The Mechanism Of The Notoginsenoside R1 Promote Keloid Fibroblasts Apoptosis In Vitro Through Wnt/?-catenin Signal Pathway

ObjectiveNotoginsenoside R1 is the most important characteristic component of panax notoginseng saponins which belongs the araliaceae perennial herb plants.Using Notoginsenoside R1 in different concentration(20?g/ml-200?g/ml)to treat keloid fibroblasts in vitro,and the optimum concentration was 120Opg/ml.To detect the expression of Wnt5a,?-catenin,GSK-3?,p-GSK-3p protein and mRNA level in 48 hours after treated by 120?g/ml notoginsenoside R1.To explore the molecular mechanism of notoginsenoside R1 induced apoptosis of keloid fibroblasts and the roles of Wnt/?-catenin signal transduction pathway.To provide the molecular and pathological theory evidences for keloid prevention and treatment.MethodsHuman normal skin and keloid tissues were collected,isolated and primary cultured by tissue explanting method into the normal skin fibroblasts and keloid fibroblasts.The fibroblasts were identified by vimentin staining.After being treated with different concentrations of Notoginsenoside R1 for 48 hours,the cell proliferation of fibroblasts were detected by CCK-8 method.The optimal concentrations were selected for the subsequent experiments.The protein expression and mRNA levels of Wnt5a,?-catenin,GSK-3? and p-GSK-3? were detected by Western Blot and RT-PCR in 48 hours after treated by 120?g/ml Notoginsenoside R1 by qualitative and semi-quantitative researches.Result1.Morphological observations of fibroblasts Tissue block method was applied to primary culture keloid fibroblasts,observing morphological and growth changes under inverted microscope,there were some spindle cells spread out of keloid tissue until the 5-7th day after inoculation.The growth of cells arranged in multiple radial,braided,shuttle-shaped.Cells appear shuttle type or irregular triangle,there was no significant difference.Keloid fibroblasts were disorder,loss of polarity,obviously they were overlaping.,2.The vimentin Immunohistochemistry staining of fibroblast showed a lot of brown-yellow particles in the cytoplasm and blue nucleus.3.CCK-8 CCK-8 showed that the OD values of cells in each group were reduced after being treated with different concentrations(20?g/ml,40?g/ml,50?g/ml,60?g/ml?70?g/ml,80?g/ml,90?g/ml,100?g/ml,110?g/ml,120?g/ml,130?g/ml,140?g/ml,150?g/ml,160?g/ml,170?g/ml,180?g/ml,190?g/ml,200?g/ml)of Notoginsenoside R1 for 48h,the OD values are 20?g/ml(3.0391±0.061277),40?g/ml(2.792133 ±0.154944),50?g/ml(2.923967±0.124839),60?g/ml(2.626233±0.120606),70?g/ml(2.833133±0.077638),80?g/ml(2.518467±0.02166),90?g/ml(2.8656±0.060383)100?g/ml(2.510367±0.093041),110?g/ml(2.477233±0.062628),120?g/ml(2.376833±0.062446),130?g/ml(2.650733±0.030904),140?g/ml(2.704233±0.103737),150?g/ml(2.411533±0.087351),160?g/ml(2.587567±0.083547),170?g/ml(2.3122±0.047022),180?g/ml(2.604867±0.106829),190?g/ml(2.2339±0.02324),200?g/ml(2.492±0.047627).Comparede with the control 0?g/ml(2.953633±0.032493),the proliferation of cells in concentration 110-180?g/ml group were suppressed(P<0.05).In the range of 170?g/ml to 180?g/ml,it had shown flaky death of fibroblasts observed by microscope.In the range of 110 ?g/ml to 160 ?g/ml,the proliferation of fibroblasts was most inhibited at 120 ?g/ml,and 120 ?g/ml was selected as the optimum drug concentration.This drug concentration of 120 ?g/ml would be used in subsequent experiment.4.Western Blot The expression of Keloid control group Wnt5a(4.054±0.0598),?-catenin(0.3503±0.005484),p-GSK-3?(1.1031±0.01276)compared with normal skin control group Wnt5a(0.3588±0.4688),?-catenin(0.1509±0.003138),p-GSK-3?(0.5878±0.009849)increased(P<0.05),the GSK-3? expression of Keloid control group(0.3440?0.0055697)compared with normal skin control group(0.3359±0.0040407)was not influenced.After being treated by 120?g/ml Notoginsenoside R1,the proteins of Keloid group Wnt5a(3.029±0.06232),?-catenin(0.225±0.005092),p-GSK-3?(0.7634±0.006712)compared with normal skin group Wnt5a(0.503436±0.005947),?-catenin(0.1489±0.0029203),p-GSK-3?(0.618845±0.00444019)expression increased(P<0.05),the GSK-3? expression of keloid group(0.3558±0.0056497)compared with normal skin group(0.301365±0.0017849)were not influenced.The expression of Keloid group Wnt5a(3.029±0.06232),?-catenin(0.225±0.005092),p-GSK-3p(0.7634±0.006712)compared with Keloid control group Wnt5a(4.054±0.0598),?-catenin(0.3503±0.005484),p-GSK-3?(1.103±0.01276)decreased(P<0.05),the GSK-3? expression of Keloid group(0.3558±0.0056497)compared with Keloid control group(0.3440±0.0055697)were not influenced.5.RT-PCR The mRNA level of keloid control group Wnt5a(45.3±0.82),P-catenin(15.97±0.202)compared with normal skin control group Wnt5a(1 ±0.18),(3-catenin(1±0.08)increased(P<0.05),the GSK-3? mRNA level(1.126±0.22)of keloid control group compared with normal skin control group(1±0.199)was not influenced.After being treated by 120?g/ml Notoginsenoside R1,the mRNA level of Wnt5a(14.07±0.59),?-catenin(1.487±0.087)of Keloid group compared with normal skin group mRNA Wnt5a(1.211±0.16),?-catenin(0.747±0.429)increased(P<0.05),the mRNA of Keloid group GSK-3?(1.555±0.24)compare with normal skin group mRNA GSK-3?(1.539±0.24)was not influenced.The mRNA Wnt5a(14.07±0.59),?-catenin(1.487±0.087)of Keloid group compare with Keloid control group mRNA Wnt5a(45.3±0.82),?-catenin(15.97±0.202)decreased(P<0.05),the mRNA of Keloid group GSK-3?(1.555±0.24)compared with Keloid control group mRNA GSK-3(3(1.126±0.22)was not influenced.6.RNA and DNA agarose gel electrophoresis The normal skin and Keloid of control group,test group,dmso group of RNA and Wnt5a,?-catenin,GSK-3?,?-Actin DNA showed a single band.Conclusion1.The phosphorylation of GSK-3p might play an important role in keloid through induce fibroblast apoptosis.Notoginsenoside R1 induce fibroblast apoptosis might through interfence phosphorylation of GSK-3?.2.Wnt/?-catenin signal transduction pathway has very important significance of keloid,Notoginsenoside R1 might induce fibroblast apoptosis through inhibit the activation of Wnt/?-catenin signal transduction pathway.3.The Notoginsenoside R1 positive drug effects might regulate phosphorylation of GSK-3? through Wnt/?-catenin signal transduction pathway.Notoginsenoside R1 might be a potential medicine to prevent and treat keloid.4.It worth to focus to the complicated relationship between GSK-3? and Wnt/?-catenin signal transduction pathway and they might be the potential treatment taget,they also worth to explore more.