TLR9在HaCaT细胞UVB光老化模型中对下游信号分子的调控作用

Role of Toll-like receptor 9 in ultraviolet B induced HaCaT cell photoaging model

摘要:

目的：探讨Toll样受体9（Toll-like receptor 9，TLR9）在中波紫外线（ultraviolet B，UVB）诱导的人永生化角质形成细胞（human immortal keratinocyte，HaCaT）光老化模型中对下游信号分子的调控作用。方法：实验分为3组：正常对照组、模型组、A151组。正常对照组予以假照射处理，模型组予以辐照剂量30 mJ/cm2 UVB照射处理，A151组予以辐照剂量30 mJ/cm2 UVB照射并加用TLR9抑制剂进行干预处理。使用衰老相关β半乳糖苷酶法观察细胞衰老。CCK-8法检测细胞增殖。Western blot检测TLR9、磷酸化核转录因子（phospho nucler factor-κB，pNF-κB）蛋白表达。RT-PCR法检测细胞肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白介素-6（interleukin-6，IL-6）mRNA转录水平。结果：Western blot结果显示模型组较正常对照组TLR9、pNF-κB蛋白表达量升高（P=0.000，P=0.000），A151组较模型组TLR9、pNF-κB蛋白表达量降低（P=0.010，P=0.000）。RT-PCR结果显示模型组较正常对照组TNF-α、IL-6mRNA转录水平上调（P=0.000，P=0.000），A151组较模型组TNF-α mRNA、IL-6 mRNA转录水平下调（P=0.000，P=0.030）。衰老相关β半乳糖苷酶染色结果显示模型组较正常对照组染色阳性细胞数增多（P=0.000），A151组较模型组染色阳性细胞数减少（P=0.000）。CCK-8结果显示模型组较正常对照组细胞增殖率降低（P=0.010），A151组较模型组细胞增殖率升高（P=0.045）。结论：UVB通过上调HaCaT中的TLR9表达，引起核转录因子磷酸化，促进细胞因子TNF-ɑ、IL-6转录水平升高，介导细胞炎性反应，从而降低细胞增殖率，诱导细胞衰老。

Abstract:

Objective：To explore the molecular mechanism of Toll-like receptor（TLR） 9 in ultraviolet B（UVB）-induced photoaging model of HaCaT cell. Methods：This experiment contains three groups：control group，model group，A151 group. The control group was given sham irradiation. The model group was established by the dose of 30 mJ/cm2 UVB irradiation. The A151 group was given the dose of 30 mJ/cm2 UVB irradiation and treated with TLR9 inhibitor. The cell senescence was evaluated by aging related beta-galac－tosidase cytochemistry staining. The cell proliferation rate was measured by CCK-8 assay. The contents of TLR9 and phosphorylation nuclear factor-κB（pNF-κB） were detected by Western blot. The mRNA expression of tumor necrosis factor-α（TNF-α） and inter－leukin-6（IL-6） were detected by RT-PCR. Results：The protein expressions of TLR9 and pNF-κB were higher in model group than control group（P=0.000，P=0.000），which were attenuated by the A151 treatment（P=0.010，P=0.000）. The mRNA levels of TNF-α，IL-6 increased in model group compared to control group（P=0.000，P=0.000），which were ameliorated by the A151 treatment（P=0.000，P=0.030）. The number of positive cells in aging related beta galactosidase staining was increased in model group than in con－trol group（P=0.000），while the number of positive cells was decreased in A151 group than in model group（P=0.000）. The cell prolif－eration rate of model group decreased markedly compared with that of control group（P=0.010），while the cell proliferation rate increased in A151 group than in model group（P=0.045）. Conclusion：The decrease of UVB-induced HaCaT cell prolifer－ation rate and senescence are mediated by phosphorylated nu－clear factor-κB and elevated mRNA of TNF-α and IL-6 via activation of the TLR9.