Erythropoietin Treatment Enhances Inhibitory Effects Of Mesenchymal Stem Cell Against TGF-β1-induced Epithelial-to-mesenchymal Transition In HK-2 Cells

Aims This study evaluated the effects of erythropoietin(EPO)pretreated on the proliferation,migration,differentiation of mesenchymal stem cells(MSCs).Furthermore,we studiedwhether or not EPO pretreatment on MSCs could better reverse Epithelial-to-Mesenchymal Transition(EMT)of human renal tubular epithelial cells(HK-2)induced by transforming growth factor-β1(TGF-β1).Methods The murine bone marrow derived MSCs were isolated,cultured with normal medium or EPO treatedby whole bone marrow methodwith normal medium or EPO added.CCK proliferation assay,migration assay,differentiation assays were performed,and immunophenotypic feature were detected by flow cytometry with normal medium or EPO pretreated.Grouping:(1)HK-2(2)HK-2+ TGF-β1(6ng/ml)(3)HK-2+TGF-β1(6ng/ml)+MSCs(10cells/200ul)(4)HK-2+TGF-β1(6ng/ml)+EPO(100IU/ml)pretreated MSCs(105/200ul).Cultured for 48h,Cell morphology of HK-2 in different groups by inverted microscope.The expression of a-SMA、E-cadherinin in HK-2 were analyzed by real time-polymerase chain reaction(RT-PCR)and western blot(WB),respectively.Results 1.BMSCs cultured stably were spindle,with great soma and large nucleus,and distributed like swirling.Cultured MSCs had the capacities for osteogenic and adipogenic differentiation,highly expressed CD105and lowly expressed CD45.2.WithEPO treated,the immunophenotypic feature of MSCs didn’t change,which was still positive for CD 105,negative for CD45.The adipogenic and osteogenic capacity had not change either after100IU/ml EPO treated.3.Compared to the control group,the CCK8assays showed the absorbance in different concentration of rhEPO(0、1、10、100、500IU/ml)were 0.230±0.030,0.301±0.012,0.343±0.038,0.348±0.029,0.306±0.027 respectively,which suggested the efficiency of EPO on MSCs proliferation increased gradually in a concentration-dependent manner with increasing concentration of rhEPO,and the ability peaked at a concentration of 100 IU/ml.The proliferation of 500IU/ml EPO treated group was inhibited to some extent.4.After different concentration of rhEPO(0、1、10、100、500IU/ml)treated with BMSCs for 8 hours,with crystal violet stained,the average number of migrating cells was calculated under the microscope.The EPO treatment increased the number of migrating BM-mMSCs in a concentration-dependent manner up to 100IU/ml(34.80±5.80),furthermore increase of the concentration of EPO(500 IU/ml)did not show a greater effect in the migratory.5.Compared to the normal group,the mRNA and the protein of a-SMA was significantly increased(P<0.05)with the E-Cadherin significantly reduced(P<0.05)in TGF-β1 induced group.Compared to TGF-β1 induced group,the mRNA and the protein of a-SMA and E-Cadherin were separately increased and reduced in both traditional MSC group and EPO pretreated MSC group(P<0.05),furthermore EPO pretreated MSC group showed a stronger protective effect than the traditional one in HK-2 cells induced by TGF-β1.Conclusion some proper concentration of rhEPO treated with BMSCs could increase the proliferation and migration of BMSCs.meanwhile,treated BMSCs had still stem cell properties.Erythropoietin Treatment could enhance inhibitory effects of MSCs against in HK-2 cells induced by TGF-β1.