Memantine attenuated E. coli K1-induced tight junction injuries through inhibition of α7 nAChR-dependent pathway in vitro

Abstract

Background: As gram-negative bacterium, E. coli K1 is notorious for causing neonatal sepsis and meningitis (NSM) which leads to high mortality and morbidity in neonates over the recent decades worldwide. Previous studies illustrated that memantine (MEM), a drug approved by FDA for the treatment of Alzheimer's disease, may be adopted as a potential novel therapeutic agent against bacterial meningitis targeting at α7nAChR. In our study, we aim to determine the effects of MEM on tight junction injury in blood-brain barrier (BBB) induced by E. coli K1 in vitro. Methods and materials: Human brain microvascular endothelial cells (HBMECs), as the important structural cells in BBB, were involved in the study. Confluent HBMECs in three 24-well plates were treated with MLA (α7 nAChR antagonist), MEM, and PBS for 1 h respectively, and then incubated with E. coli K1 (0.5× CFU/well) for 2.5 h. After that, adhesion assays were carried out to determine the protective effects of MEM. In order to evaluate the integrity of tight junctions of HBMECs, western blot and immunofluorescence staining were used to detect the expressions of Occludin and ZO-1 in different groups. Results: Results show that MEM inhibited the adhesion of E. coli K1 to HBMECs (p < 0.05) dose-dependently, while MLA also blocked the bacterial adhesion (p < 0.05). The western blot result shows that MEM markedly up-regulated the expression of Occludin and ZO-1 through blocking α7nAChR signaling (p < 0.05) as MLA performed. Furthermore, immunofluorescence staining shows that E. coli K1 impaired the integrity of the tight junction, while MEM and MLA both ameliorated tight junction damage induced by E. coli E44 in the study. Conclusion: Our study suggests that MEM enhances the expression of the tight junction- associated proteins (Occludin and ZO-1) and alleviates the tight junction damage through inhibiting α7 nAChR signaling way. (Acknowledgements: *Corresponding author: Hong Cao. The study was supported by National Natural Science Foundation of China, No. 81871198 to H.C.,and Undergraduate Training Program for Innovation and Entrepreneurship of SMU, China, No.201912121238)