The Mechanism Of MKP-5 In Regulating Palmitic Acid-induced Neuron Dysfunction

Background:Alzheimer’s disease（Alzheimer’s disease,AD）is the most common progressive neurodegenerative disease,which is mainly manifested as cognitive dysfunction.Its etiology and pathogenesis are not fully understood,and there is no effective treatment method.The main pathological features of AD are neurofibrillary tangles（NFTs）,a large amount of amyloidβ（Aβ）deposition and so on.Studies have shown that obesity is an important inducement to increase the risk of AD.The lipotoxicity caused by high saturated free fatty acid levels in the brain is one of the key factors that increase the risk of AD,but the mechanism of lipotoxicity in the brain has not been fully elucidated.Mitogen-activated protein kinase phosphatase-5（MKP-5）is a dual specificity phosphatase（DUSP）that plays a regulatory role in diabetes,pulmonary fibrosis and cancer.Studies have shown that MKP-5 affects the differentiation of oligodendrocytes and provides the possibility of treating diseases related to myelin deposition,and myelin abnormalities are related to a variety of neurodegenerative diseases.However,the mechanism of MKP-5 in neuronal injury has not been reported yet.In this study,palmitic acid（PA）was used to establish a PC12 lipotoxic cell model.By observing apoptosis,endoplasmic reticulum stress,oxidative stress,inflammation,and Aβproduction,we explored the effects of MKP-5 on lipotoxicity-induced neurons The regulation and possible mechanism of injury.Purpose:By regulating the expression of MKP-5 in PC12 cells,the effect of MKP-5 on the process of neuronal lipotoxic injury was analyzed,and its possible mechanism was explored.Method:1.A high fat diet（HFD）mouse model was constructed,the cerebral cortex and hippocampus were separated,and the expression of MKP-5 protein was detected by Western blot.2.PC12 cells were treated with different concentrations of PA（75,150,300,400,500μM）,and detect the changes in cell proliferation activity by the CCK-8 method.PC12 cells were treated with PA（300μM）for different time（0,1,3,6,9,12,18 h）,Western blot method was used to detect the protein of apoptosis indicators（cleaved Casepase 3,cleaved Casepase 9）Level.3.PC12 cells were transfected with MKP-5 si RNA（si-MKP-5）and scramble si RNA（si-SC）for 48 h,then PC12 cells were treated with 300μM PA for 6 h.The cell proliferation viability was detected by CCK-8 method;Cell apoptosis and endoplasmic reticulum stress-related protein expression were detected by Western blot;Amyloid production,neuroinflammatory factors and oxidative stress-related m RNA expression were detected by Real-time PCR;The contents of ROS,MDA,CAT,T-AOC,SOD and GSH were detected by corresponding kits.4.PC12 cells were further infected with Retro-MKP-5 overexpressing MKP-5interference sequence and its control Retro-PLN_CX2 for 48 h,respectively.After 300μM PA treatment for 6 h,Western blot was used to detect the expression of apoptosis and endoplasmic reticulum stress-related proteins;Amyloid production,neuroinflammatory cytokines and oxidative stress related indicators were detected by Real-time PCR.Results:1.The protein levels of MKP-5 was up-regulated by HFD in the cerebral cortex and hippocampus of miceWestern blot results showed that the body weight and blood sugar of obese mice increased significantly,and the protein expression of MKP-5 in the cerebral cortex and hippocampus increased significantly.2.PA inhibits PC12 cell proliferation and induces apoptosisCCK-8 test results showed that PC12 cell proliferation activity decreased in a dose-dependent manner after PA treatment,and cell viability decreased by about 50%under 300μM PA treatment.Western blot results showed that apoptosis-related cleaved Casepase 3 and cleaved Casepase 9 proteins expressed the highest levels after PA treatment for 6 h.3.Interference with the expression of MKP-5 inhibits PA-induced apoptosis of PC12 cellsCCK-8 results showed that interference with the expression of MKP-5significantly inhibited the decrease in cell proliferation induced by PA.Western blot results showed that interference with the expression of MKP-5 significantly inhibited the cleavage activation of the apoptotic effector enzyme Caspase 3 in the Caspase family of proteases induced by PA.In addition,down-regulating the expression of MKP-5 significantly down-regulated the PA-induced death receptor apoptosis pathway related protein cleaved Caspase 8 and the mitochondrial apoptosis pathway related protein cleaved Caspase 9 and Bax.In addition,the overexpression of MKP-5 further promotes PA-induced cleavage activation of Caspase 8,Caspase 3 and Caspase 9,and Bax further increases.The results show that interference with MKP-5 expression can inhibit PA-induced apoptosis in the death receptor pathway and mitochondrial pathway.4.Interference with the expression of MKP-5 relieves PA-induced endoplasmic reticulum stressWestern blot results show that PA can significantly up-regulate the phosphorylation levels of IRE1 and EIF2αproteins,while down-regulating MKP-5 can inhibit the phosphorylation of the above-mentioned proteins induced by PA.At the same time,PA induces the expression of endoplasmic reticulum stress apoptosis-related protein cleaved Caspase 12,and interference with the expression of MKP-5 can significantly down-regulate the high expression of cleaved Caspase 12 induced by PA.The overexpression of MKP-5 can further increase the phosphorylation level of IRE-1αand EIF2α,promote the splicing activation of Caspase 12 and the expression of CHOP,thereby exacerbating the PA-induced endoplasmic reticulum stress damage in PC12 cells.The results show that silencing MKP-5 has the effect of inhibiting PA-induced endoplasmic reticulum stress.5.Interference with MKP-5 expression inhibits PA-induced amyloid production and oxidative stressReal-time PCR results showed that PA can induce the expression of Aβproduction related genes APP and BACE1,while inhibiting the expression of MKP-5 significantly down-regulates APP and BACE1.Overexpression of MKP-5 can further up-regulate the expression of APP and BACE1.The results of oxidative stress analysis showed that PA can induce an increase in the generation of ROS,increase the production of lipid peroxidation product MDA,inhibit the levels of antioxidant enzymes SOD,CAT and non-enzymatic antioxidant GSH,and reduce cell T-AOC content.After knocking down MKP-5,ROS production was significantly reduced,MDA level was down-regulated,and the antioxidant capacity of PC12 cells was up-regulated.At the same time,Real-time PCR results showed that PA can increase the m RNA levels of p22phox and NOX-4,while silencing MKP-5 significantly inhibited the stimulating effect of PA on the above-mentioned NADPH enzyme-related genes.At the same time,the overexpression of MKP-5 aggravated the up-regulation of p22phox.Interference with the expression of MKP-5 can inhibit the oxidative stress response of cells.6.Interference with MKP-5 expression inhibits PA-induced inflammation and STAT3 phosphorylationReal-time PCR further confirmed that PA can up-regulate the m RNA levels of pro-inflammatory cytokines IL-6,IL-1βand i NOS,and overexpression of MKP-5 can further promote the expression of the above-mentioned inflammatory factors.Interference with the expression of MKP-5 significantly inhibits the expression level of neuroinflammatory factors and inhibits the inflammatory response induced by PA.Western blot results showed that PA increased the phosphorylation level of STAT3,but interference with the expression of MKP-5 would inhibit the activation of STAT3.Overexpression of MKP-5 further exacerbated the increase in PA-induced phosphorylation of STAT3.Concussion:Inhibition of MKP-5 expression can alleviate PA-induced PC12 cell proliferation inhibition,endoplasmic reticulum stress,oxidative stress,apoptosis and neuronal inflammation,and reduce lipotoxic-related neuronal Aβdeposition.