Activation Of Melanocortin-1 Receptor By NDP-MSH Attenuates Oxidative Stress And Neuronal Apoptosis Following Intracerebral Hemorrhage In Mice Via PI3K/Akt/Nrf2 Signaling Pathway

Background: Intracerebral hemorrhage(ICH)is a disease with high morbidity and mortality in acute cerebrovascular disease and the most common cause of permanent disability in adults.At present,the specific mechanism of brain damage caused by cerebral hemorrhage is not completely clear.Inflammation,oxidative stress,apoptosis and autophagy are currently considered as one of molecular mechanisms of brain damage after ICH.It is of great significance to study the molecular mechanism of brain injury and help to find effective therapeutic targets.Recent studies have showed that NDP-MSH(Nle4-D-Phe7-α-MSH)binding to melanocortin receptor-1(Mc1r)exert anti-inflammatory,anti-apoptotic and anti-oxidative stressthrough various signaling pathways in subarachnoid hemorrhage,experimental brain trauma and other diseases.However,the neuroprotective effect of NDP-MSH on brain injury after ICH,especially the effects of anti-apoptosis and anti-oxidative stress response and related molecular mechanisms are still unclear.Objective: To study the effect of Mc1 r agonist NDP-MSH on post-ICH neurological function,degree of cerebral edema,and oxidative stress response,and explore its related molecular mechanisms.Methods: 25 g adult C57 BL / 6 mice were selected to establish ICH model by blood injection.The brain tissues around the hematoma were harvested at 6,12,24,72 and 7 days after ICH and the dynamic changes of MC1 R expression were detected by Western blot.In addition,the brains were cut off at 24 h after ICH,frozen sections were made,and the expression of Mc1 r in neurons was detected by double standard immunofluorescence staining(double immunfluorescence staining).In order to explore the neuroprotective effect of MC1 R agonist ndp-msh on brain injury of mice with ICH and determine the optimal dose of ndp-msh,three different doses(1.5 μ g /mouse,5 μ g /mouse,15 μ g/mouse)of ndp-msh were injected intraperitoneally one hour after modeling.At 24 h after ICH,we used modified Garcia score and Beam balance to evaluate the neurological function of mice,and measured the water content of bilateral brain by dry and wet method.The levels of MDA,CAT and SOD were detected by Enzyme linked immunosorbent assay(ELISA).In order to explore the molecular mechanism of MC1 R neuroprotection,MC1 R si RNA or PI3 K specific inhibitor LY294002 was injected into lateral ventricle 48 hours before the establishment of the model;NDP-MSHwas injected intraperitoneally one hour after ICH model was established.The levels of MDA,cat and SOD were detected by ELISA 24 hours after ICH;Western blot was used to detect the expression of MC1 R,p-pi3 k,PI3K,p-Akt,Akt,p-nrf2,Nrf2,Bcl-2,c-caspase 3 and caspase 3;Apoptosis was detected by terminal deoxynucleotidyl transfer mediated d UTP biotin nick end labeling(TUNEL).Results: Compared with sham operation group,Western blot showed that MC1 R expression increased significantly at 24 hours after ICH,continued to 72 hours after ICH,and returned to normal level at 7 days after ICH;double immunofluorescence showed that MC1 R was mainly expressed in neurons after ICH.After ICH,the mice showed severe neurological dysfunction,improved Garcia and Beam balance scores decreased,brain water content increased,oxidative stress related molecules MDA expression level increased,SOD and CAT expression levels decreased.After low dose of NDP-MSH(1.5 μg /mouse),the neurological score,brain edema and antioxidant stress were not significantly improved,but after medium and high dose of NDP-MSH(5 μ g /mouse,15 μ g /mouse),the neurological score,brain water content and antioxidant stress were significantly improved.In addition,there was no significant difference between the middle dose group and the high dose group.Therefore,5μg/mouse NDP-MSH was selected as the next treatment dose.After treatment with NDP-MSH(5μg/mouse),the neurological function,the degree of brain edema and the antioxidant stress state of ICH mice were significantly improved.The expression of downstream related factors and anti apoptotic related proteins increased,the expression of Pro apoptosis related proteins decreased,and the apoptotic cells of neurons decreased.However,when MC1 R was knocked down or PI3 K was inhibited,the neuroprotective effect of NDP-MSH decreased,the expression of downstream related factors and anti apoptosis related proteins decreased,the expression of apoptosis related proteins increased,and neuron apoptosis cells increased.Conclusion: MC1 R expression increased in the early stage after ICH,and the protein was mainly expressed in neurons.NDP-MSH combined with MC1 R can improve the neurological dysfunction,reduce brain edema,reduce oxidative stress response and neuronal apoptosis after ICH in mice.At least part of its neuroprotective function is related to the activation of PI3 K / Akt / Nrf2 signal pathway.