Effects Of EBOV Glycoprotein On The Outer Blood-Retinal Barrier Function And Therapeutic Agent

Background:Ebola virus(EBOV)is a virulent infectious disease that can cause Ebola haemorrhagic fever(EBHF)in humans and other primates.Since the first outbreak of EBOV in 1976,more than 40 outbreaks have been confirmed.The most severe EBOV outbreak occurred in West Africa from 2013 to 2016,resulting in more than 28,000 infections and 11,000 deaths.During the global COVID-19 pandemic,there are repeatedly EBOV outbreaks in Africa.The latest outbreak began on 20 September 2022 and was declared over on 11 January 2023,with 142 confirmed cases(22 suspected)and 55 confirmed deaths.These small outbreaks remind us that the EBOV epidemic is ongoing and requires constant vigilance.In a large number of Ebola virus disease(EVD)survivors,the persistence of EBOV has been reported to cause a variety of sequela,including joint pain,cognitive impairment,headaches,hearing loss and myalgia,a phenomenon known as post-Ebolavirus disease syndrome(PEVDS).Eye disease is one of the most common sequelae among EVD survivors.Among them,about 35%of EVD survivors have different kinds of ocular complications,including blurred vision,ocular inflammation,optic nerve and retinopathy,etc.If untreated,the ocular sequelae of EVD survivors can lead to visual impairment or blindness,which could seriously affect the life quality of EVD survivors.In our previous study,it was found that HIV and its envelope protein gp120 could destroy the outer blood-retinal barrier by induction of a large number of inflammatory factors in retinal pigment epithelial cells.This finding suggests that free type I transmembrane glycoproteins derived from other viruses might also influence the function of the outer blood-retinal barrier.EBOV can infect the retinal pigment epithelial cells,but there are no reports of EBOV mediated destruction of outer blood-retinal barrier by non-infectious paracellular pathway.In view of this,we intend to explore whether EBOV GP could also damage the outer blood-retinal barrier and thus affect the function of blood-retinal barrier?What is the mechanism?Does it cause further bacterial co-infection or damage to the optic nervous system after breaking the blood-retinal barrier?Are there any drugs that can protect against this damage?These studies have not been reported and are worth further discussion.In order to further explore the destructive effect of EBOV GP on the outer blood-retinal barrier function and clarify its related mechanism,this study will explore the role of EBOV GP in disrupting the outer blood-retinal barrier,identify the key signaling pathways involved,which will provide theoretical basis and therpeutcial target for the prevention and treatment of EVD-related retinopathy.Objectives:In this study,retinal pigment epithelial cell line D407 cells were used to construct an outer blood-retinal barrier model in vitro and EBOV GP was injected into mouse vitreous cavity to construct an animal model in vivo.We aimed to explore the effect of EBOV GP on the function of outer blood-retinal barrier in vitro;to explore the effect of EBOV GP on bacterial/virus translocation and subsequent infection of optic nervous system due to the desrupted blood-retinal barrier;to explore the molecular mechanism of by which EBOV GP damaging the blood-retina barrier and the key domain of EBOV GP;to explore the effect of EBOV GP on blood-retinal barrier function in mice;to evaluate the protective effect of the natural compound Resveratrol on EBOV GP-induced destruction of the blood-retinal barrier.The results are expected to reveal the pathogenesis of EVD-associated retinopathy1.Establish an outer blood-retinal barrier system in vitro,and evaluate the integrity and stability of this barrier by measuring TEER value.2.TEER value measurement,NaF leakage assay,RT-qPCR,Western Blot and indirect immunofluorescence assay were use to explore the effects of EBOV GP on the function of outer blood-retinal barrier in vitro.3.The effects of EBOV GP on D407 cell viability were detected by MTT assay.4.Bacterial invasion experiment and HIV-1 trans-infection of optic glial cells experiment was conducted to explore the effects of EBOV GP on bacterial/viral co-infection of optic nervous system after the destruction of outer blood-retinal barrier in vitro.5.RNA-Seq analysis,Western Blot,RT-qPCR,ELISA,Reactive oxygen species(ROS)experiment,Nrf2 inhibitor and activator experiment,Nrf2 overexpression and knockdown experiment,MTT assay were applied to explore the molecular mechanism of by which EBOV GP disrupts the outer blood-retinal barrier in vitro and potential drug therapeutic target,the key domain of EBOV GP disrupting the outer blood-retinal barrier.6.The blood-retinal barrier model in mice was established,HE staining,Evans Blue leakage assay,immunohistochemistry and Western Blot assay to explore the effects of EBOV GP on the blood-retinal barrier function in mice and the changes in the signaling pathway.7.HE staining,Evans Blue leakage assay and Western Blot assay were used to evaluate the protective effect of Resveratrol on the destruction of blood-retinal barrier function induced by EBOV GP in mice.Results:1.The measurement of TEER value,NaF leakage assay,RT-qPCR calculation results,Western Blot and indirect immunofluorescence showed that EBOV GP could downregulate the expression of tight junction proteins,resulting in decrease of TEER value and increase cell monolayer permeability of D407 cells,thus disrupting the function of outer blood-retinal barrier in vitro,and this effect was not correlated with cytotoxicity.2.Bacterial invasion experiment and HIV-1 infection of optic glial cells experiment results indicated that EBOV GP could further lead to potential bacterial co-infection and optic nervous system damage after destroying the outer blood-retinal barrier in vitro.3.The results of RNA-Seq analysis indicated that the antioxidant-related signaling pathway and PI3K/Akt signaling pathway were related to the damage of EBOV GP to the outer blood-retinal barrier.4.Western Blot,RT-qPCR calculation results,ELISA,Reactive oxygen species(ROS)experiments and MTT experiments showed that EBOV GP could activate PI3K/Akt signaling pathway,inhibit Nrf2/HO-1 antioxidant signaling pathway,induce oxidative stress and increase production of ROS,thus disrupting the function of outer blood-retinal barrier.5.Nrf2 inhibitor and activator experiments,Nrf2 overexpression and knockdown experiments indicated that Nrf2 may be a potential drug target for the treatment of EVD-associated retinopathy.6.Western Blot,reactive oxygen species(ROS)and MTT experiments indicated that GP2 subunit may be the key domain in which EBOV GP destroys the outer blood-retina barrier.7.HE staining,Evans Blue leakage assay,immunohistochemistry and Western Blot showed that EBOV GP damaged the retina of mice by destroying BRB,and PI3K/Akt-Nrf2 signaling pathway was involved in the damage process of EBOV GP on BRB.8.HE staining,Evans Blue leakage assay and Western Blot showed that Resveratrol had a protective effect on EBOV GP-induced blood-retinal barrier disruption,which regulated the expression of retinal tight junction proteins through the regulation of PI3K/Akt-Nrf2 signaling pathway,alleviated the retinal structure destruction and permeability increase,thus improve the function of mouse blood-retinal barrier.Conclusion:1.EBOV GP can destroy the outer blood-retinal barrier in vitro,and this effect is independent of cytotoxicity.2.EBOV GP mediates the destruction of blood-retinal barrier in vitro and in vivo through the PI3K/Akt-Nrf2 signaling pathway,and Nrf2 can be used as a potential drug therapeutic target.3.The GP2 subunit may be the key domain in which EBOV GP destroys the outer blood-retinal barrier.4.The destruction of outer blood-retinal barrier in vitro by EBOV GP can further lead to potential bacterial or virus co-infection and optic nervous system damage.5.Resveratrol plays a protective role in EBOV GP-induced destruction of blood-retina barrier.