Experimental Study on the Inhibition of Lipopolysaccharide-induced Inflammatory Response in Human Lacrimal Gland Epithelial Cells by Pouzolzia Zeylanica Extract
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 n-butanol,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(t48 h=6.827,t72 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 20 μg/mL being suitable for the extracts.(3)Substance identification in Pouzolzia zeylanica chloroform extract:LF-1 was(-)-Epicatechin,LF-2 was 8-(2-Pyrrolidinone-5-yl)-(-)-epicatechin,LF-4 was 3'-Demethylicariside E3,and LF-3 and LF-5 were newly discovered.(4)Medicine target screening:Among the five chloroform extracts,there were five common targets,including arachidonate 5-lipoxygenase activating protein(ALOX5),interleukin(IL)-2,IL-6,nuclear factor κB1(NF-κB1),and vascular endothelial growth factor A(VEGFA).(5)Inflammatory proteins:The chloroform extracts of Pouzolzia zeylanica could inhibit the levels of inflammatory proteins to varying degrees,with LF-3 showing the best inhibitory effect.CONCLUSIONS Pouzolzia zeylanica extract can inhibit the inflammatory response of LPS-induced human lacrimal gland epithelial cells,with LF-3 in the chloroform extract showing the best pharmacological activity.
human lacrimal gland epithelial cellsPouzolzia zeylanica extractinflammationactive ingredientschloroform
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