The Role Of RhoA In Crossing The Blood-Brain Barrier In The Form Of "Trojan Horse" In Listeria Monocytogenes

Objective:Listeria monocytogenes(Lm)crossing the blood-brain barrier in the form of "Trojan horse" is of great significance in the occurrence and development of bacterial encephalitis and meningitis.Cell migration and breakthrough of the blood-brain barrier are very important to the process.The RhoGTPase family is a key factor regulating cell migration.This study was to investigate the expression of RhoA in this process and to observe the effects of RhoA on biological behavior such as cell migration and expression of immune factors.At the same time,the effect of RhoA in regulating macrophages on the blood-brain barrier was studied.Methods:1.The Transwell chamber assay was used to detect the migration ability of RAW264.7 infected by Lm.Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)and ELISA were used to detect the changes of IL-1β,IL-6,TNF-α and IL-10 in mRNA and protein levels after RAW264.7 infected by Lm.qRT-PCR and Western blot were used to detect the difference in expression of RhoGTPase family in mRNA and protein levels after RAW264.7 infected by Lm.2.Downregulated the expression of RhoA in RAW264.7,and its efficiency was verified by qRT-PCR and Western blot.Transwell chamber assay was used to detect changes in migration ability of control and interference groups in RAW264.7.Flow cytometry(FCM)was used to detect the ability of antigen presentation and cell cycle changes in control and interference groups.CCK8 and phagocytosis assays were used to detect cell proliferation and phagocytosis.qRT-PCR and ELISA were used to detect changes in mRNA and protein levels of IL-1β,IL-6,TNF-α,and IL-10 in the control and interference groups.The expression of ROCK was detected by Western blot.3.The blood-brain barrier is constructed by rat brain microvascular cells and astrocytes.After stimulated by the supernatants of RAW264.7 infected by Lm,HRP leakage test was used to detect blood-brain barrier permeability.Wound-Healing assay and Transwell chamber assay was used to detect the migration of endothelial cell.Real-time quantitative PCR was used to detect changes in tight junctions and ICAM-1 of endothelial cells.Result:1.Compared with the control group,the migration ability and the expression of IL-1β,IL-6,TNF-α,IL-10 in mRNA level were increased with the increase of Lm amount.The expression of IL-1β,IL-6,TNF-α in protein level was increased,however IL-10 expression did not change.2.The results of qRT-PCR and Western blot showed that the expression of RhoGTPase family in infected cells was different compared with the control group,and the difference of RhoA was the most obvious.Its downstream effector molecule ROCK2 also showed a consistent change with RhoA.3.Downregulated the expression of RhoA in infected RAW264.7,it could significantly inhibit the migration of RAW264.7 cells and the expression of IL-6,TNF-α and IL-1β,but it had no effect on IL-10.It has no effect on cell proliferation and phagocytosis,and can reduce the antigen presentation ability of RAW264.7 cells.4.Downregulated the expression of RhoA in infected RAW264.7,it could reduce the destructive effect of Lm on the blood-brain barrier.After RhoA downregulated,BBB permeability was decreased,endothelial cell migration ability was increased,tight junction protein occludin and claudin5 were increased,ICAM-1 was decreased,and the number of bacteria carried by macrophage acrossing BBB was reduced.Conclusion:RhoA plays an important role on Lm crossing the blood-brain barrier in the form of "Trojan horse".It may regulate IL-6,IL-1β to disrupt tight junction which increasing blood-brain permeability and to upregulate ICAM-1 which promoting adhesion of macrophages and endothelial cells to promote cell migration for crossing the blood-brain barrier.This process may be regulated by RhoA/ROCK2 signaling pathway and has positive effects on the treatment of the bacterial encephalitis and cerebral edema.