薄芝糖肽对人角质细胞增殖和黏附功能的影响及其治疗银屑病的机制

Effects of Bozhi glycopeptide on proliferation and adhesion function of human keratinocytes and its mechanism in treatment of psoriasis

目的:探讨薄芝糖肽对人角质细胞(KC)增殖和黏附因子β-连环蛋白(β-catenin)表达的影响,阐明薄芝糖肽治疗银屑病的可能机制。方法:增殖功能实验中,对数生长期的人永生化角质形成细胞(HaCaT细胞)分为对照组和不同浓度(0.011719、0.023438、0.046875、0.093750、0.187500、0.375000、0.750000和1.500000 g·L^(-1))薄芝糖肽组,分别处理24 h后,采用CCK-8法检测各组细胞增殖率,确定薄芝糖肽作用的有效浓度;对数生长期HaCaT细胞分为对照组和不同浓度(0.625、1.250、2.500、5.000、10.000、20.000、40.000和80.000 mg·L^(-1))薄芝糖肽组,分别处理24和48 h后,采用CCK-8法检测各组细胞增殖率。黏附功能实验中,对数生长期HaCaT细胞分为对照组和不同浓度(10和20 mg·L^(-1))薄芝糖肽组,分别处理24和48 h后,实时荧光定量PCR(RT-qPCR)法检测各组细胞中β-catenin mRNA表达水平,Western blotting法检测各组细胞中β-catenin蛋白表达水平。结果:增殖功能实验,与对照组比较,作用24 h后,0.011719~0.093750 g·L^(-1)薄芝糖肽组细胞增殖率均明显降低(P<0.01),该浓度区间为薄芝糖肽有效浓度区间。与对照组比较,作用24和48 h后,1.250、2.500、5.000、10.000、20.000、40.000和80.000 mg·L^(-1)薄芝糖肽组细胞增殖率均明显降低(P<0.05或P<0.01),且20 mg·L^(-1)薄芝糖肽组作用48 h时细胞增殖率降低最明显(P<0.01)。黏附功能实验,与对照组比较,作用24 h时10和20 mg·L^(-1)薄芝糖肽组细胞中β-catenin mRNA表达水平明显降低(P<0.01),作用48 h时10和20 mg∙L-1薄芝糖肽组细胞中β-catenin mRNA表达水平降低更明显(P<0.01);与对照组比较,作用24 h时10 mg∙L-1薄芝糖肽组细胞中β-catenin蛋白表达水平明显降低(P<0.01),作用48 h时10和20 mg∙L-1薄芝糖肽组细胞中β-catenin蛋白表达水平有增高趋势,但差异无统计学意义(P>0.05)。结论:薄芝糖肽在一定的浓度和作用时间内可以抑制KC增殖,并可通过降低β-catenin表达抑制KC黏附功能,可能是薄芝糖肽治疗银屑病的作用机制之一。

Objective:To investigate the effects of Bozhi glycopeptide on the proliferation of the human keratinocytes(KC)and the expression ofβ-catenin,and to elucidate the possible mechanism of Bozhi glycopeptide in the treatment of psoriasis.Methods:In the proliferation function experiment,the HaCaT cells in the logarithmic growth phase were divided into control group and different concentrations(0.011719,0.023438,0.046875,0.093750,0.187500,0.375000,0.750000 and 1.500000 mg·L^(-1))of Bozhi glycopeptide groups.After the cells were treated for 24h,the CCK-8 method was used to detect the proliferation rates of the cells in various groups to determine the effective concentration of Bozhi glycopeptide.The HaCaT cells in the logarithmic growth phase were divided into control group and different concentrations(0.625,1.250,2.500,5.000,10.000,20.000,40.0000 and 80.000 mg·L^(-1))of Bozhi glycopeptide groups.After the cells were treated for 24 and 48 h,the cell proliferation rates in various groups were detected by CCK-8 method.In the adhesion function experiment,the HaCaT cells in the logarithmic growth phase were divided into control group and different concentrations(10 and 20 mg·L^(-1))of Bozhi glycopeptide groups.After the cells were treated for 24 and 48 h,the RT-qPCR method was used to detect the expression levels ofβ-catenin mRNA in the cells in various groups;Western blotting method was used to detect the expression levels ofβ-catenin protein in the cells in various groups.Results:In the proliferation function experiment,compared with control group,the cell proliferation rates in 0.011719-0.093750 g·L^(-1)Bozhi glycopeptide groups were significantly reduced after treated for 24 h(P<0.05),which was the effective concentration range of Bozhi glycopeptide.Compared with control group,the proliferation rates of cells in 1.250,2.500,5.000,10.000,20.000,40.000,and 80.000 mg·L^(-1)Bozhi glycopeptide groups were significantly reduced after treated for 24 and 48 h(P<0.05 or P<0.01);the proliferation rate was decreased significantly(P<0.01)when the cells were treated with 20 mg·L^(-1)Bozhi glycopeptide for 48 h.In the adhesion function experiment,compared with control group,the expression levels ofβ-catenin mRNA in the cells in 10 and 20 mg·L^(-1)Bozhi glycopeptide groups were decreased significantly after treated for 24 h(P<0.01),and the expression levels ofβ-catenin mRNA were decreased significantly after treated for 48 h(P<0.01).Compared with control group,after 24 h of treatment,the expression level ofβ-catenin protein in 10 mg·L^(-1)Bozhi glycopeptide group was decreased significantly;after 48 h of treatment,the expression levels ofβ-catenin protein in 10 and 20 mg·L^(-1)Bozhi glycopeptide groups tended to increase,but there were no significant differences(P>0.05).Conclusion:Bozhi glycopeptide can inhibit the proliferation of KC at a certain concentration and action time,and can inhibit KC adhesion function by reducing the expression ofβ-catenin.This may be one of the possible mechanisms of Bozhi glycopeptide in the treatment of psoriasis.