The Mechanism Of NLRP3 Inflammasome Activated By VDAC1-Mediated Mitophagy In Diabetic Retinopathy

ObjectivesDiabetes mellitus is one of the most important chronic non-communicable diseases threatening human health worldwide.As a highly specific microvascular complication of diabetes mellitus,diabetic retinopathy(DR)has become the first irreversible blindness disease in working-age population.Proliferative diabetic retinopathy(PDR)is the main type of severe visual impairment.The etiology of DR is complex.Epidemiological investigation shows that DR has significant racial differences and family cohabitation,suggesting that genetic factors play an important role in the susceptibility of DR.In view of this,in the first part of this study,the adult patients with type 2 diabetes mellitus in the Han population of South China were selected as the subjects,the patients with PDR as the case group,and the patients with type 2diabetes mellitus without retinopathy(NDR)as the control group.The single-nucleotide polymorphism(SNP)associated with the occurrence of PDR was screened by whole exome sequencing(WES).SNPs and related susceptibility genes were verified by SnaPshot technique.Inflammatory response is an important factor mediating retinal microvascular damage in DR.NLRP3(nucleotide-binding oligomerization domain-like receptors 3)inflammasome,as an important component of innate immunity,has been proved to play an important role in the occurrence and development of DR.Our previous studies found that the expression of NLRP3 mRNA and protein in peripheral blood mononuclear cells of DR patients increased,suggesting that NLRP3 inflammasome may participate in the occurrence of DR.Mitophagy has also been shown to play an important role in diabetes and its complications.In recent years,many studies have shown that mitophagy is a balanced regulator of NLRP3 inflammasome.Voltage-dependent anion channel 1(VDAC1)protein is a porous protein with abundant content on the outer membrane of mitochondria,which controls the entry and exit of metabolites such as reactive oxygen species(ROS)in mitochondria and plays an important role in regulating the metabolism and energy transport of mitochondria.It has been found that VDAC1 also participates in the activation of NLRP3 inflammasome,but the mechanism is still unclear.We speculate that "VDAC1 mediated mitophagy regulates the activation of NLRP3 inflammasome which participates in the occurrence of DR".In order to verify this hypothesis,the second part of this study,which makes human retinal vascular endothelial cells(HRCECs)as the research object,high glucose as the condition,will gradually explored the effects of high glucose in DR on retinal vascular injury,NLRP3 inflammatory bodyinflammasome activation,mitophagy and VDAC1 expression,and reveal the role of VDAC1 in mediating NLPR3 inflammasome activation through drug intervention and cell transfection studies.This study will lay a basis for elucidating the pathogenesis of DR and provide a new target for the prevention and treatment of DR.Methods1.Prospective study design included type 2 diabetic patients diagnosed in Dongguan Eye Study from September 2011 to February 2012 and type 2 diabetic patients with or without PDR diagnosed and treated in ophthalmology department of Guangdong People's Hospital from July 2017 to March 2018.Single nucleotide polymorphism(SNP)and InDel were used to identify DNA samples from peripheral blood samples of NDR and PDR patients by WES technology.Significant differences were screened out among them.SnaPshot technology was used to verify the differences between the two groups.2.HRCECs cell lines with good growth in vitro were treated with normal glucose concentration(control group,8.3 mM D-glucose),high mannitol concentration(HM group,8.3 mM D-glucose + 21.7 mM D-mannitol)and high glucose concentration(HG group,30 mM D-glucose)for 48 hours respectively.Cell proliferation was measured by MTT method,cell migration ability was measured by scratch test and tubule formation test was performed.ELISA kit was used to detect the levels of VEGF and IL-6 in cell supernatant.The expression of NLRP3 inflammasome-related protein,VDAC1 protein,mitophagy-related protein Mfn2,Drp1,PINK1 and Parkin were detected by Western Blot.The morphology and quantity of mitochondria and autophages were detected by transmission electron microscopy,and reactive oxygen species were detected by MitoSOX kit.3.HRCECs were treated with control group,control+25uM Mdivi-1 group,HG group and HG+25uM Mdivi-1 group for 48 hours respectively.PINK1,LC3 B,VDAC1 and NLRP3 protein expression were detected by Western Blot.HRCECs were treated with control group,lentivirus empty vector group,lentivirus overexpression VDAC1 group,HG group and HG+lentivirus overexpression VDAC1 group,respectively.MTT method was used to detect the proliferation of cells in each group and scratch test was fine.Cell migration ability,tubular formation assay and Western Blot were used to detect the expression of VDAC1,PINK1 and NLRP3 proteins.Results1.Seventy-five SNP loci detected by WES were associated with PDR,involving 53 genes,11 of which were located in exon region,and 7 were non-synonymous mutations.The protein encoded by NLRP5 gene located in exon region is a member of NLR family.Nine loci were screened and verified by SnaPshot SNP typing.The results showed that there was no significant difference in genotype and allele frequencies between PDR group and NDR group(P > 0.05).2.HRCECs were treated with high glucose for 48 hours.Cell proliferation,migration and tubular formation were significantly enhanced,and the expression of VEGF and IL-6increased.The expression of NLRP3 inflammasome-related protein NLRP3,Caspase-1 and IL-1beta increased.And the expression of mitochondrial mitotic protein Drp1 increased,while the expression of fusion protein Mfn2,mitochondrial autophagy protein Parkin,PINK1 and VDAC1 decreased(P < 0.05).3.25 uM Mdivi-1 inhibited mitophagy and increased the expression of NLRP3 protein(P <0.05),but there was no significant change in the expression of VDAC1 protein(P > 0.05).In high glucose environment,after overexpressing VDAC1,the expression of PINK1 and Parkin protein increased significantly,while the expression of NLRP3 protein decreased,and the cell proliferation,migration and tube formation ability decreased significantly.Conclusions1.PDR is a polygenic genetic disease.Its occurrence and development are the result of the interaction of genetic factors and the external environment.2.High glucose can reduce the expression of HRCECs VDAC1 protein,inhibit mitophagy,activate NLRP3 inflammasome,and enhance cell proliferation,migration and tubular formation,stimulate the secretion of inflammatory factors.3.Inhibiting mitophagy can activate NLRP3 inflammasome,but has no significant effect on the expression of VDAC1 protein.Overexpressing VDAC1 in high glucose condition enhanced the level of mitophagy,inhibited the activation of NLRP3 inflammasome and protected the biological function of cells.This mechanism may be involved in the occurrence of DR in high glucose condition.