烟草烟雾提取物通过Klotho-CHRNA5轴诱发皮肤衰老的机制

doi:10.6040/j.issn.1671-7554.0.2025.0937

摘要/Abstract

摘要： 目的探讨烟草烟雾导致抗衰老蛋白Klotho与烟碱型乙酰胆碱受体CHRNA5表达变化调控皮肤衰老的机制。方法采用烟草烟雾提取物(cigarette smoke extract, CSE)处理HaCaT角质形成细胞,通过β-半乳糖苷酶染色及Western blotting检测细胞衰老相关表型及CHRNA5、Klotho蛋白表达变化。随后,在3~5月龄、6~7月龄及14月龄小鼠中,利用HE染色、免疫组化、Western blotting及RT-qPCR分析皮肤组织中CHRNA5、Klotho及衰老相关标志物的年龄依赖性变化。进一步地,在野生型与Klotho基因敲低小鼠中,通过免疫组化、Western blotting及RT-qPCR验证Klotho敲低效率,并检测Klotho基因对CHRNA5及衰老标志物的表达的影响。最后,对HaCaT细胞分别进行Klotho或CHRNA5的siRNA干扰,采用Western blotting及RT-qPCR检测两者及相关衰老标志物的表达,以验证其相互关系。结果 CSE处理可诱导HaCaT细胞衰老,表现为β-半乳糖苷酶染色阳性细胞显著增加(P=0.000 4),同时伴随CHRNA5蛋白水平显著上调(P=0.000 2)及Klotho蛋白水平下调(P=0.002 5)。在14月龄小鼠衰老小鼠相较于3–5月龄小鼠,皮肤表皮厚度减少(P<0.000 1),CHRNA5升高(P=0.000 9),而Klotho降低(P=0.000 1)。在Klotho基因敲低小鼠中,皮肤组织出现加速衰老表型,并伴随CHRNA5上调(P=0.010 7)。在Klotho敲低的HaCaT细胞中,同样观察到衰老标志物上升及CHRNA5表达增强(P=0.034 7)。相反,CHRNA5敲低可显著降低p16和p21等衰老相关标志物的表达(P=0.002 3; P=0.005 4)。结论烟草提取物可以下调Klotho表达,及上调CHRNA5表达促进角质形成细胞衰老,进而促进皮肤衰老。

关键词: 烟草烟雾提取物, Klotho, CHRNA5, 角质形成细胞, 皮肤衰老

Abstract: Objective To investigate how cigarette smoke accelerates skin aging by focusing on the interaction between the anti-aging protein Klotho and the tobacco-associated receptor CHRNA5, and explore potential targets for skin rejuvenation strategies. Methods HaCaT cells were treated with CSE(cigarette smoke extract). Cellular senescence and the expression of CHRNA5 and Klotho proteins were assessed by β-galactosidase staining and Western blotting. Subsequently, skin samples from mice aged 3-5, 6-7, and 14 months were analyzed using H&E staining, immunohistochemistry, Western blotting, and RT-qPCR to determine age-dependent changes in CHRNA5, Klotho and senescence-associated markers. Furthermore, in Klotho-knockdown mice, we assessed the efficiency of Klotho silencing and the effects of Klotho on both CHRNA5 expression and senescence markers using immunohistochemistry, Western blotting and RT-qPCR. Finally, HaCaT cells were transfected with siRNAs targeting Klotho or CHRNA5, and the expression of both genes and related senescence markers was measured by Western blotting and RT-qPCR to verify their regulatory relationship. Results CSE exposure induced senescence in HaCaT cells, as evidenced by a significant increase in β-galactosidase-positive cells(P=0.000 4), accompanied by the upregulation of CHRNA5(P=0.000 2)and the downregulation of Klotho(P=0.002 5). In 14-month-old mice, compared with 3-5-month-old controls, the epidermis was thinner(P<0.000 1), CHRNA5 was elevated(P=0.000 9), and Klotho was markedly decreased(P=0.000 1). In Klotho-knockdown mice, accelerated skin aging phenotypes were observed along with increased CHRNA5 expression(P=0.010 7). Similarly, Klotho-knockdown HaCaT cells exhibited enhanced senescence markers and elevated CHRNA5 expression(P=0.034 7). In contrast, CHRNA5 knockdown significantly reduced the expression of senescence-associated markers p16 and p21 in HaCaT cells(P=0.002 3; P=0.005 4). Conclusion CSE downregulates Klotho and upregulates CHRNA5, thereby promoting keratinocyte senescence andaccelerating skin aging.

Key words: Cigarette smoke extract, Klotho, CHRNA5, HaCaT, Skin aging

中图分类号:

R574

王艺博,王颖超,徐琳琳,黄淑红,党宁宁. 烟草烟雾提取物通过Klotho-CHRNA5轴诱发皮肤衰老的机制[J]. 山东大学学报 (医学版), 2026, 64(1): 88-98.

WANG Yibo, WANG Yingchao, XU Linlin, HUANG Shuhong, DANG Ningning. Mechanism of skin aging induced by cigarette smoke extract via the Klotho-CHRNA5 axis[J]. Journal of Shandong University (Health Sciences), 2026, 64(1): 88-98.

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