Effect Of Lithium Chloride Mediated Canonical Wnt/β-catenin Signaling On Proliferation And Osteogenic Differentiation Of Rat Adipose-derived Stem Cells

Objective To investigate the effect of different concentrations of lithium chloride onproliferation and osteogenic differentiation of rat adipose-derived stem cell(ADSCs) invitro culture and its possible mechanism.Methods①The ADSCs were harvested from the fat pad in the groin of3-week-old rats,then maintained in Dulbecco’s modified Eagle’s medium supplemented with5ml10%Fetal bovine serum after digesting with0.1%collagenase type Ⅰ;Confluent passaged-3cells were cultured with osteogenic medium in6-well culture plates. Medium wasrefreshed every other two days.Cells were stained with alizarin red after7days;ADSCswere cultured to third passage,then plated at1×105/well in6-well plates. After reachingnear80%confluence,cells were cultured in adipogenic medium. Adipose differentiationof the cells were examined by oil Red-O staining after7days.②ADSCs were exposedto lithium chloride at0,2.5,5,10,20,40mmol/L for24,48and72hours.The effect oflithium chloride on cells proliferation was determined by MTT assay.③Alkalinephosphatase(AKP) activity in cells was detected after ADSCs cultured for7days inosteogenic differentiation medium in2.5,5,10,20,40mmol/L or with no lithium chloride.④The ADSCs were treated with different concentrations of lithium chloride for7daysafter treating with osteogenic differentiation medium for3days, and the expressions ofβ-catenin, glycogen synthase kinase-3β,AKP were detected by using RT-PCT method.Results ADSCs’ Alizarin red staining and Oil red O staining were positive.In vitro,low doses of lithium chloride (2.5,5and10mmol/L) sitimulated ADSCsproliferation,whereas high doses caused a inhibition of proliferation.Lithium chloride atthe concentration of5mmol/L effectively improved proliferation of ADSCs higher thanthe other concentrations.5mmol/L lithium chloride induced AKP activity in ADSCswhich were induced the differentiation towards osteolasts,however40mmol/Lsignificantly inhibited AKP activity.Meanwhile,lithium chloride upregulated the geneexpression of internal components of Wnt/β-catenin signaling pathway as well asosteogenesis-related genes.Conclusions In vitro, rat ADSCs can be differentiated into the osteogenic andadipogenic lineages.Lithium chloride has a dual effect on ADSCs proliferation in vitro,and it improves AKP activity as well as promotes osteogenic differentiation in adose-dependent manner.5mmol/L lithium chloride can be used as the optimumconcentration for stimulating cell proliferation and promoting osteogenic differentiationin rat ADSCs.