Study On Osteogenesis Mechanism Of Bone Marrow Mesenchymal Stem Cells Induced By Left And Right Pills By MAPK / TGF-β1 / Smads Signal Cascade

PurposeBasing on the theory of “kidney governing bones， generating marrow” to explore therepresentative formulas Zuogui Wan and Yougui Wan based on the theory of nourishingkidney-Yin and tonifying kidney-Yang can promote osteogenesis of bone marrowmesenchymal stem cells (BMSCs), to compare the osteogenesis of BMSCs by Zuogui Wanand Yougui Wan, and to expound the relationship of bone formation and the representativeformulas Zuogui Wan based on the theory of nourishing kidney-Yin and Yougui Wan by theeffect of MAPK/TGF-β1/Smads signaling cascade on the osteogenesis of BMSCs.Materials and methodsTwo hundreds and ten special pathogen free Sprague-Dawley rats（210±10g）wererandomly distributed into seven groups. There are Zuogui Wan group, Yougui Wan group,mutual drugs of Zuogui Wan and Yougui Wan group, Nourishing kidney-Yin drugs group,Tonifying kidney-Yang drugs group, positive control drug (Estradiol Valerate, BJL) group andcontrol group. Thirty rats were in each group, male and female were divided equally betweengroups. The method of calculation accords to the principle of1kg rat intaking in6.3timesdrugs of the same weight of a human. Four days later, we got the medicated serum from theaorta abdominalis, centrifuged (2500r·min-1,4oC,20min) and inactivated in the water of56oC,gathered them respectively at last. Firstly, we used the chromatographic column Thermo(4.6×250mm,5μm) with the mixed solvents of acetonitrile: water=11:89. The flow rate was1.0mL·min-1. Column temperature was30℃. The eluate was monitored with UV absorptionat240nm．Under the condition, the loganin of Zuogui Wan and Yougui Wan and theirdisassembled prescriptions was tested both in vitro and vivo. Next all groups of the medicatedserum were separately adding inducer (dexamethasone, vitamin C, sodium β-glycerophosphate) to every group and the other group of the inducer group to simultaneouslyintervene the BMSCs. Then, to test the alkaline phosphatase (ALP) activity by the means ofmodified calcium-combat staining; to count the mineralization nodules with alizarin red staining, to detect the expressing of Cbfα1and COLⅠmRNA by RT-PCR, to show theexpression of core binding factor alpha1(Cbfα1)and typeⅠcollagen(COLⅠ) protein withthe method of Western blot.Then we got the better results of Zuogui Wan group, Yougui Wangroup and Nourishing kidney-Yin drugs group, adding the control group to go on the study.There were eight groups in the experiment of the study on ERK signal pathway: Control,Control+PD98059group (Control+PD), Zuogui Wan group (ZGW), Zuogui Wan+PD98059group (ZGW+PD), Yougui Wan group (YGW), Yougui Wan+PD98059group (YGW+PD),Nourishing kidney-Yin drugs group (ZYY), Nourishing kidney-Yin drugs+PD98059group(ZYY+PD). Also there were eight groups in the experiment of the study on JNK signalpathway: Control, Control+SP600125group (Control+SP), Zuogui Wan group (ZGW),Zuogui Wan+SP600125group (ZGW+SP), Yougui Wan group (YGW), Yougui Wan+SP600125group (YGW+SP), Nourishing kidney-Yin drugs group (ZYY), Nourishingkidney-Yin drugs+SP600125group (ZYY+SP). Then the P4BMSCs were seeded andcultured with starvation media for about24h.After24h, the a-MEM media was instead ofrelevant medicated serum with or without10μM PD98059or10μM SP600125, theduration was9/days. To test the alkaline phosphatase (ALP)activity by the means ofmodified calcium-combat staining； to oget the mineralization nodules with alizarin redstaining, to detect the expression of TGF-β1、 Smad4、 Cbf α1、 COL Ⅰ mRNA byRT-PCR,moreover,to show the expression of TGF-β1、Smad4、Cbfα1、COLⅠ、p-ERK1/2and p-JNK protein with the method of Western blot and to use immunofluoresent assay toobserve the expression of p-ERK1/2.Results1The loganin of Zuogui Wan and Yougui Wan and their disassembled prescriptions wastested both in vitro and vivo.The results showed that the peak time of loganin was13.057and the loganin could befound in the groups of decoction and medicated serum of ZGW, YGW, GTY and ZYY groups,but could not be got in the decoction and medicated serum of BYY group and medicatedserum of control group. 2Research on Zuogui Wan, Yougui Wan and their disassembled prescriptions on osteogenic differenti ati on of BM S C s.2.1Effect of Zuogui Wan, Yougui Wan and their disassembled prescriptions on osteogenesis in bone marrow mesenchymal stem cells.Enhancement of ALP activity and mineralized nodule formation were early and late signs of bone formation, Thus, what the effect of Zuogui Wan, Yougui Wan and their disassembled prescriptions on BMSCs ALP activity and mineralized nodule, can reflect the osteogenic capacity of BMSCs by Zuogui Wan, Yougui Wan and their disassembled prescriptions.2.1.1BMSCs ALP production induced by serum containing Zuogui Wan, Yougui Wan and their disassembled prescriptions.The results showed that ZGW+YDJ>ZYY+YDJ groups had a significant increase in ALP activity of BMSCs as compared with the control and YDJ groups (p<0.05); Meanwhile, ZGW+YDJ group promoting the expression of BMSCs ALP activity than the other groups, but no significant difference ZYY+YDJ group.2.1.2BMSCs mineralization induced by serum containing Zuogui Wan, Yougui Wan and their disassembled prescriptions.The results showed that ZGW+YDJ> ZYY+YDJ groups had a significant increase in mineralization of BMSCs as compared with the control and YDJ groups (p<0.05); Meanwhile, ZGW+YDJ group promoting the expression of BMSCs mineralization than the other groups, but no significant difference ZYY+YDJ group.2.2Zuogui Wan and Yougui Wan and their disassembled prescriptions effect on BMSCs Cbf a1and COL I.Cbf a1is a key transcription factor of BMSCs osteogenic differentiation,also is the earliest and most specific marker of bone formation, and COL I is the only type of collagen of the mineralized bone, also is an important indicator of bone formation.It could been seen that Zuogui Wan and Yougui Wan and their disassembled prescriptions have an impact on BMSCs Cbf a1and COL I, and also reflects the Zuogui Wan and Yougui Wan and their disassembled prescriptions effect on BMSCs osteoblast differentiation.2.2.1BMSCs Cbf a1mRNA and protein expression induced by serum containing Zuogui Wan,Yougui Wan and their disassembled prescriptions. The results showed that ZGW+YDJ、ZYY+YDJ groups had a significant increase in Cbfα1mRNA and protein expression of BMSCs as compared with the control and YDJ groups（p<0.05）；Meanwhile, ZGW+YDJ group promoting the expression of BMSCs Cbfα1mRNA and protein expression than the other groups, but no significant difference ZYY+YDJgroup.2.2.2BMSCs COLⅠmRNA and protein expression induced by serum containing ZuoguiWan, Yougui Wan and their disassembled prescriptionsThe results showed that ZGW+YDJ、ZYY+YDJ groups had a significant increase inCOLⅠmRNA and protein expression of BMSCs as compared with the control and YDJgroups（p<0.05）；Meanwhile, ZGW+YDJ group promoting the expression of BMSCs COLⅠmRNA and protein expression than the other groups, but no significant difference ZYY+YDJ group.3Osteogenesis of Bone Mesenchymal Stem Cells Induced by serum containing Zuigui Wanand Yougui Wan via ERK/TGF-β1/Smads Signal Cascade.3.1The osteogenic differentiation of BMSCs induced by Zuogui Wan and Yougui Wancontaining serum via ERK signaling pathway.3.1.1Expression of phosphorylation of ERK protein of BMSCs by serum containing ZuoguiWan, Yougui Wan.The result showed: As compared with the control group, the ZGW, YGW and ZYYgroups showed enhanced levels of p-JNK. Compared with the ZGW group, the YGW groupshowed enhanced levels of p-JNK, but was much lower than the ZGW group. The expressionof p-JNK protein of BMSCs was also weakened after adding PD98095to each group (P<0.01or P<0.05).3.1.2BMSCs ALP production induced by serum containing Zuogui Wan, Yougui Wan byERK signaling pathway.The results showed that the ZGW, YGW and ZYY groups showed a significant increasein ALP activity of BMSCs as compared with the control group. The effect of augmenting ALPactivity in BMSCs by the YGW treated group was inferior to the ZGW treated group.Moreover, the ALP activity was markedly weakened after adding PD98059to all groups(P<0.01or P<0.05). 3.1.3BMSCs mineralization induced by serum containing Zuogui Wan,Yougui Wan by ERK signaling pathway.The results showed that The ZGW, YGW and ZYY groups significantly promoted mineral nodule formation of BMSCs as compared with the control group. The effect of promoting mineral nodule formation in BMSCs following treatment by YGW and ZYY groups was inferior to the ZGW treated group. Moreover, the level of forming mineral nodules decreased after adding PD98059in all groups (P<0.01or P<0.05).3.1.4Expression of Cbf a1mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by ERK signaling pathway.The results showed that the expression of Cbf a1mRNA and protein was increased in the ZGW, YGW and ZYY groups, as compared with the control group. The enhanced expression of Cbf a1mRNA and protein of BMSCs in the YGW group was inferior to the ZGW group. Additionally, the expression of Cbf a1mRNA and protein of BMSCs was weakened after adding PD98095to each group (P<0.01or P<0.05).3.1.5Expression of COL I mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by ERK signaling pathway.The results showed that The expression levels of COLI mRNA and protein were increased by the ZGW, YGW and ZYY groups as compared with the control group. The YGW group showed weakenly improved COLI mRNA and protein expression in BMSCs and the ZYY group only weakenly improved COLI mRNA. Additionally, the expression of COLI mRNA and protein in BMSCs was weakened after adding PD98059to each group (P<0.01or P<0.05).3.2The osteogenic differentiation of BMSCs induced by Zuogui Wan and Yougui Wan containing serum via ERK/TGF-β1/Smads signaling cascade.3.2.1Expression of TGF-β1mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by ERK/TGF-β1/Smads signaling cascade.The results showed that the TGF-β1mRNA and protein expression of BMSCs. As compared with the control group,the ZGW, YGW and ZYY groups showed enhanced TGF-β1mRNA and protein expression in BMSCs. However, as compared with the ZGW group, the enhanced levels of TGF-β1mRNA and protein seen in the YGW group and TGF-β1protein seen in the ZYY group were decreased. Furthermore, the expression of TGF-β1mRNA andprotein in BMSCs was dampened after adding PD98059to each group (P<0.01or P<0.05).3.2.2Expression of Smad4mRNA and protein of BMSCs by serum containing Zuogui Wan,Yougui Wan by ERK/TGF-β1/Smads signaling cascadeThe results showed that the Smad4mRNA and protein expression of BMSCs. The ZGW,YGW and ZYY groups were showed to enhance Smad4mRNA and protein expression inBMSCs as compared with the control group. However, as compared with the ZGW group, theenhanced levels of Smad4mRNA and protein expression seen in the YGW group weredecreased. With respect to the ZYY group, only the enhanced level Smad4mRNA wasinferior as compared with the ZGW group. Furthermore, the expression of Smad4mRNA andprotein in BMSCs was weakened after adding PD98059to each group (P<0.01or P<0.05).4Osteogenesis of Bone Mesenchymal Stem Cells Induced by serum containing Zuigui Wanand Yougui Wan via JNK/TGF-β1/Smads Signal Cascade.4.1The osteogenic differentiation of BMSCs induced by Zuogui Wan and Yougui Wancontaining serum via JNK signaling pathway.4.1.1Expression of phosphorylation of JNK protein of BMSCs by serum containing ZuoguiWan, Yougui Wan by JNK signaling pathway.The result showed: As compared with the control group, the ZGW, YGW and ZYYgroups showed enhanced levels of p-JNK. Compared with the ZGW group, the YGW andZYY groups showed enhanced levels of p-JNK, but was much lower than the ZGW group.The expression of p-JNK protein of BMSCs was also weakened after adding SP600125toeach group (P<0.01or P<0.05).4.1.2BMSCs ALP production induced by serum containing Zuogui Wan,Yougui Wan by JNKsignaling pathway.The results showed that the ZGW, YGW and ZYY groups showed a significant increasein ALP activity of BMSCs as compared with the control group. The effect of augmenting ALPactivity in BMSCs by the YGW treated group was inferior to the ZGW treated group.Moreover, the ALP activity was markedly weakened after adding SP600125to all groups(P<0.01or P<0.05). 4.1.3BMSCs mineralization induced by serum containing Zuogui Wan,Yougui Wan by JNK signaling pathway.The results showed that The ZGW, YGW and ZYY groups significantly promoted mineral nodule formation of BMSCs as compared with the control group. The effect of promoting mineral nodule formation in BMSCs following treatment by YGW was inferior to the ZGW treated group. Moreover,the level of forming mineral nodules decreased after adding SP600125in all groups (P<0.01or P<0.05).4.1.4Expression of Cbf a1mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by JNK signaling pathway.The results showed that the expression of Cbf a1mRNA and protein was increased in the ZGW, YGW and ZYY groups, as compared with the control group. The enhanced expression of Cbf a1mRNA and protein of BMSCs in the YGW and ZYY groups was inferior to the ZGW group. Additionally, the expression of Cbf a1mRNA and protein of BMSCs was weakened after adding SP600125to each group (P<0.01or P<0.05).4.1.5Expression of COL I mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by JNK signaling pathway by JNK signaling pathway.The results showed that The expression levels of COLI mRNA and protein were increased by the ZGW, YGW and ZYY groups as compared with the control group. The YGW group showed improved COLI mRNA and protein expression in BMSCs and the ZYY group only showed improved COLI mRNA expression in BMSCs. Additionally, the expression of COLI mRNA and protein in BMSCs was weakened after adding SP600125to each group (P<0.01or P<0.05).4.2The osteogenic differentiation of BMSCs induced by Zuogui Wan and Yougui Wan containing serum via JNK/TGF-β1/Smads signaling cascade.4.2.1Expression of TGF-β1mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by JNK/TGF-β1/Smads signaling cascade.The results showed that the TGF-β1mRNA and protein expression of BMSCs. As compared with the control group, the ZGW, YGW and ZYY groups showed enhanced TGF-β1mRNA and protein expression in BMSCs. However, as compared with the ZGW group, the enhanced levels of TGF-β1mRNA and protein seen in the YGW and ZYY group were decreased. Furthermore, the expression of TGF-β1mRNA and protein in BMSCs was dampened after adding SP600125to each group (P<0.01or P<0.05).4.2.2Expression of Smad4mRNA and protein of BMSCs by serum containing Zuogui Wan, Yougui Wan by JNK/TGF-β1/Smads signaling cascade.The results showed that the Smad4mRNA and protein expression of BMSCs. The ZGW, YGW and ZYY groups were showed to enhance Smad4mRNA and protein expression in BMSCs as compared with the control group. However, as compared with the ZGW group, the enhanced levels of Smad4mRNA and protein expression seen in the YGW group were decreased. With respect to the ZYY group, only the enhanced level Smad4mRNA was inferior as compared with the ZGW group. Furthermore, the expression of Smad4mRNA and protein in BMSCs was weakened after adding SP600125to each group (P<0.01or P<0.05).Conclusions1Zuogui Wan and Yougui Wan and their disassembled prescriptions contained serum still retains loganin that is one of prototype components, which showed the partly consistency between the decoction and medicated serum of Zuogui Wan and Yougui Wan and their disassembled prescriptions.2Serum containing Zuogui Wan, Yougui Wan and their disassembled prescriptions can help inducer on osteogenesis in BMSCs,and Zuogui Wan and Yin-nourishing drugs of Zuogui Wan are better, which indicates that Zuogui Wan and Yin-nourishing drugs of Zuogui Wan based on the theory of nourishing kidney-Yin is superior to Yuogui Wan and Yang-tonifying drugs of Yuogui Wan based on the theory of tonifying kidney-Yang.3ERK、JNK MAPK were taken part in the process of osteogenesis of BMSCs by Serum containing Zuogui Wan, Yougui Wan.4Zuogui Wan based on the theory of nourishing kidney-Yin and Yuogui Wan based on the theory of tonifying kidney-Yang can induce the osteogenesis of BMSCs via ERK、 JNK/TGF-β1/Smads signal cascade.