Left To Pill Containing Medicine Serum Intervention Mc3t3 E1 Cell Erk1/2 Research Of Signaling Pathways

Purpose:Based on the theory that kidney stores the essence, control bone and generate marrow, we were to study the role of Zuogui pill in the proliferation and differentiation of MC3T3-E1cell of mice osteogenesis precursor cells through the serum pharmacology method.We were required to explore the interaction mechanisms of Zuogui Pill to osteoblasts via ERK1/2signaling pathway. Furthermore, We wanted to clarify the cross linking role between ERK1/2signaling pathway and TGF-β1/Smads signaling pathway in the intervention of Zuogui pill to osteoblasts.Material and method:One hundred and fifty four SD female rats, SPF class,180～200g, were randomly stratified by weight, and each layer was divided into5groups according to quantity:Control group with30rats, Zuogui pill high, medium and low dose group [ZG (H, M, L)] with31each, BML group with31. According to the clinical dose conversion, the rats were orally administered medicine for7days and twice a day:Zuogui groups with different dose of Zuogui pill suspension liquid, BML group with Conjugated Estrogens suspension liquid, and control group with distilled water. Two hours after the eighth day’s irrigating, rats were anesthetized by10%hydration chlorine aldehyde intraperitoneal injection. And then, the medicated serum was separated by abdominal aortic blood, stalled for2h at room temperature, centrifuged for20min at4℃and2500rpm,and the supernate was collected.Following, the medicated serum was inactivated for30min in56℃water bath, aseptic filtrated by0.22um membrane filter, packed in1.5m! EP tube, and preserved at-86℃. Before using, the medicated serum was diluted to necessary concentration with α-MEM, and saved at4℃. MC3T3-E1cell proliferation was tested by MTT assay. Modified calcium and cobalt dyeing method was applied to detect alkaline phosphatase (ALP) production of MC3T3-E1cell. The calcified nodules of MC3T3-E1cell were observed by the way of alizarin red dyeing. The Cbf a1, COL Ⅰ, TGF-β1, Smad4and Smad2mRNA expression of MC3T3-E1cell were detected by Real Time RT-PCR. The Cbf a1, COL Ⅰ, TGF-β1, Smad4, ERK1/2and p-ERK1/2protein production were analyzed with Western blotting. Results:1Effects of Zuogui Pill Medicated Serum on MC3T3-E1cell proliferationThe effect of Zuogui Pill Medicated Serum on MC3T3-E1cell proliferation was depended on the dose and interaction time. Therein, compared with that of10%or20%, the effect of15%(v/v) ZG(H、M、L) were all significant. Compared with the rest of15%groups, treated with15%ZG(L) for48h, MC3T3-E1cell proliferation increased significantly (P<0.01). Unfortunately, there was no significant difference detection compared with BML (P>0.05).Therefore,15%ZG (L)(marked with ZG (L)) would be used for experiment latar.Incubation with15%ZG (L) for48h, MC3T3-E1cell proliferation increased significantly vs control (P<0.01), but there was no significant difference vs BML. PD98059significantly reversed the effect of ZG (L) on MC3T3-E1cell proliferation (P<0.01).2Effects of Zuogui Pill Medicated Serum on MC3T3-E1cell differentiation induced by ERK1/2pathway2.1The effect of Zuogui Pill Medicated Serum on MC3T3-E1cell ALP production induced by ERK1/2pathwayAfter treated by modified calcium and cobalt dyeing, ALP of MC3T3-E1cell was dyed brownish black tiny particles except for the negative control group. The result shows that, after exploring to15%ZG (L) or BML for14days, the cell density and brownish black tiny particles increased, and the cell synapses became more and longer in contrast with control. The characterization was significantly against by the addition of PD98059.2.2The effect of Zuogui Pill Medicated Serum on MC3T3-E1cell calcified nodules induced by ERK1/2pathwayThe calcified nodules are dyed red by alizarin red dyeing. After exploring to15%ZG (L) or BML for14days, calcified nodules of MC3T3-E1cell obviously increased compared with control. The phenomena were antagonized by PD98059in a certain extent.2.3The effect of Zuogui Pill Medicated Serum on MC3T3-E1cell Cbf α1and COL Ⅰ mRNA and protein expression induced by ERK3/2pathwayCompared with control, Cbf α1and COL Ⅰ mRNA and protein expression of MC3T3-E1cell were increased (P<0.01, P<0.05) by15%ZG (L) or BML.The increase was induced after the addition of PD98059vs that without PD98059(P<0.05).3Effects of Zuogui Pill Medicated Serum on ERK1/2and TGF-β1/Smads signaling pathway of MC3T3-E1cell3.1The level of ERK1/2protein phosphorylation of MC3T3-E1cell induced by Zuogui Pill Medicated SerumCompared with control, ERK1/2protein phosphorylation was obviously inducted by ZG (L) or BML, and that of ZG(L) was stronger than BML group (P<0.01). The level of ERK1/2protein phosphorylation was obviously weakened after the addition of PD98059compared with that of no PD98059(P<0.05).3.2The effect of Zuogui Pill Medicated Serum on MC3T3-E1cell TGF-β1、Smad4mRNA and protein, Smad2mRNA expression induced by ERK1/2pathwayIn contrast with control, both ZG (L) and BML significantly raise TGF-β1, Smad4mRNA and protein expression and Smad2mRNA expression (P<0.05or P<0.01). Compared with that of no PD98059, all of the above were decreased rather than TGF-β1mRNA and protein expression(P<0.05or P<0.01). In detail, TGF-β1protein production was elevated by PD98059,but there was no significant differences. However, TGF-β1mRNA expression was marked increased by PD98059(P<0.05).Conclusion:1. Zuogui Pill Medicated Serum can promote MC3T3-E1cell proliferation and differentiation.2. ERK1/2signaling pathway involves in the proliferation and differentiation of MC3T3-E1cell.3. Zuogui Pill regulates osteoblast proliferation and differentiation via ERK1/2and TGF-β1/Smads signaling pathways, which could be one of the mechanisms that Zuogui Pill effectively prevents and treats osteoporosis.