The Function Of MiR-200a/KEAP1/NRF2 Antioxidant Signaling On Arterial Endothelium Of Diabetic Artery And The Mechanism Study

Diabetes mellitus(DM)is a chronic disease that is increasing dramatically,posing a severe threat to public health worldwide.DM-induced arterial lesion is a direct mechanismfor amputation caused by diabetic foot,resulting in high mortality and morbidity.As a result,this greatly hampers the health of inhabitants,leaving a tremendous burden to social economy.However,the mechanism of diabetic arterial lesionis unclear,and there is a lack of effective approaches for its intervention.DM-induced arterial endothelial injury is the critical first step of diabetic arterial lesion.Therefore,it is of great importance to understand the mechanism of the pathogenesis of DM-induced arterial endothelial injury,and discover viable targets for intervention.Oxidative stress contributes to DM-induced arterial endothelial injury.Nuclear factor erythroid 2-related factor 2(NRF2)is a master regulator of the cellular antioxidant defense system.As a transcription factor,NRF2 translocates to the nucleus to active the transcription of various antioxidant genes,enhancing the cellular antioxidant capacity that fights against DM-induced oxidative stress.Hence,activation of NRF2 is a promising strategy for the amelioration of DM-induced arterial endothelial injury.Kelch-like ECH-associated protein 1(KEAP1)is a key negative regulator of NRF2.In the cytoplasm,KEAP1 combines NRF2,restricting NRF2 from nuclear translocaton and facilitating the proteasomal degradation of NRF2.Thus,inhibition of KEAP1 is an effective approach to activate NRF2.Previously,we found that dimethyl fumarate–a small molecule inhibitor of KEAP1–was able to activate NRF2,enhancing the cellular antioxidant capacity.In addition to the inhibition of KEAP1 protein activity,regulation of Keap1 gene expression at the m RNA level– which is supposed to lower KEAP1 protein level –may be another strategy to activate NRF2.However,it is still unclear how Keap1 m RNA expression is regulated.Micro RNAs(miRNAs)are highly conserved,single-stranded non-coding RNAs of 18 to 25 bp in length,exerting regulating effects at the post-transcriptional level.By binding the 3'untranslated region(3'UTR)of the target m RNA,an miRNA is able todegrade the target m RNA or inhibit the process of the protein translation.Our previous study has demonstrated that miR-200 a activates NRF2 antioxidant signaling by degrading Keap1 m RNA in diabetic nephropathy,a microvascular complication of DM.However,the effect of miR-200 aon DM-induced arterial endothelial injury and the underlying mechanism is still unknown.Objectives:1.Observe the role of miR-200a/Keap1/NRF2 pathway in DM-induced arterial endothelial injury.2.Explore the role of KEAP1 and NRF2 in mediating the effects of miR-200 a in DM-induced arterial endothelial injury.Methods:1.To observe the impact of high glucose(HG)on miR-200a/KEAP1/NRF2 pathway and the levels of oxidative stress and inflammation,arterial endothelial cells(AECs)were removed from the aortas of the wild-type(WT)mice and were challenged by HG for 1 h,6 h,12 h,24 h and48 h.2.To determine the effect of miR-200 a on the expression of KEAP1/NRF2 signaling and its impact on oxidative stress and inflammation,HG-challenged AECs were co-treated with miR-200a's mimic(miR-200a-M)or inhibitor(miR-200a-I),as well as their respective negative controls,for 48 h.3.To investigate the role of KEAP1 in mediating miR-200a's efficaciesinthe regulation of KEAP1/NRF2 signaling,oxidative stress and inflammation,AECs were co-treated with HG,Keap1 siRNA and miR-200a-M,for 48 h.4.To further test whetheror not NRF2 is indispensible for miR-200a's actions,AECs isolated from WT and Nrf2 gene knockout(KO)mice were treated with miR-200a-M,for 48 h.5.To verify the effect of miR-200 a supplementation in vivo,WT and Nrf2 KO diabetic mice– which were induced by multiple low dose injection of streptozotoxin– were treated with miR-200a-M or its negative control,for 20 weeks,with the expression of miR-200a/Keap1/NRF2 pathway and the levels of oxidative stress and inflammation determined in the aortas.6.In order to test the effect of miR-200a-M on arterial endothelial dysfunction,the contraction and relaxation were evaluated using the aortas of the WT and Nrf2 KO mice.Results:1.HG time-dependently decreased miR-200 a,increased Keap1 gene expression,and lowered the level of total cellular NRF2 protein(t-NRF2).After 48 h of HG incubation(endpoint of the time course),the amounts of nuclear NRF2(n-NRF2),and m RNAs of Ho1 and Nqo1 was decreased to the lowest levels,whereas oxidative stress indicators ROS and MDA,and inflammatory markers Vcam-1 and Mcp-1 m RNAs were brought to the highest levels.2.MiR-200a-M significantly elevated miR-200 a level,inhibited Keap1 expression,activated NRF2 antioxidant signaling,and attenuated oxidative stress and inflammation in HG-treated WT AECs.On the contrary,miR-200a-I had adverse impacts on these indices.3.MiR-200a-M was not able to further enhance NRF2 antioxidant signaling,and inhibit HG-induced oxidative stress and inflammation in the presence of the Keap1 siRNA.4.In Nrf2 KO AECs,miR-200a-M completely lost the capability to enhance the transcription of Ho1 and Nqo1,and alleviate HG-induced oxidative stress and inflammation.5.MiR-200 a up-regulated arterial miR-200 a,inhibited Keap1 expression,enhanced NRF2 antioxidant signaling,and diminished oxidative stress and inflammation in the WT diabetic mice,the effects of which were completely abolished in the Nrf2 KO mice.6.MiR-200 a improved DM-induced arterial endothelial dysfunction in the WT,but not Nrf2 KO,mice.Conclusions:1.MiR-200a/KEAP1/NRF2 pathway controlled arterial endothelial antioxidant capacity under the DM/HG conditions.2.KEAP1 and NRF2 completely mediated miR-200a's actions in HG-treated AECs.3.NRF2 was required for MiR-200a's protective effects on DM-induced arterial endothelial injury or HG-induced endothelial oxidative stress and inflammation.