premarin pharmacological serum in48hand7days incubation was opposed by SB203580(P＜0.01).
2.3p38MAPKsignaling mediates ZGP pharmacological serum dependent increase ofmineralized deposition
As compared with the control group, ZGP and premarin group significantly promotedmineralized deposition in14days incubation of MC3T3-E1cells. Mineralized depositioninduced by ZGP and premarin pharmacological serum in the fourteenth day incubation wasopposed by SB203580.
2.4p38MAPKsignaling mediates ZGP pharmacological serum dependent increase of Runx2protein expression
As compared with the control group, ZGP and premarin group markedly promoted Runx2protein expression in48h incubation of MC3T3-E1cells. Runx2protein expression inducedby ZGP and premarin pharmacological serum in48h incubation was opposed by SB203580.3.JNK/p38MAPK/Smads signaling cascade mediates ZGP pharmacological serum dependentincrease of cell functions
3.1TGFβ1mRNA expression was up-regulation induced by ZGP and premarinpharmacological serum in MC3T3-E1cells
As compared with the control group, ZGP and premarin group markedly promoted TGFβ1mRNA expression in48h incubation of MC3T3-E1cells (P＜0.01).
3.2phosphorylated level of JNK and p38could be increased by ZGP pharmacological serumsignificantly
The results showed that phosphorylated level of JNK and p38could be promoted by ZGP andpremarin pharmacological serum compared with controls significantly. The phosphorylatedlevel of JNK and p38induced by ZGP pharmacological serum and premarin were inhibited bySP600125or SB203580.
3.3JNK and p38MAPK signaling mediates ZGP pharmacological serum dependent increaseof proliferation
The results showed that cell proliferation could be stimulated by ZGP and premarinpharmacological serum compared with controls significantly (P<0.01). The effect induced byZGP was better than premarin (P＜0.01). Cell proliferation induced by ZGP pharmacologicalserum and premarin was inhibited by SP600125or SB203580(P＜0.05or P＜0.01).
3.4JNK and p38MAPK signaling mediates ZGP pharmacological serum dependentregulation of different cell cycle phases
The cell cycle results were analyzed and showed that ZGP reduced the percentage of“G1-Period” Cells and dramatically increased the percentage of “G2/M-Period” Cells in CellCycle (P＜0.01), while the percentage of “S-Period” was unaffected (P>0.05). The regulationof different cell cycle phases induced by ZGP pharmacological serum and premarin wasinhibited by SP600125or SB203580(P＜0.01).
3.5JNK and p38MAPK signaling mediates ZGP pharmacological serum dependent increaseof phosphorylated level of Smad2/3and ColI protein expressionAs compared with the control group, ZGP and premarin group markedly promotedphosphorylated level of Smad2/3and ColI protein expression in48h incubation ofMC3T3-E1cells (P＜0.01). phosphorylated level of Smad2induced in48h incubation byZGP pharmacological serum was better than premarin pharmacological serum (P＜0.01) andColI protein expression induced by ZGP pharmacological serum was better than premarin pharmacological serum (P＜0.05). phosphorylated level of Smad3induced by ZGPpharmacological serum was not better than premarin pharmacological serum（P>0.05).phosphorylated level of Smad2and phosphorylated level of Smad3induced by ZGP andpremarin pharmacological serum were opposed by SP600125or SB203580（P＜0.01). ColIprotein expression induced by ZGP pharmacological serum was opposed by SP600125orSB203580(P＜0.01). ColI protein expression induced by premarin pharmacological serumwas opposed by SP600125(P＜0.01), while ColI protein expression induced by premarinpharmacological serum was opposed by SB203580(P＜0.05).
3.6JNK and p38MAPK signaling mediates ZGP pharmacological serum dependent increaseof Smad4, Runx2and ColI mRNA expression
As compared with the control group, ZGP and premarin group markedly promoted Smad4,Runx2and ColI mRNA expression in48h incubation of MC3T3-E1cells (P＜0.01). Smad4and Runx2mRNA expression induced by ZGP was not better than premarin. ColI mRNAexpression induced by ZGP was better than premarin (P＜0.01). Smad4and Runx2mRNAexpression induced by ZGP pharmacological serum were unaffected (P>0.05), while ColImRNA expression induced by ZGP pharmacological serum was opposed by SP600125(P＜0.01). ColI mRNA expression induced by premarin pharmacological serum was unaffected(P>0.05), while Smad4and Runx2mRNA expression induced by premarin pharmacologicalserum were opposed by SP600125(P＜0.05or P＜0.01). Smad4mRNA expression inducedby ZGP pharmacological serum was unaffected (P>0.05), while Runx2and ColI mRNAexpression induced by ZGP pharmacological serum were opposed by SB203580(P＜0.01).Smad4mRNA expression induced by premarin pharmacological serum was unaffected(P>0.05), while Runx2and ColI mRNA expression induced by premarin pharmacologicalserum were opposed by SB203580(P＜0.05or P＜0.01).
Conclusion：
1.Cell proliferation, differentiation and mineralization in MC3T3-E1cells could be inducedby ZGP pharmacological serum.
2.JNK/p38MAPK/Smads signaling cascade were associated with cell proliferation anddifferentiation in MC3T3-E1cells.
3.JNK/p38MAPK signaling mediates ZGP pharmacological serum dependent increase of cellfunctions.
4.JNK/p38MAPK/Smads signaling cascade mediates ZGP pharmacological serum dependentpromotion of bone formation.
材料与方法：采用血清药理学方法，将SPF级SD雌性大鼠60只（180~220g）按体质量随机分为空白对照组、左归丸组和倍美力组，每组各20只，依据人与大鼠等剂量换算公式折算大鼠左归丸等临床剂量为1.6g·Kg-1，大鼠倍美力结合雌激素片的等临床剂量为56.25μg·kg-1，使用前用蒸馏水将药物制成混悬液，空白对照组给予蒸馏水，灌服10ml·Kg-1体质量，2次·d-1，连续灌胃7d，末次给药2h后，麻醉大鼠，腹主动脉取血，室温静置2h，2500r·min-1，4℃离心20min，收集上清，同组血清混匀，56℃水浴灭活30min，制备大鼠含药血清。采用MC3T3-E1成骨细胞系体外研究模型，用含10%FBS的α-MEM培养液（100U·ml-1青霉素和100μg·ml-1链霉素）培养。细胞实验分为空白对照组（control）、SP600125组（SP）、SB203580组（SB）、左归丸组（ZG）、左归丸+SP600125组（ZG+SP）、左归丸+SB203580组（ZG+SB）、倍美力组（BML）、倍美力+SP600125组（BML+SP）、倍美力+SB203580组（BML+SB）。细胞接种24h后，换用含0.2%FBS的α-MEM培养液（100U·ml-1青霉素和100μg·ml-1链霉素），饥饿24h，吸弃旧培养液，加入含或不含JNK、p38MAPK特异抑制剂SP600125、SB203580(10μmol·L-1，溶于DMSO，体积分数<0.1%)的α-MEM培养液（100U·ml-1青霉素和100μg·ml-1链霉素），预处理60min后，各组加入相应血清（阴性对照孔、control组、SP组、SB组加入空白对照大鼠血清，ZG组、ZG+SP组、ZG+SB组加入左归丸大鼠含药血清，BML组、BML+SP组、BML+SB组加入倍美力大鼠含药血清），体积分数为15%，孵育48h。采用蛋白质印迹Western blot测定JNK和p38MAPK磷酸化蛋白表达水平，采用免疫荧光染色法进一步验证JNK和p38MAPK磷酸化蛋白表达水平。采用MTT法检测MC3T3-E1细胞增殖；采用流式细胞术分析细胞周期。采用对硝基苯磷酸盐(PNPP)法和改良钙钴染色法检测MC3T3-E1细胞碱性磷酸酶（ALP）活性；采用茜素红染色法检测MC3T3-E1细胞钙化结节形成。采用Western blot分析OB分泌蛋白情况，检测核结合因子（Runx2）、ALP、Ⅰ型胶原（ColI）、Smad2/3、 p-Smad2和p-Smad3蛋白表达水平。采用Q-PCR技术测定相关基因mRNA表达水平，检测MC3T3-E1细胞转化生长因子β1（TGF-β1）、Smad4、Runx2和ColI mRNA表达水平。
结果：
1.JNK信号通路参与左归丸含药血清诱导MC3T3-E1细胞分化的调控
1.1左归丸含药血清能够诱导MC3T3-E1细胞JNK信号通路活化
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞JNK蛋白磷酸化；ZG显著优于BML；加入SP600125后，显著抑制ZG和BML对JNK蛋白磷酸化的诱导作用。
1.2JNK信号通路主要在分化晚期参与左归丸含药血清对MC3T3-E1细胞ALP活性的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞48h和7d的ALP活性（P＜0.01）；对于48h的ALP活性的影响，ZG组显著优于BML组（P＜0.01）；加入SP600125后，显著抑制BML对48h和7d的ALP活性的刺激作用（P＜0.01），对ZG诱导48h的ALP活性的影响没有统计学意义（P>0.05），但显著抑制了ZG诱导7d的ALP活性。
1.3JNK信号通路参与左归丸含药血清促进MC3T3-E1细胞矿化作用的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞14d的矿化作用；加入SP600125后，显著抑制ZG和BML对矿化作用的诱导。
1.4JNK信号通路对左归丸含药血清干预Runx2和ALP蛋白表达的影响
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞48h的Runx2和ALP蛋白的表达；加入SP600125后，未能对抗ZG诱导48h的Runx2和ALP蛋白表达，也未能对抗BML诱导的Runx2蛋白的表达，但显著抑制了BML诱导的ALP蛋白表达。
2.p38MAPK信号通路参与左归丸含药血清诱导MC3T3-E1细胞分化的调控
2.1左归丸含药血清能够诱导MC3T3-E1细胞p38MAPK信号通路活化
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞p38磷酸化蛋白的表达；加入SB203580后，显著抑制ZG和BML对p38蛋白磷酸化的诱导作用。
2.2p38MAPK信号通路参与左归丸含药血清对MC3T3-E1细胞分化不同阶段ALP活性的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞48h和7d的ALP活性（P＜0.01）；加入SB203580后，显著抑制ZG和BML对48h和7d的ALP活性的刺激作用（P＜0.01）。
2.3p38MAPK信号通路参与左归丸含药血清促进MC3T3-E1细胞14d矿化作用的调控
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞14d矿化作用；加入SB203580后，显著抑制ZG和BML对细胞矿化的诱导作用。
2.4左归丸含药血清通过p38MAPK信号通路的活化上调Runx2蛋白的表达
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞Runx2蛋白的表达；加入SB203580后，显著抑制ZG和BML对Runx2蛋白表达的诱导。
3.JNK、p38MAPK/Smads信号级联参与左归丸含药血清对MC3T3-E1细胞功能的调控
3.1左归丸含药血清上调MC3T3-E1细胞TGFβ1mRNA表达
结果显示，与control组比较，ZG组和BML均组能显著上调MC3T3-E1细胞TGFβ1mRNA的表达（P＜0.01）。
3.2左归丸含药血清诱导MC3T3-E1细胞磷酸化JNK和磷酸化p38蛋白表达
结果显示，与control组比较，ZG组和BML组均能显著诱导MC3T3-E1细胞磷酸化JNK和p38蛋白的表达；ZG组明显优于BML组；加入SP600125或SB203580后，显著抑制ZG和BML对磷酸化JNK或p38蛋白的诱导作用。
3.3JNK和p38MAPK信号通路参与左归丸含药血清干预MC3T3-E1细胞增殖的调控
结果显示，与control组比较，ZG和BML均能显著促进MC3T3-E1细胞增殖（P＜0.01）；ZG组显著优于BML组（P＜0.01）；加入SP600125或SB203580后，抑制ZG组和BML组对MC3T3-E1细胞增殖的诱导作用（P＜0.05或P＜0.01）。
3.4JNK和p38MAPK信号通路参与左归丸含药血清干预MC3T3-E1细胞周期分布的调控
细胞周期分析结果显示，与control组比较，ZG组G1期的细胞百分率显著下降（P＜0.01），S期的细胞百分率变化不显著（P>0.05），G2/M期的细胞百分率显著增加（P＜0.01）；与ZG组比较，ZG+SP组和ZG+SB组显著抵抗了ZG对细胞周期的调控作用（P＜0.01）。
3.5左归丸含药血清活化JNK和p38MAPK信号通路有助于磷酸化Smad2/3和上调ColI蛋白表达
结果显示，与control组比较，ZG和BML均能显著诱导MC3T3-E1细胞Smad2/3蛋白磷酸化和上调ColI蛋白表达水平（P＜0.01）；对p-Smad2的诱导作用，ZG组显著优于BML组（P＜0.01），对ColI蛋白表达的上调作用，ZG组优于BML组（P＜0.05），对p-Smad3的诱导作用，两组比较无统计学意义（P>0.05）；加入SP600125或SB203580后，显著抑制ZG和BML对MC3T3-E1细胞Smad2/3蛋白磷酸化的诱导作用（P＜0.01），显著抑制ZG对MC3T3-E1细胞ColI蛋白表达的上调作用（P＜0.01），加入SP600125后显著抑制BML诱导的ColI蛋白表达（P＜0.01），加入SB203580后抑制BML诱导的ColI蛋白的表达（P＜0.05）。
3.6左归丸含药血清干预Smad4、Runx2和ColI mRNA表达作用及JNK和p38MAPK通路对其影响
结果显示，与control组比较，ZG和BML均能显著上调MC3T3-E1细胞Smad4、Runx2和ColI mRNA的表达（P＜0.01）；对Smad4和Runx2mRNA表达诱导作用ZG组与BML组比较无统计学意义（P>0.05），对ColI mRNA诱导作用ZG组显著优于BML组（P＜0.01）；加入SP600125后，对ZG诱导Smad4和Runx2mRNA表达的抑制作用没有统计学意义（P>0.05），可以显著抑制ZG诱导的ColI mRNA表达（P＜0.01），对BML诱导的ColI mRNA表达的抑制作用没有统计学意义（P>0.05），可以抑制BML诱导的Smad4和Runx2mRNA的表达（P＜0.05或P＜0.01）；加入SB203580后，对ZG诱导Smad4mRNA表达的抑制作用没有统计学意义（P>0.05），可以显著抑制ZG诱导的Runx2和ColI mRNA表达（P＜0.01），对BML诱导的Smad4mRNA表达的抑制作用没有统计学意义（P>0.05），可以抑制BML诱导的Runx2和ColI mRNA的表达（P＜0.05或P＜0.01）。
结论：
1.左归丸含药血清具有诱导MC3T3-E1细胞增殖、分化和矿化作用。
2.JNK、p38MAPK/Smads信号级联参与MC3T3-E1细胞的增殖与分化。
3.左归丸含药血清可能部分通过活化JNK和p38MAPK通路调控MC3T3-E1细胞功能。
4.左归丸含药血清可能部分通过对JNK、p38MAPK/Smads信号级联的调节而达到促骨形成作用。
Purpose：To explore the mechanisms of ZuoGui pill to promote bone formation and themediation of JNK/p38MAPK/Smads signaling cascade on it.
Material and method：Sixty special pathogen free female Sprague-Dawley rats (180~220g) were randomly distributed into three groups. Rats were orally administratedphysiological saline (negative group), ZGP (1.6g/kg) and Premarin suspension (56.25μg/kg,positive group) twice a day for7days. The serum was collected from femoral artery at thetime of2h after last orally administration, then the serum was inactivated at56°C for30min.Murine MC3T3-E1subclone14preosteoblast cell lines (MC3T3cells) were cultured inα-Minimum Essential Medium (α-MEM) supplemented with10%fetal bovine serum (FBS)containing100U/mL penicillin and100μg/mL streptomycin. Cells were assigned to ninegroups, including control, SP600125, SB203580, Zuogui Pills, Zuogui Pills plus SP600125,Zuogui Pills plus SB203580, premarin, premarin plus SP600125and premarin plus SB203580group. MC3T3cells growth arrested for24h in α-MEM containing0.2%FBS andpenicillin-streptomycin, then cells were preincubated in the absence or presence of a specificinhibitors of JNK or p38MAPK (SP600125or SB203580) for60min, then cultured inmedium containing15%control serum, ZGP and Premarin pharmacological serum in theabsence or presence of10μmol/L SP600125or SB203580for48h. Then the effects of ZGPpharmacological serum on cells were observed. phosphorylated level of JNK and p38wereassessed by western blot analysis and immunofluorescence. Cell viability was assayed byMTT method after the cell cultured with ZGP pharmacological serum for48h. Cell cycle wasanalyzed by flow cytometry after propidium iodide staining. The test for alkaline phosphatase(ALP) activity was carried out with PNPP for48h and ALP staining method on the seventhday. Alizarin red staining method was applied to the detection of the mineralizationdeposition. Runx2, ALP, type I collagen (ColI), Smad2/3, p-Smad2and p-Smad3proteinexpression were assayed by western blot analysis. TGFβ1, Smad4, Runx2and ColI mRNAexpression were evaluated by the method of real-time quantitative reverse transcriptase polymerase chain reaction (Q-PCR).
Results：
1.JNK signaling mediates ZGP pharmacological serum dependent increase of celldifferentiation
1.1phosphorylated level of JNK could be increased by ZGP pharmacological serumsignificantly
The results showed that phosphorylated level of JNK could be promoted by ZGP andpremarin pharmacological serum compared with controls significantly. The effect induced byZGP was better than premarin. The phosphorylated level of JNK induced by ZGPpharmacological serum and premarin was inhibited by SP600125.
1.2JNK signaling mediates ZGP pharmacological serum dependent increase of ALP activityin telophase
As compared with the control group, ZGP and premarin group markedly promoted ALPactivity in48h and7days incubation of MC3T3-E1cells (P＜0.01). ALP activity inducedin48h incubation by ZGP pharmacological serum was better than premarin pharmacologicalserum (P＜0.01). ALP activity induced by ZGP pharmacological serum in48h incubationwas unaffected by SP600125(P>0.05), but ALP activity induced by ZGP pharmacologicalserum in the seventh day incubation was opposed by SP600125.
1.3JNK signaling mediates ZGP pharmacological serum dependent increase of mineralizeddeposition
As compared with the control group, ZGP and premarin group significantly promotedmineralized deposition in14days incubation of MC3T3-E1cells. Mineralized depositioninduced by ZGP and premarin pharmacological serum in the fourteenth day incubation wasopposed by SP600125.
1.4Effects of ZGP pharmacological serum on Runx2and ALP protein expression ofMC3T3-E1cells
As compared with the control group, ZGP and premarin group markedly promoted Runx2andALP protein expression in48h incubation of MC3T3-E1cells. Runx2and ALP proteinexpression induced by ZGP pharmacological serum and Runx2protein expression induced by premarin pharmacological serum in48h incubation was unaffected by SP600125, but ALPprotein expression induced by premarin pharmacological serum in the seventh day incubationwas opposed by SP600125.
2.p38MAPK signaling mediates ZGP pharmacological serum dependent increase of celldifferentiation
2.1phosphorylated level of p38could be increased by ZGP pharmacological serumsignificantly
The results showed that ZGP and premarin pharmacological serum significantly promotedphosphorylated level of p38compared with controls. SB203580inhibited the phosphorylatedlevel of p38induced by ZGP pharmacological serum and premarin via blocking the p38.
2.2p38MAPK signaling mediates ZGP pharmacological serum dependent increase of ALPactivity
As compared with the control group, ZGP and premarin group markedly promoted ALPactivity in48h and7days incubation of MC3T3-E1cells (P＜0.01). ALP activity induced in48h incubation by ZGP pharmacological serum was better than premarin pharmacologicalserum (P＜0.01). ALP activity induced by ZGP and premarin pharmacological serum in48hand7days incubation was opposed by SB203580(P＜0.01).
2.3p38MAPKsignaling mediates ZGP pharmacological serum dependent increase ofmineralized deposition
As compared with the control group, ZGP and premarin group significantly promotedmineralized deposition in14days incubation of MC3T3-E1cells. Mineralized depositioninduced by ZGP and premarin pharmacological serum in the fourteenth day incubation wasopposed by SB203580.
2.4p38MAPKsignaling mediates ZGP pharmacological serum dependent increase of Runx2protein expression
As compared with the control group, ZGP and premarin group markedly promoted Runx2protein expression in48h incubation of MC3T3-E1cells. Runx2protein expression inducedby ZGP and premarin pharmacological serum in48h incubation was opposed by SB203580.3.JNK/p38MAPK/Smads signaling cascade mediates ZGP pharmacological serum dependentincrease of cell functions
3.1TGFβ1mRNA expression was up-regulation induced by ZGP and premarinpharmacological serum in MC3T3-E1cells
As compared with the control group, ZGP and premarin group markedly promoted TGFβ1mRNA expression in48h incubation of MC3T3-E1cells (P＜0.01).
3.2phosphorylated level of JNK and p38could be increased by ZGP pharmacological serumsignificantly
The results showed that phosphorylated level of JNK and p38could be promoted by ZGP andpremarin pharmacological serum compared with controls significantly. The phosphorylatedlevel of JNK and p38induced by ZGP pharmacological serum and premarin were inhibited bySP600125or SB203580.
3.3JNK and p38MAPK signaling mediates ZGP pharmacological serum dependent increaseof proliferation
The results showed that cell proliferation could be stimulated by ZGP and premarinpharmacological serum compared with controls significantly (P<0.01). The effect induced byZGP was better than premarin (P＜0.01). Cell proliferation induced by ZGP pharmacologicalserum and premarin was inhibited by SP600125or SB203580(P＜0.05or P＜0.01).
3.4JNK and p38MAPK signaling mediates ZGP pharmacological serum dependentregulation of different cell cycle phases
The cell cycle results were analyzed and showed that ZGP reduced the percentage of“G1-Period” Cells and dramatically increased the percentage of “G2/M-Period” Cells in CellCycle (P＜0.01), while the percentage of “S-Period” was unaffected (P>0.05). The regulationof different cell cycle phases induced by ZGP pharmacological serum and premarin wasinhibited by SP600125or SB203580(P＜0.01).
3.5JNK and p38MAPK signaling mediates ZGP pharmacological serum dependent increaseof phosphorylated level of Smad2/3and ColI protein expressionAs compared with the control group, ZGP and premarin group markedly promotedphosphorylated level of Smad2/3and ColI protein expression in48h incubation ofMC3T3-E1cells (P＜0.01). phosphorylated level of Smad2induced in48h incubation byZGP pharmacological serum was better than premarin pharmacological serum (P＜0.01) andColI protein expression induced by ZGP pharmacological serum was better than premarin pharmacological serum (P＜0.05). phosphorylated level of Smad3induced by ZGPpharmacological serum was not better than premarin pharmacological serum（P>0.05).phosphorylated level of Smad2and phosphorylated level of Smad3induced by ZGP andpremarin pharmacological serum were opposed by SP600125or SB203580（P＜0.01). ColIprotein expression induced by ZGP pharmacological serum was opposed by SP600125orSB203580(P＜0.01). ColI protein expression induced by premarin pharmacological serumwas opposed by SP600125(P＜0.01), while ColI protein expression induced by premarinpharmacological serum was opposed by SB203580(P＜0.05).
3.6JNK and p38MAPK signaling mediates ZGP pharmacological serum dependent increaseof Smad4, Runx2and ColI mRNA expression
As compared with the control group, ZGP and premarin group markedly promoted Smad4,Runx2and ColI mRNA expression in48h incubation of MC3T3-E1cells (P＜0.01). Smad4and Runx2mRNA expression induced by ZGP was not better than premarin. ColI mRNAexpression induced by ZGP was better than premarin (P＜0.01). Smad4and Runx2mRNAexpression induced by ZGP pharmacological serum were unaffected (P>0.05), while ColImRNA expression induced by ZGP pharmacological serum was opposed by SP600125(P＜0.01). ColI mRNA expression induced by premarin pharmacological serum was unaffected(P>0.05), while Smad4and Runx2mRNA expression induced by premarin pharmacologicalserum were opposed by SP600125(P＜0.05or P＜0.01). Smad4mRNA expression inducedby ZGP pharmacological serum was unaffected (P>0.05), while Runx2and ColI mRNAexpression induced by ZGP pharmacological serum were opposed by SB203580(P＜0.01).Smad4mRNA expression induced by premarin pharmacological serum was unaffected(P>0.05), while Runx2and ColI mRNA expression induced by premarin pharmacologicalserum were opposed by SB203580(P＜0.05or P＜0.01).
Conclusion：
1.Cell proliferation, differentiation and mineralization in MC3T3-E1cells could be inducedby ZGP pharmacological serum.
2.JNK/p38MAPK/Smads signaling cascade were associated with cell proliferation anddifferentiation in MC3T3-E1cells.
3.JNK/p38MAPK signaling mediates ZGP pharmacological serum dependent increase of cellfunctions.
4.JNK/p38MAPK/Smads signaling cascade mediates ZGP pharmacological serum dependentpromotion of bone formation.
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