Ascorbic Acid Protects Retinal Pigment Epithelial Cells From High Glucose By Inhibiting NF-κB Signal Pathway Through MALAT1/IGF2BP3 Axis

Objectives: The common complication of diabetes is diabetic retinopathy(DR)with severe blindness,but its current treatment and prevention is limited.The pathogenesis of DR is closely related to the intraocular oxidative stress induced by hyperglycemia,but its detailed mechanism still unclear.Therefore,this study intends to take “the oxidative stress of DR” as the entry point to explore whether the antioxidant ascorbic acid(vitamin C,AA)is beneficial to relieve DR oxidative stress and its mechanism.In view of the fact that AA can inhibit NF-κB signaling in retinal progenitor cells,Lnc RNA MALAT1 can regulate antioxidant defense in diabetic retinopathy through NF-κB signaling pathway,and MATAL1 may have RNA protein binding site(RBP)with IGF2BP3,this study proposes the hypothesis that AA inhibits NF-κB signaling pathway by regulating MALAT1/IGF2BP3 to alleviate diabetic retinopathy.The purpose of this study was to investigate the regulatory role and mechanism of AA and MALAT1/IGF2BP3 in high glucose(HG)-induced RPE cell injury.Method: 1)ARPE-19 cells were treated with high glucose(HG)to establish a cell damage model induced by HG.The binding interaction of MALAT1 with insulin-like growth factor 2 m RNA-binding protein 3(IGF2BP3)was verified by RIP-q PCR assay.Gene expression levels and protein density were assessed by q RT-PCR or Western Blot.Immunofluorescence detection of protein translocation status.Inflammatory factor levels were measured by ELISA assay.Apoptosis was measured by flow cytometry;2)40 DR patients and 10 healthy volunteers were collected;DR patients were divided into a control group and a treatment group.The treatment group was given AA,2g,intravenously for 3 consecutive days,5m L blood samples were drawn from the healthy group,the treatment group and the negative control group,and the expression level of MALAT1 and IGF2BP3 genes was detected by the methods described above.Results: 1)(1)RIP-q PCR electrophoresis map and RIP-q PCR analysis data map showed that there was binding between IGF2BP3 protein and lnc RNA-MALAT1 in ARPE-19 cells.Compared with NG group,the binding ability between MALAT1 and IGF2BP3 in HG group was improved,and there was a statistically significant difference compared with the NG group(P<0.01):(2)q PCR showed that HG could increase the m RNA expression of IGF2BP3(P<0.001),AA treatment reduced the level of MALAT1(P<0.01)and m RNA level of IGF2BP3(P<0.001)in HC-treated RPE cells.The inhibitory effect of AA administration on IGF2BP3 m RNA expression was terminated by overexpression of MALAT1(P<0.001).(3)Western Blot results showed that HG treatment increased IGF2BP3 protein expression,AA application decreased IGF2BP3 protein expression level(P<0.001),overexpression of MALAT1 increased IGF2BP3 protein expression(P<0.001),and silencing MALAT1 decreased IGF2BP3 protein expression(P<0.001).Overexpression of IGF2BP3 promotes p-p65/p65,p-IκBα/IκBα,IGF2BP3 silencing promoted the transport of p65 protein to the cytoplasm.There was a statistical difference in the expression of P65 between IGF2BP3 silencing group and negative control group(P<0.01)(4)ELISA test suggested that AA application reduced HG induced inflammatory factors release(including IL-1β,IL-6,TNF-α and MCP-1),the difference is statistically significant(IL-1β Group and TNF-α Group P<0.01;IL-6 group P<0.05;In MCP-1group,P<0.001),while the combination of MALAT1 overexpression reversed this trend,with statistically significant differences(IL-1β Group P<0.05;IL-6 group and TNF-αGroup P<0.01;MCP-1 group(P<0.001).(5)IF detection showed that IGF2BP3 silencing inhibited p65 from entering the nucleus.(6)Flow cytometry showed that overexpression of MALAT1 increased apoptosis(P<0.01),Overexpression of IGF2BP3 promotes apoptosis of HG-treated ARPE-19 cells and silencing IGF2BP3 inhibited the apoptosis of ARPE-19 cells(P<0.01).2)The gene expression of MALAT1 and IGF2BP3 was higher in blood samples from DR patients,and the gene expression of MALAT1 and IGF2BP3 in blood samples from DR patients was significantly decreased after AA administration.Conclusion: Ascorbic acid inhibits the NF-κB signaling pathway through the MALAT1/IGF2BP3 axis,and plays a regulatory role in relieving cell damage.Induced by HG.