雾水葛提取物抑制脂多糖诱导的人泪腺上皮细胞炎症反应的实验研究  

Experimental Study on the Inhibition of Lipopolysaccharide-induced Inflammatory Response in Human Lacrimal Gland Epithelial Cells by Pouzolzia Zeylanica Extract

作　　者：吴国海 邵永晴 梁丽金 李丹雨 汪腊一 谢玮玮 李真 郁琪华 刘小天 WU Guohai;SHAO Yongqing;LIANG Lijin;LI Danyu;WANG Layi;XIE Weiwei;LI Zhen;YU Qihua;LIU Xiaotian(Ningbo Eye Hospital,Ningbo 315157,China)

机构地区：[1]宁波市眼科医院,宁波315157 [2]宁波瑞瑧生物科技有限公司,宁波315157 [3]萌润堂中药有限公司,中国香港999077

出　　处：《中国中医眼科杂志》2024年第4期301-308,共8页China Journal of Chinese Ophthalmology

基　　金：浙江省中医药科技计划(2021ZB267);宁波市医疗卫生高端团队重大攻坚项目(2023030716)。

摘　　要：目的 观察雾水葛提取物对人泪腺上皮细胞活力和炎症的影响,鉴定活性单体并探索药理机制。方法 选取宁波市眼科医院收治的一位25岁女性泪腺炎患者,分离培养其人泪腺上皮细胞,免疫荧光法检测细胞标志物表达以鉴定细胞,不同剂量脂多糖(LPS)诱导细胞炎症反应,四甲基偶氮唑盐比色(MTT)检测细胞活力筛选LPS最佳剂量。使用水、氯仿、乙酸乙酯、正丁醇分别提取雾水葛活性成分,MTT法检测不同溶剂和剂量雾水葛提取物对人泪腺上皮细胞活力的影响。通过电喷雾质谱和核磁共振谱图确定氯仿提取物中活性成分的结构,将鉴定得到的5种化合物(LF1~5)进行药物靶标的预测,Western Blot法检测5种化合物的雾水葛提取物对人泪腺上皮细胞炎症相关蛋白表达的影响。结果 (1)细胞鉴定:角蛋白(CK)14、CK7、E-钙黏蛋白(E-cadherin)等阳性表达确定得到的是人泪腺上皮细胞和少量肌上皮细胞。(2)剂量筛选:处理48 h和72 h后,当LPS浓度为80 mg/L时,细胞活力开始低于对照组,差异有统计学意义(t_(48 h)=6.827、t_(72 h)=6.222,均P=0.002),故LPS浓度80 mg/L、处理时间为24 h为最佳诱导方法。高浓度下,不同雾水葛提取物都具有对细胞活力的抑制作用,2.5、5、10、20μg/mL为提取物的适宜浓度。(3)雾水葛氯仿提取物中物质鉴定:LF-1为(-)-Epicatechin,LF-2为8-(2-Pyrrolidinone-5-yl)-(-)-epicatechin,LF-4为3’-Demethylicariside E3,LF-3、LF-5为首次发现。(4)药物靶标筛选:5种氯仿提取物中,一共有5个共同靶点,分别为花生四烯酸5-氧化酶活化蛋白(ALOX5)、白细胞介素(IL)-2、IL-6、核因子κB1(NF-κB1)和血管内皮生长因子A(VEGFA)。(5)炎症相关蛋白:5种雾水葛的氯仿提取物都能不同程度地抑制炎症相关蛋白的水平,其中以LF-3的抑制效果最佳。结论 雾水葛提取物能够抑制LPS诱导的人泪腺上皮细胞的炎症反应,其中以氯仿提取物中的LF-3成分的药�OBJECTIVE To observe the effects of Pouzolzia zeylanica extract on the viability and inflammation of human lacrimal gland epithelial cells,identify active monomers,and explore the pharmacological mechanism.METHODS Human lacrimal gland epithelial cells were isolated and cultured from a 25-year-old female patient with dacryoadenitis admitted to Ningbo Eye Hospital.Isolate and culture human lacrimal gland epithelial cells.Immunofluorescence staining was used to detect cell biomarker expression to identify cells.Different doses of lipopolysaccharide (LPS) were used to induce cell inflammatory responses,and the best dose of LPS was screened using the MTT assay.Pouzolzia zeylanica active ingredients were extracted using water,chloroform,ethyl acetate,and nbutanol,and the effects of different solvents and doses of Pouzolzia zeylanica extract on the viability of human lacrimal gland epithelial cells were detected using the MTT method.The structure of active ingredients in chloroform extract was determined by electrospray mass spectrometry and nuclear magnetic resonance spectroscopy.A total of five compounds identified(LF1~5) were predicted for drug targets,and the effects of Pouzolzia zeylanica extract on the expression of inflammatory proteins in human lacrimal gland epithelial cells were detected by Western blot.RESULTS (1) Cell identification:Positive expression of cytokeratin (CK) 14,CK7,and E-cadherin confirmed the isolation of human lacrimal gland epithelial cells and a small number of myoepithelial cells.(2) Dose screening:After treatment for 48 h and 72 h,cell viability began to decrease compared to the control group when the LPS concentration was 80 mg/L,and the difference was statistically significant (t_(48 h)=6.827,t_(72 h)=6.222,both P=0.002).Therefore,the optimal induction method was LPS concentration of 80 mg/L and treatment time of 24 h.At high concentrations,different Pouzolzia zeylanica extracts had inhibitory effects on cell viability,with concentrations of 2.5,5,10,and 20μg/mL being suitable for the

关 键 词：人泪腺上皮细胞 雾水葛提取物 炎症 活性成分 氯仿 

分 类 号：R285.5[医药卫生—中药学]