| ObjectsKeloid is a pathological healing secondary after skin trauma and inflammation.The mechanism still keep unknown.Previous studies showed phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)signal transduction pathwayplayed a role in development of hypertrophic scar,and mitochondrial stress might be one of the core events for keloid formation.As notoginsenoside R1(NR1)had the function of anti-hepatocellular fibrosis.Did it suppose also to influense the proliferation of keloid?what the possible mechanism is?In this research,PI3K/AKT pathway and its downstream proteinl were been detected after treated by NR1 for 48 hours,LY294002 for 24 hours,NAC for 1 hour.At the same time,the products of oxidation reduction reaction ROS also be checked.To investigate the influence of NR1 exert in primary cultured fibroblasts.Providing experimental evidences for mechanism research of keloid and pharmacological development.Methods1.Primary cultured fibroblasts(Fibroblast,FB)were isolated by tissue block method-Experimental groups divided:keloid control,keloid experimental group:NR1 group,LY294002 group,NAC group.Normal skin control,normal skin experimental group:NR1 group,LY294002 group,NAC group.After treated by different concentrations of LY294002 for 24 hours,120ug/ml NR1 for 48 hours[2],the cell proliferation of keloid fibroblasts were detected by CCK-8 method,the optimal concentration of LY294002 were selected.After treated by different concentrations of NAC for 1 hour,ROS of fibroblasts were detected by flow cytometry,the optimal concentration of NAC were selected.2.The proteins expression of AKT、AKT[pS473]、AKT[pT308]、AKR1-C3 were detected by Western Blot after fibroblasts treated by NR1,LY294002 and NAC in different time period.3.The localization and expression of AKT、AKT[pS473]、AKT[pT308]、AKR1-C3 in keloid and normal skin were detected on immunohistochemistry staining.4.mRNA levels of AKT、AKT[pS473]、AKT[pT308]、AKR1-C3 were detected by qPCR after fibroblasts treated by NR1,LY294002 and NAC.5.ROS of fibroblasts were detected by flow cytometry after fibroblast treated by NR1,LY294002.Result1.Morphological observations of fibroblasts Tissue block method was applied to primary culture fibroblasts,observed morphological and growth changes under inverted microscope,there were some shuttle-type cells climb out of keloid tissue until the 5-7th day,and same celles climbed out from normal skin at 8-10th day.The immunocytochemical staining of vimentin.showed that a lot of brown-yellow particles in fibroblast cytoplasm.The result of identification was that fibroblast cultured in vitro succeed.2.CCK8 The keloid fibroblasts were treated by different concentrations(5mmol/L,10mmol/L,15mmol/L,20mmol/L,25mmol/L,30mmol/L,35mmol/L,40mmol/L,45mmol/L,50mmol/L)of LY294002 for 24h,the ODs were 5mmol/L(1.49±0.11),10mmol/L(1.51 ±0.08),15mmol/L(1.37 ± 0.10),20mmol/L(1.39±0.06),25mmol/L(1.37 ± 0.10),30mmol/L(1.23 ± 0.09),35mmol/L(1.23 ± 0.07),40mmol/L(1.18±0.10),45mmol/L(1.14 ± 0.08),50mmol/L(1.18 ± 0.09),compared with the control(1.54 ± 0.08),the proliferation of cells in concentration 15-50mmol/L group were suppressed(P<0.05),that there were dose-response relationships between drugs and cells proliferation,selected 15mmol/L as the final LY294002 concentration to the subsequent experiments.Keloid fibroblasts were treated by NR1 at a concentration of 120ug/ml for 48h[2],the OD was significantly lower(1.31 ± 0.06)than the control(1.53 ± 0.06)(P<0.05).3.The fluorescence intensity of ROS in the keloid control(0.86 ± 0.01)were significantly higher than the normal skin control(0.79 ± 0.01)(t=7.839,P<0.01).After keloid fibroblasts were treated by different concentrations(4mM,6mM,8mM,lOmM,12mM))of NAC for 1 hour.The fluorescence of ROS were 4mM(0.79 ± 0.02),6mM(0.72 ±0.03),8mM(0.78 ± 0.01),10mM(0.77 ± 0.02),12mM(0.75 ± 0.06).When NAC concentration was 6mM(F=5.347,P<0.01),the intracellular ROS was the lowest,it was the optimal concentration.4.Western Blot Compared with the keloid control(0.73 ± 0.07),the expression of AKR1-C3 in the normal skin control(0.31 ± 0.05)were decreased(t=7.949,p<0.01),the expression AKR1-C3 in the keloid experimental group NAC(0.33 ± 0.06),LY294002(0.23 ±0.06)and NR1(0.34 ± 0.04)were lower than that the control(F=47.18,P<0.001).Normal skin experimental group NAC(0.28 ± 0.06),LY294002(0.30 ± 0.07)and NR1(0.26 ± 0.04)compared with the control had no significant difference(F=0.298,P>0.05).The expression of keloid control AKT(0.93 ± 0.06)were higher than normal skin control(0.31 ± 0.04)(t=12.90,P<0.001).The expression of AKT in the keloid experimental group NAC(0.74 ±0.06),LY294002(0.71 ± 0.06)and NR1(0.78 ± 0.03)were lower than that the control(F=11.32,P<0.001).The expression of AKT in normal skin experimental group NAC(0.29 ±0.03),LY294002(0.30 ± 0.07)and NR1(0.31 ± 0.03)compared with the control had no significant difference(F=0.057,P>0.05).The expression of AKT[pT308]in keloid control(0.63 ± 0.02)were significantly higher than normal skin control(0.23 ± 0.02)(t=12.54,P<0.001).the expression of AKT[pT308]in the keloid experimental group NAC(0.51 ±0.07),LY294002(0.12 ± 0.03),NR1(0.32 ± 0.05)were lower than that the control(F=59.13,P<0.001).The expression of AKT[pT308]of normal skin experimental group NAC(0.23 ±0.04),LY294002(0.22 ± 0.06)and NR1(0.21 ± 0.05)compared with the control had no significant difference(F=0.083,P>0.05).The expression of AKT[pS473](0.79 ± 0.05)were higher than normal skin group(0.25 ± 0.06)(t=12.00,P<0.001),The expression of AKT[pS473]in the keloid experimental group NAC(0.48 ± 0.08),LY294002(0.14 ± 0.04)and NR1(0.23 ± 0.04)were lower than that the control(F=119.0,P<0.001).The expression of AKT[pS473]in normal skin experimental group NAC(0.22 ± 0.06),LY294002(0.23 ± 0.04)and NR1(0.24±0.05)compared with the control had no significant difference(F =0.088,P>0.05).5.Immunohistochemistry The optical density of keloid AKT(27.29 ± 1.97)were higher than the normal skin(4.97 ± 1.9)(t=12.78,P<0.001).The optical density of keloid AKT[pS473](16.75 ±3.3)were higher than the normal skin(4.02±1.5)(t=10.47,P<0.001).The optical density of keloid AKT[pT308](16.2±1.56)were higher than normal skin(1.82±0.5)(t=13.80,P<0.001).The optical density of keloid AKR1-C3(26.69±2.50)were higher than normal skin(1.47± 1.07)(t=13.06,p<0.001).All proteins were localized in cytoplasm.6.q-PCR Compared with the keloid group(1.40 ± 0.05),the AKR1-C3 mRNA level were decreased in the normal skin group(0.98 ± 0.04)(t=6.702,p<0.01).The AKR1-C3 mRNA level of keloid experimental group NAC(0.43 ± 0.04),LY294002(0.72 ± 0.03)and NR1(0.89 ±0.03)were lower than control(F=94.32,P<0.001).The AKR1-C3 mRNA level of normal skin experimental group NAC(0.86 ± 0.03),LY294002(0.85 ± 0.06)and NR1(0.80 ± 0.05)compared with the control had no significant difference(F=2.321,P>0.05).The AKT mRNA level of keloid control group(1.38 ±0.08)were higher than normal skin control group(0.98 ± 0.11)(t=4.977,P<0.01).The AKT mRNA level of keloid experimental group NAC(0.96 ± 0.11),LY294002(0.73 ± 0.06)and NR1(0.90 ± 0.03)were lower than the control(F=45.56,P<0.001).The AKT mRNA level of normal skin experimental group NAC(0.92 ± 0.08),LY294002(0.89 ± 0.06),NRl(0.84 ± 0.02)compared with the control had no significant difference(F = 5.843,P>0.05).7.The ROS fluorescence value of keloid group were LY294002 treated group(0.78±0.004),NR1 treated group(0.82±0.003)significantly lower than the control(P<0.05).The ROS fluorescence value of the normal skin group were LY294002(0.75 ± 0.003),R1(0.74±0.005)compared with the control had no significant difference(P>0.05).Conclusion1.PI3K/AKT signal transduction pathway involved in keloid,and its specific inhibitor LY294002 inactivated PI3K/AKT pathway in a dose-dependent method.Co-phosphorylation AKT[pS473]and AKT[pT308]might be one of the molecular mechanisms of keloid.2.Increasing ROS in keloid fibroblasts hint the role of oxidative stress played in keloid.ROS regulated activation of PI3K/AKT signal transduction pathway,at the same time,PI3K/AKT signal transduction pathwayalso exert influence in ROS expression.It worth to advanced research in mutual regulation between ROS and PI3K/AKT signal transduction pathway.3.The expression of protein AKR1-C3 regulated by ROS and PI3K/AK pathway and played roles in keloid.4.NR1 might effectively inhibit the proliferation of keloid fibroblasts in vitro through dephosphorylation of PI3K/AKT signal transduction pathway.5.NR1 suppression partially ROS expression in fibroblasts of keloid,might be regulated by multiple signal transduction pathways and related to the complex regulated mode in mitochondrial.It would provide clues in further investigations. |
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