The Effect Of Artemisia Annua Pollen On Tight Junction Of Nasal Mucosal Epithelial Cells And Its Mechanism Analysis

Objective:To analyze the effect of Artemisia annua pollen on the tight junction of human nasal epithelial cells(HNEpC),study the effect of Artemisia annua pollen on the expression and localization of Occludin and Claudin-1 protein in HNEpC membrane,clarify the relationship between Occludin and Claudin-1 protein and p38 MAPK signal pathway activation,and to find out the possible mechanism of Artemisia annua pollen inducing and aggravating allergic rhinitis,so as to provide a new target and treatment for the future clinical prevention and treatment of allergic diseases sensitized by pollen.Methods:1.HNEpC cell lines were cultured in vitro,and the cell viability was detected by CCK-8 method;Immunofluorescence staining was used to observe the expression and distribution of Occludin and Claudin-1 in cell membrane;the levels of p38 MAPK and its phosphorylated protein p-p38 MAPK were detected by Western Blot;Western blot and Quantitative real-time PCR(qPCR)were used to detect the protein and mRNA levels of Occludin and Claudin-1,respectively.2.On the basis of the above experiments,the cells were pretreated with 50 ?mol/L p38 MAPK inhibitor(SB203580)for 1 h,and then treated with 200 ?g/mL Artemisia annua pollen for 24 h.Finally,Western blot,real-time fluorescence quantitative PCR and immunofluorescence chemical staining were used to detect the expression of Occludin and Claudin-1,and the inhibition of SB203580 on tight junction damage was clarified.Results:1.The results of CCK-8 showed that the cell viability of Artemisia annua at different concentrations(20,40,80,100,160,200 ?g/mL)was increased 6 hours after the intervention of HNEpC(P<0.05);after 12 h of intervention,the cell viability of 20,40,80,100,160 ?g / mL group did not change significantly(P>0.05),the cell viability of200 ?g/mL group was decreased(P<0.05);after 24 hours of intervention,no significant changes in cell viability were observed in the 20 and 40 ?g/mL group(P>0.05),cell viability of 80,100,160,and 200 ?g/mL groups decreased(P<0.05).Immunofluorescence staining showed that normal Occludin and claudin-1 proteins were located on the cell membrane and were highly expressed,forming a ring structure around the cell membrane.However,the expression levels of Occludin and Claudin-1 protein decreased significantly when artemisia annua was interfered with 200 ?g/mL and 40,80,100,160,200 ?g/mL respectively,and fracture,ambiguity uneven distribution were observed.The results of Western blot showed that the expression of p-p38 MAPK in hnepc cells was significantly increased after pollen treatment.The results of Western blot and Quantitative real-time PCR showed that the expression level of HNEpC Claudin-1protein and its mRNA in the pollen(40,80,100,160,200 ?g/mL)intervention group was lower than that in the control group(P< 0.05),while the expression level of Occludin protein and mRNA in the pollen treatment group was only lower than that in the pollen treatment group(P<0.05)and increased in the other treatment group(P<0.05).2.The results of immunofluorescence,Western blot and real-time quantitative PCR showed that SB203580 could inhibit the decreased expression of Occludin caused by pollen of artemisia annua,but had no effect on the expression of Claudin-1.Conclusion:1.Different concentrations of artemisia annua pollen had different effects on the activity of nasal epithelial cells,and the expression and distribution of Occludin and Claudin-1 with tight connections were also different.High concentration of artemisia annua pollen could reduce the expression of tight junction protein and mRNA in HNEpCcells,caused increased epithelial permeability,which in turn disrupts cell barrier function,making it easier for allergens to enter the body.This may be an important mechanism of allergic rhinitis induced by pollen of artemisia annua.2.By activating the p38 MAPK signaling pathway,the pollen of artemisia annua could down-regulate the expression of the close connection between HNEpC cells and aggravate the occurrence of allergic rhinitis.Inhibition of p38 MAPK signaling pathway can enhance the function of nasal mucosal epithelial barrier,which provides experimental support for the treatment of airway allergic diseases caused by pollen of artemisia annua at the cellular and molecular level.