A Preliminary Study On The Effect Of Leositirizine Hydrochlorid On The Growth Of Human Dermal Papilla Cells

Objective:The study is to research the effect of levocetirizine hydrochloride on the growth,oliferation and the expression of prostaglandin related signal molecules of human dermal papilla cells?hDPCs?in vitro,to preliminarily evaluate the effect of levocetirizine hydrochloride at different concentrations on the growth of in vitro cultured hDPCs,in order to explore and provide scientific basis for the treatment of androgenetic alopecia?AGA?.Methods:Human dermal papilla cells were cultivated to the third generation,identified and observed by alkaline phosphatase staining and?-SMA immunofluorescence staining.hDPCs were divided into several groups to be cultured with Dulbecco?s modified eagle medium?DMEM?containing 0?control group?,1,10,100,1 000,10 000ng/ml levocetirizine hydrochloride respectively for 48 hours.Immunofluorescence staining was performed to observe the growth of the dermal papilla cells,and methyl thiazolyl tetrazolium?MTT?assay to evaluate the proliferative activity of the dermal papilla cells.Real-time fluorescence-based quantitative PCR was conducted to measure the mRNAexpressionofcyclooxygenase2?COX-2?,prostaglandinD2synthase?PTGDS?,prostaglandin E2?PGE2?,prostaglandin F2alpha?PGF2??,G protein-coupled receptor 44?GPR44?,protein kinase B?AKT?and glycogen synthase kinase 3??GSK3??,and Western blot analysis to determine the protein expression of PTGDS,pAKT and pGSK3?.After 24-hour culture with DMEM containing levocetirizine hydrochloride at different concentrations,enzyme-linked immunosorbent assay?ELISA?was performed to detect the levels of prostaglandin D2?PGD2?and PGD2R receptor in the culture supernatant of the hDPCs.Statistical analysis was carried out with SPSS17.0 software using one-way analysis of variance for the comparison of the above indices among the groups,and least significant difference?LSD?-t test for multiple comparisons.Statistical charts were drawn with Graphpad prism 6 software.Results:1.The AKP staining and?-SMA immunofluorescence staining positive could be found.Immunofluorescence staining showed that hDPCs grew well and reached over 90%confluence in the 100ng/ml levocetirizine hydrochloride group.MTT assay revealed that there were significant differences in the proliferation rate among all the groups?F=42.22,P<0.05?,and the proliferation rate was significantly higher in the 100 ng/ml levocetirizine hydrochloride group?115.80%±5.10%?than in the control group?100%,t=28.26,P<0.05?.2.The mRNA expression(2^-??Ct)of COX-2,PGF2a,PTGDS,GPR44 and AKT all significantly differed among these groups?F=1.972,3.66,2 2.172,2.658 and 7.325 respectively,all P<0.05?,while no significant difference in the mRNA expression of PGE2 and GSK3?was observed among these groups?F=0.872 and 1.189 respectively,both P>0.05?.The 100ng/ml levocetirizine hydrochloride group showed significantly decreased mRNA expression of COX-2,PTGDS and GPR44?0.84±0.08,0.81±0.10 and 0.85±0.09respectively?compared with the control group?t=1.972,2.172 and 2.658respectively,all P<0.05?,but significantly increased mRNA expression of PGF2?and AKT?1.96±0.25 and 1.74±0.32 respectively?compared with the control group?t=3.662,7.325 respectively,both P<0.05?.Moreover,the protein expression of PTGDS,pAKT,pGSK3?,PGD2 and PGD2R significantly differed among these groups?F=11.84,3.892,4.065,66.15,44.33respectively,all P?27?0.05?.When levocetirizine hydrochloride concentration was100ng/ml,the levels of PTGDS,PGD2 and PGD2R protein?0.32±0.05,141.62±5.44,215.08±9.55?were significantly lower than the control group?0.73±0.06,180.08±6.15,273.24±3.18,t=5.667,45.07,92.05 respectively,all P<0.05?,and the expression of pAKT and pGSK3?protein?0.59±0.05,0.46±0.03?were significantly higher than the control group?0.46±0.02,0.35±0.042,t=16.598,7.734 respectively,both P?27?0.05?.Conclusion:1.100ng/ml concentration of levocetirizine hydrochloride can promote the growth of hDPCs in vitro.Levocetirizine hydrochloride can inhibit its growth at the concentration higher than 1000ng/ml.2.Levocetirizine hydrochloride can promote the in vitro growth of human dermal papilla cells,likely by inhibiting the PGD2-GPR44 pathway and activating the AKT signal pathway.