| Background and ObjectiveWound healing is the key problem in the treatment of burn,in order to shorten the healing time of the wound and improve the quality of wound healing,besides the existing drug therapy,surgery therapy,etc.,looking for better therapeutic agents or other means to achieve the high quality of wound healing is always the goal and direction of scientific research of clinicians especially burns physician.Previous studies showed that estrogen may play an important role in many critical cells of wound healing,for instance,estrogen can promote keratinocyte proliferation,but the downstream molecular mechanism of the effect of estrogen on the proliferation of keratinocyte is not completely understood.It is necessary to further study the specific signal transduction pathways of estrogen promotes keratinocyte proliferation,and then clarify the mechanisms of estrogen accelerate wound healing on the cellular and molecular level,which will not only provide a further theoretical basis for the clinical application of estrogen therapy,but also provide a new research ideas and directions about estrogen.Therefore,the main purpose of this study is to explore the molecular mechanism of estrogen promotes human keratinocytes via observe the effect of estrogen deficiency on skin development and wound healing in mice and the effect of estrogen on the proliferation of human immortal keratinocyte line(HaCa T).Methods(1)5 adult C57BL/6 mice(8 weeks old)which were selected by the method of vaginal shedding cytology were set as estrus group,5 adult C57BL/6 mice(8 weeks old)which had ovarian resection before sexual development(4 weeks old)were set as the ovariectomized group.The tail root 1 cm wide full-thickness skin were collected from the two mouse groups,the distribution of epidermal proliferating cell nuclear antigen(PCNA)positive cells were observed and counted by immunohistochemical method,and the skin thickness was observed and measured by HE staining.(2)5 adult C57BL/6 mice(8 weeks old)which were selected by the method of vaginal shedding cytology were set as estrus group,5 adult C57BL/6 mice(8 weeks old)which had ovarian resection before sexual development(4 weeks old)were set as the ovariectomized group.The full-thickness skin incision with a length of 1 cm was made on both sides of the back spine of the two mouse groups.In the 0,1,3,5,7 days after injury,the wound healing rate was observed and measured by taking picture,then the skin around the wound was taken from the mice in the 7 days after injury and the length of new epithelium of the wound was observed and measured by HE staining.(3)16 adult C57BL/6 mice(8 weeks old)was divided into proestrus group,estrus group,metestrus group,diestrus group by the method of vaginal shedding cytology,with 4 mice in each group.5 adult C57BL/6 mice(8 weeks old)which had ovarian resection before sexual development(4 weeks old)were set as the ovariectomized group.The normal abdominal skin tissue of the five mouse groups were collected,and the expression levels of p-ERK,p-Akt and PCNA were detected by Western blotting.(4)HaCa T cells in logarithmic growth phase were cultured in RPMI1640 medium containing 10% FBS(the same culture method below).Cells were divided into negative control group,estradiol group,protein kinase B(Akt)inhibitor group,extracellular signal regulated kinase(ERK)inhibitor group according to the random number table,with 25 wells in each group.In each culture liquid,1 μL dimethyl sulfoxide was added in negative control group,1 μL 100 nmol/L of 17β-estradiol was added in estradiol group,Akt inhibitor group and ERK inhibitor group were separately added with 10 μmol/L LY294002 and 30 μmol/L PD98059 along with same dose and volume of 17β-estradiol previous mentioned.At post culture hour(PCH)0(immediately),24,48,72,96 the proliferation level of cells were detected by cell counting kit and microplate reader(denoted as absorbance value),with 5 wells each group.(5)HaCa T cells in logarithmic growth phase were collected,the grouping and processing methods of cells were the same as(4),with 3 wells each group.At PCH 72,cell cycle distribution was detected by flow cytometry,and then calculating proliferation index(PI)and s phase fraction(SPF)according to formula.(6)Ha CaT cells in logarithmic growth phase were collected,and cells were divided into 7 groups according to the time that Ha CaT cells were treated with estrogen(17-beta estradiol),respectively is 0min group(without estrogen treatment),5min group,15 min group,30 min group,60 min group,120 min group and 240 min group.At the time of the corresponding culture,the cells were collected and the protein of each group was extracted,and then protein levels of phosphorylated ERK(p-ERK),phosphorylated Akt(p-Akt)and PCNA was determined with Western blotting.(7)HaCaT cells in logarithmic growth phase were collected and the grouping and processing methods of cells were the same as(4),with 3 wells each group.At PCH 72,protein levels of phosphorylated ERK(p-ERK),phosphorylated Akt(p-Akt)and PCNA was determined with Western blotting.(8)HaCaT cells in logarithmic growth phase were collected and the grouping and processing methods of cells were the same as(4),with 3 wells each group.At PCH 72,the cells were collected and the RNA of each group was extracted,and then the gene expression levels of glycogen synthase kinase 3 beta(GSK-3 β)and mammalian target of rapamycin(mTOR)were detected by real-time fluorescence quantitative PCR.(9)Data were processed with t test,one-way analysis of variance,analysis of variance of factorial design and LSD test.Results(1)The PCNA positive cells were mainly concentrated in the basal layer of epidermis of the two mouse groups,and the number of PCNA positive cells of epidermis in the ovariectomized group(37?12)was significantly declined than the estrus group(96?15)(t=15.3,P<0.01),Compared with the estrus group(51.4?3.1)μm,the skin thickness of the mice in the ovariectomized group(33.5?3.0)μm was significantly decreased(t=20.7,P<0.01).(2)At the 5th day after injury,compared with the estrus group wound healing rate(59.0?2.2)%,the ovariectomized group wound healing rate(49.9?2.1%)was delayed(P<0.05).At the 7th day after injury,compared with the estrus group wound healing rate(85.5?3.0)%,the ovariectomized group wound healing rate(73.0?0.9%)was significantly delayed(P<0.01).(3)In the different estrous cycle and ovariectomized mice,the expression levels of p-Akt,p-ERK and PCNA were not consistent in normal skin.And the expression level of p-ERK,p-Akt and PCNA protein in the skin of the mice in proestrus and estrus was higher than that in metestrus,diestrus and ovariectomized mice.(4)During PCH 0 to 48,the level of cells proliferation was no significantly difference between the estradiol group and the negative control group(P>0.05).At PCH 72,Compared with that of negative control group(0.983?0.060),the proliferation of HaCa T cells in estradiol group was significantly increased(2.092?0.177,P<0.01),and the proliferation of Ha Ca T cells in Akt inhibitor group and ERK inhibitor group respectively were 0.325?0.031 and 0.359?0.063,all of them were significantly lower than previous two group(P<0.01).At PCH 96,the proliferation of Ha CaT cells in estradiol group was 1.888 ? 0.022,which still higher than the negative control group 0.669 ? 0.032(P<0.01),and the proliferation of Ha Ca T cells in Akt inhibitor group and ERK inhibitor group respectively were 0.367 ? 0.019 and 0.474 ? 0.011,all of them were significantly lower than previous two group(P<0.01).(5)At PCH 72,Compared with the PI of negative control group(51.6?1.1)%,the PI of HaCa T cells in estradiol group was obviously elevated [(58.5?0.8)%,P<0.05];and the PI of HaCa T cells in Akt inhibitor group and ERK inhibitor group respectively were(34.9?0.8)% and(48.2?0.4)%,all of them were significantly lower than previous two groups(P<0.01).Compared with the SPF of negative control group(31.7 ? 1.2)%,the SPF of HaCa T cells in estradiol group was obviously elevated [(40.2 ? 0.5)%,P<0.01];and the SPF of Ha CaT cells in Akt inhibitor group was(15.7 ? 0.7)%,which was significantly lower than previous two group(P<0.01),and the SPF of HaCa T cells in ERK inhibitor group was(33.0 ? 0.1)%,which had not significantly difference compared with the negative control group(P>0.05),but was significantly lower than estradiol group(P<0.01).(6)At post culture minute(PCM)5,p-ERK started significantly increased,and compare with the protein expression of p-ERK in 0min group(0.037 ? 0.017),the protein expression of p-ERK in 5,15,30 min group respectively were 3.306 ? 0.042,1.847 ? 0.053,0.545 ? 0.053,and all of them were significant higher than 0min group(P<0.01).But then the protein expression of p-ERK of Ha CaT cells gradually decreased,the difference of 0min group and 60,120,240 min group was not significant(P>0.05).At PCM 15,compare with 0min group(0.100 ? 0.007),the protein expression of p-Akt in 15,30,60,120 min group respectively were 0.153 ? 0.007,0.188 ? 0.009,0.201 ? 0.007,0.220 ? 0.006,and all of them were significant higher than 0min group(P<0.05 or P<0.01),But the difference of the protein expression of p-Akt in 0min group and 240 min group was not significant(P>0.05).Compared with the 0min group,the protein expression of PCNA was not significantly changed in 240 min of 17β-estradiol treated HaCa T cells.(7)At PCH 72,Compared with that of negative control group 0.566?0.034,the protein expression of p-Akt in estradiol group was significantly increased(1.048?0.077,P<0.01),and the protein expression of p-Akt in Akt inhibitor group and ERK inhibitor group respectively was 0.682?0.095,0.672?0.019,both of them were significantly lower than estradiol group(P<0.01).At PCH 72,Compared with that of negative control group 0.469?0.013,the protein expression of p-ERK in estradiol group,Akt inhibitor group and ERK inhibitor group were all significantly increased(respectively 1.064?0.089,1.010?0.038,0.778?0.065,P<0.01),and the protein expression of p-ERK in ERK inhibitor group was obviously lower than estradiol group(P<0.01).At PCH 72,Compared with that of negative control group 0.386?0.053,the protein expression of PCNA was significantly increased in estradiol group(0.743?0.043,P<0.01),and the protein expression of PCNA in Akt inhibitor group and ERK inhibitor group respectively were 0.264?0.019 and 0.223?0.065,all of them were significantly lower than previous two group(P<0.01).(8)At PCH 72,compared with the mTOR gene expression level in negative control group(1.003 ? 0.058),estradiol group was significantly increased(1.803 ? 0.292,P<0.05),and Akt inhibitor group(1.255 ? 0.081)and ERK inhibitor group(0.987 ? 0.042)had not significant difference with negative control group(P>0.05).Compared with the mTOR gene expression level in estradiol group,the Akt inhibitor group had not significant difference with it(P>0.05),and the ERK inhibitor group was significantly lower(P<0.05).Compared with the GSK-3β gene expression level in negative control group(1.015 ? 0.124),estradiol group was significantly decreased(0.043 ? 0.003,P<0.01),and both Akt inhibitor group(0.526 ? 0.0042)and ERK inhibitor group(0.276 ? 0.040)were significant decreased(P<0.01).Compared with the GSK-3β gene expression level in estradiol group,the ERK inhibitor group had not significant difference with it(P>0.05),and the Akt inhibitor group was significantly increased(P<0.01).ConclusionLack of estrogen will damage the ability of epidermis growth of mice and slow the rate of wound healing.Estrogen may promote Ha Ca T cells proliferation by increasing the expression of PCNA via activating ERK/AKT signaling pathway,this study provides a molecular mechanism for estrogen promoting skin wound re-epithelization and accelerating wound healing from the perspective of the proliferation. |
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