Based On JNK, P38 / Smads Signal Cascade Research Zuoguiwan Of MC3T3-E1 Cells Containing Serum Regulatory Mechanism

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 celldifferentiation1.1phosphorylated level of JNK could be increased by ZGP pharmacological serumsignificantlyThe 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 telophaseAs 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 mineralizeddepositionAs 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-E1cellsAs 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 celldifferentiation2.1phosphorylated level of p38could be increased by ZGP pharmacological serumsignificantlyThe 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 ALPactivityAs 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 depositionAs 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 expressionAs 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-E1cellsAs 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 serumsignificantlyThe 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 proliferationThe 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 phasesThe 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 expressionAs 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.