鸦胆子苦醇通过Toll样受体4介导对尖锐湿疣角质形成细胞免疫调节的研究  

Study on immune regulation mechanism of Brusatol on keratinocytes of condyloma acuminatum mediated by Toll-like receptor 4

作　　者：吴伟棋 杨娟[1] 卢秀仪 温柳演 袁绍萍 白艳 吴启文 WU Wei-qi;YANG Juan;LU Xiu-yi;WEN Liu-yan;YUAN Shao-ping;BAI Yan;WU Qi-wen(Department of Dermatology,The Fourth Affiliated Hospital of Guangzhou Medical University,Guangzhou 511300,Guangdong Province,China;Clinical Laboratory,The Fourth Affiliated Hospital of Guangzhou Medical University,Guangzhou 511300,Guangdong Province,China;Department of Infectious Diseases,The Fourth Affiliated Hospital of Guangzhou Medical University,Guangzhou 511300,Guangdong Province,China)

机构地区：[1]广州医科大学附属第四医院皮肤科,广东广州511300 [2]广州医科大学附属第四医院检验科,广东广州511300 [3]广州医科大学附属第四医院感染科,广东广州511300

出　　处：《中国临床药理学杂志》2023年第10期1436-1440,共5页The Chinese Journal of Clinical Pharmacology

基　　金：广东省医学科学技术研究基金资助项目(A2020433);广州市科技计划基金资助项目(202102080594,202201011802);广州市增城区科技计划基金资助项目(ZCKJ2019-005);广州市中医药和中西医结合科技基金资助项目(20222A010088)。

摘　　要：目的探究鸦胆子苦醇(BRU)对尖锐湿疣(CA)角质形成细胞免疫调节的机制。方法收集本院尖锐湿疣组织,并分离CA角质形成细胞。用0、5.0、10.0和20.0 nmol·L^(-1)的BRU处理CA角质形成细胞,分别标记为空白组和低、中、高剂量实验组。将本院分离的CA角质形成细胞分为对照组(正常培养的CA角质形成细胞)、高剂量实验组(用20.0 nmol·L^(-1)的BRU处理细胞)、BRU+pcDNA-NC组(转染pcDNA-NC+20.0 nmol·L^(-1)的BRU)、BRU+pcDNA-TLR4组(转染pcDNA-TLR4+20.0 nmol·L^(-1)的BRU)。用CCK-8法和EdU法检测细胞的增殖情况,用实时荧光定量聚合酶链反应法检测Toll样受体4(TLR4)mRNA表达量,用蛋白质印迹法检测各组细胞TLR4和细胞核相关抗原Ki67(Ki67)的表达水平,用酶联免疫吸附试验法检测各组细胞γ干扰素(IFN-γ)和白细胞介素-8(IL-8)含量。结果高剂量实验组和空白组的TLR4 mRNA表达量分别为0.32±0.03和1.00±0.08,TLR4蛋白相对表达水平分别为0.36±0.04和1.07±0.11,差异均有统计学意义(均P<0.05)。对照组、高剂量实验组、BRU+pcDNA-NC组和BRU+pcDNA-TLR4组的EdU阳性细胞率分别为(42.36±3.92)%、(21.70±2.42)%、(20.89±1.85)%和(40.43±4.07)%,TLR4蛋白相对表达水平分别为1.02±0.09、0.34±0.03、0.38±0.04和0.79±0.09,Ki67蛋白相对表达水平分别为0.99±0.11、0.44±0.05、0.46±0.04和0.88±0.06,IFN-γ分别为(98.79±7.68)、(165.53±11.57)、(168.01±12.73)和(119.93±8.03)pg·mL^(-1),IL-8分别为(116.41±11.89)、(243.72±19.60)、(248.42±28.17)和(128.11±13.19)pg·mL^(-1)。对照组的上述指标与高剂量实验组比较,BRU+pcDNA-NC组的上述指标与BRU+pcDNA-TLR4组比较,差异均有统计学意义(均P<0.05)。结论BRU可通过调控TLR4的表达,进而抑制CA角质形成细胞增殖,从而参与调控CA的进展。Objective To investigate the mechanism of Brusatol(BRU)on the immune regulation of keratinocytes of genital warts(CA).Methods The genital warts tissue of our hospital was collected and CA keratinocytes were isolated.The CA keratinocytes were treated with 0,5.0,10.0 and 20.0 nmol·L^(-1)BRU and labeled as blank group and experimental-L,-M,-H groups,respectively.The CA keratinocytes isolated by our hospital were divided into a control group(normally cultured CA keratinocytes),BRU group(cells treated with 20.0 nmol·L^(-1)BRU)and BRU+PCDNA-NC group(transfected with pcDNA-NC+20.0 nmol·L^(-1)BRU),BRU+pcDNA-TLR4(transfected with PCDNA-TLR4+20.0 nmol·L^(-1)BRU).The proliferation of cells was detected by CCK-8 method and EdU method.The expression of Toll-like receptor 4(TLR4)mRNA was detected by real-time quantitative polymerase chain reaction.The expression levels of TLR4 and nucleus-associated antigen Ki67(Ki67)were detected by Western blot.The contents ofγinterferon(IFN-γ)and interleukin-8(IL-8)were detected by enzyme-linked immunosorbent assay.Results The expression levels of TLR4 mRNA in the experimental-H and blank groups were 0.32±0.03 and 1.00±0.08;the relative expression levels of TLR4 protein were 0.36±0.04 and 1.07±0.11,and the differences were statistically significant(all P<0.05).The EdU-positive cell rates of the control,experimental-H,BRU+pcDNA-NC and BRU+pcDNA-TLR4 groups were(42.36±3.92)%,(21.70±2.42)%,(20.89±1.85)%and(40.43±4.07%);the relative expression levels of TLR4 protein were 1.02±0.09,0.34±0.03,0.38±0.04 and 0.79±0.09;the relative expression levels of Ki67 proteins were 0.99±0.11,0.44±0.05,0.46±0.04 and 0.88±0.06;the IFN-γcontents were(98.79±7.68),(165.53±11.57),(168.01±12.73)and(119.93±8.03)pg·mL^(-1);the IL-8 contents were(116.41±11.89),(243.72±19.60),(248.42±28.17)and(128.11±13.19)pg·mL^(-1),respectively.The differences of above indexes were statistically significant between the control group and the experimental-H group,and between the BRU+pcDNA-NC group and the

关 键 词：鸦胆子苦醇 尖锐湿疣 角质形成细胞 TOLL样受体4 细胞增殖 

分 类 号：R28[医药卫生—中药学]