The Study On Application Of Neuroprotective Nanodrugs In Acute Ocular Hypertension Glaucoma

Purpose:Acute primary angle-closure glaucoma(APACG)is a blinding ocular disease with rapid onset and progressive irreversible loss of vision.The specific irreversible death of retinal ganglion cells(RGCs)and the loss of their axons are the characteristic pathological changes of APACG.However,the mechanism of RGCs damage in APACG has not been elucidated yet.Therefore,in this study,we firstly analyzed the transcriptome sequencing data of retina from mice and rats at different time points in acute ocular hypertension glaucomatous model(AOH)to reveal the molecular mechanism and dynamic changes of RGCs death,laying the foundation of finding suitable targets for intervention,and then constructed novel neuroprotective drugs based on nanomaterials,and finally clarified the protective effects and the specific mechanism of the constructed nanodrugs on RGCs in AOH model.Methods:(1)Firstly,we clarified the essential signaling pathways and cell death patterns in different species and during different pathological progression of glaucoma by differentially expressed genes and enrichment analysis of retinal transcriptome sequencing data from different time points in AOH of mice and rats from public databases.(2)The polymer P1was constructed using reactive oxygen species(ROS)sensitive response monomer,ROS depleting monomer and dihydroxycholesterol unit as the main body,and P1 was equipped with necrostatin-1(NEC)based on titration vortex method to construct the nanoparticle NP1.The morphology and particle size and zeta potential of NP1 were examined by transmission electron microscopy(TEM)and Malvern nanoparticle size potentiometry.Then,the oxygen and glucose deprivation(OGD)R28 cell model and mouse AOH model were established,and the survival of each group of cells was detected using CCK8 and Calcein AM/PI double-staining method after the intervention of NP1 and NEC,respectively.The expression of NP1(loaded with Cy5.5),DCFH-DA fluorescently labeled ROS,and necroptosis-related proteins were examined using confocal fluorescence microscopy and flow cytometry.The structure of the mouse retina was examined using hematoxylin-eosin staining(HE).The number of RGCs was observed by immunofluorescence of retinal mounts,and the visual electrical activity of mice was recorded by electroretinogram(ERG)and visual evoked potential(VEP).(3)The nano-drug ASX-NP was constructed by ultrasonic emulsification using thermosensitive responsive polymers,astaxanthin polymers and STAT3 inhibitor(stattic)as the main components,and then investigated the protective effects of ASX-NP on acute glaucoma model of cells and mice by cell proliferation toxicity assay,apoptosis assay,HE staining of mouse eyeball,immunofluorescence staining of retinal mounts and electrophysiological assay and so on.Results:(1)Analysis of sequencing data revealed that oxidative stress and inflammatory signaling pathways were significantly upregulated in mice and rats at different time points in AOH models;JAK-STAT signaling pathways were significantly upregulated;necroptosis pathway was significantly enriched and the expression of related molecules such as RIPK1/3 were also significantly overexpressed in AOH models.(2)TEM and Malvern nanoparticle size potentiometry showed that NP1 had a more homogeneous circular morphology and a negatively charged surface.Confocal fluorescence microscopy and flow cytometry analysis showed that NP1 had started to accumulate intracellularly at an earlier time,and that the level of ROS production was reduced in R28 cells of the OGD model with NP1 intervention.Expression of proteins related to the necroptosis pathway was also significantly decreased.Furthermore,in the AOH mouse model,both HE staining and retinal mounts staining results showed increased survival of RGCs after NP1 intervention,besides,significantly increased the wave amplitudes of a and b waves in ERG and N1-P1 in VEP.(3)The results of ~1H NMR showed that the astaxanthin macromolecule had the typical characteristic peaks of astaxanthin.TEM and Malvern nanoparticle size potentiometry showed that ASX-NP had a homogeneous circular structure and satisfactory dispersion stability.The proliferation toxicity and apoptosis assays showed that ASX-NP significantly increased the survival and decreased the apoptosis of R28cells after OGD modeling.Both HE staining and retinal mounts staining showed a significant reduction in the loss of RGCs in the AOH mice model after ASX-NP intervention,and ERG and VEP results showed a significant increase in wave amplitudes.Conclusions:(1)The necroptosis and JAK-STAT signaling pathways play important roles in the pathological damage of AOH models.(2)ROS-responsive and depleted and cell membrane-targeted nanodrug NP1 is successfully constructed,which can effectively protect RGCs as well as visual function in AOH mouse model by inhibiting necroptosis.(3)The constructed novel drug delivery system ASX-NP can protect RGCs as well as visual function of mice after AOH modeling by inhibiting STAT3expression,anti-inflammation and anti-oxidation,and reducing apoptosis,which laid the theoretical and practical foundation for clinical development of novel neuroprotective drugs.