Roles Of FNDC5 In Alleviating Inflammation,Oxidative Stress And Vascular Smooth Muscle Cell Migration Of Hypertension

Type ? domain-containing protein 5(FNDC5)is a glycosylated transmembrane protein with glycosylation sites at 39K and 84A.The structure mainly includes two fibronectin structure domain,a signal peptide and a hydrophobic C-terminal domain inserted into the cell membrane.Under the action of proteolytic enzymes,it cleaves and releases the polypeptide fragment irisin.FNDC5/irisin was originally discovered in muscle tissue,and can promote the browning of white fat.It has the effect of improving energy consumption and metabolic diseases,so it plays an important role in the pathogenesis of metabolic diseases.FNDC5/irisin reverses the phenotypic transformation of vascular smooth muscle cells(VSMCs)induced by platelet-derived growth factor via activating Signal Transducer and Activator of Transcription 3(STAT3).However,the role and underlying mechanisms of FNDC5/irisin in inflammation,oxidative stress and migration of VSMCs has not yet been reported.This thesis consists of two parts:(1)the effects and mechanisms of FNDC5 on nucleotide-binding oligomerization domain-like receptors family pyrin domain containing 3(NLRP3)inflammasome and oxidative stress in Ang ?-induced mouse hypertension model;(2)Effects and mechanisms of miR-31-5p on FNDC5 expression,migration and oxidative stress in Ang ?-treated VSMCs.Part 1 Roles and mechanisms of FNDC5 in inhibiting oxidative stress and NLRP3 inflammasome activity in vascular smooth muscle cellBackgroundHypertension is a common chronic cardiovascular and cerebrovascular disease characterized by persistent high blood pressure.Chronic inflammation and excessive oxidative stress play a key role in the development of hypertension.Therefore,the inhibition of vascular inflammation and oxidative stress may become a therapeutic method to lower blood pressure.VSMCs are the main cellular constituent of the vascular media,and the abnormal function of VSMCs play an important role in the course of cardiovascular diseases such as hypertension,atherosclerosis,coronary heart disease and aneurysms.Intracellular reactive oxygen species(ROS)level are sensitive receptor for oxidative stress level.Excessive oxidation products lead an increase of inflammation level,which promotes the abnormal proliferation,migration,apoptosis and extracellular matrix deposition of VSMCs and eventually leads to vascular remodeling.However at present,the effects and underlying mechanisms of FNDC5 on NLRP3 inflammasome activation and oxidative stress in VSMCs has not been reported.ObjectiveThe aim of this study was to investigate the role and molecular mechanisms of exogenous FNDC5 in Ang ?-induced inflammation and oxidative stress,and the effects of subcutaneously implanted micro-pump sustained-release Ang ? on inflammation and oxidative stress of thoracic aorta in FNDC5^-/-mice(FNDC5 gene knockout were used C57BL/6J mice as background).MethodsIn this experiment,A7R5 cells were cultured in DMEM medium containing 10%FBS,100 units/ml penicillin,and 100?g/ml streptomycin.Primary mouse VSMCs were cultured in a special medium for primary VSMCs.The medium contains 10%cytokine,10%FBS and 1%dual antibiotics,and all cells were cultured in a cell incubator at 37oC and 5%CO2.The animals in this experiment were purchased from the Animal Experimental Center of Nanjing Medical University,8-week-old male C57BL/6J mice and FNDC5^-/-mice were used.After a week of acclimation,the mice were randomly divided into four groups,six mice per group.The normal control group was subcutaneously embedded with a microinjection pump containing PBS,the normal experimental group was subcutaneously embedded with a microinjection pump containing Ang ?,FNDC5^-/-control group was subcutaneously implanted with a microinjection pump containing PBS,and FNDC5^-/-experimental group was implanted subcutaneously with a microinjection pump containing Ang ?.Blood pressure and heart rate of mice were recorded during the period.After 2 weeks,mice were anesthetized,sampled and subjected to acute experiments.Western blot was used to detect the protein expression level of NLRP3,FNDC5,Silent mating type information regulation 2 homologl(SIRT1),apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),Nicotinamide adenine dinucleotide phosphate oxidase2(NOX2),adenosine monophosphate-activated protein kinase(AMPK),p-AMPK,mature and precursor Cysteinyl aspartate specific proteinase 1(Caspase-1),mature and precursor of interleukins and mature Interleukin-1?(IL-1?),Nicotinamide adenine dinucleotide phosphate oxidase4(NOX4)and?-actin.Real-time PCR(RT-PCR)was used to detect FNDC5 m RNA levels.Masson's trichrome staining was used to detect fibrosis and stenosis of thoracic aorta in mice.Dihydroethidium(DHE)staining was used to detect reactive oxygen level in mouse primary VSMCs and A7R5 cells.Immunofluorescence technology was used to detect the expression of NLRP3 in A7R5 cells.Enzyme-linked immunosorbent assay(ELISA)was used to determine the level of SIRT1 enzyme activity.Results(1)In a hypertensive mouse model induced by subcutaneous implantation of an osmotic pump and administration of Ang ? for 2 weeks,compared with wild-type mice,the blood pressure in FNDC5 gene knockout mice were significantly increased,the thickness and area of the thoracic aorta of the mice were also severely increased.Meanwhile,administration of Ang ? for 2 weeks significantly inhibited the expression of FNDC5 in the thoracic aorta of wild type mice.(2)WT and FNDC5^-/-mice were treated Ang ? for 2 weeks by subcutaneous implanted osmotic pump.In the aorta of WT and FNDC5^-/-mice,the production of ROS and protein expression of NOX2,NLRP3,pro-IL-1?and IL-1?were all increased.Compared with wild-type mice,this effect was more significant in the FNDC5^-/-mice.(3)Ang ? treatment for 24 h increased the protein expression of NOX2,NOX4 and ROS production,while exogenous FNDC5 inhibited these effect caused by Ang ?.Interestingly,Ang ? treatment for 24 h had no significant effect on FNDC5 expression in A7R5 cells.(4)Ang ? treatment for 24 h significantly increased NLRP3,ASC,procaspase-1,Caspase-1 and pro-IL-1?protein expression,and also promoted IL-1?production.Moreover,exogenous FNDC5 inhibited the expression of NLRP3,Caspase-1 and IL-1?induced by Ang ?.(5)In Ang ?-treated A7R5 cells,exogenous FNDC5 inhibited Ang ? induced up-regulation of ROS production,the increased protein expression of NOX2,NLRP3 and IL-1?production by promoting AMPK?phosphorylation.However,this effect can be eliminated by the inhibitor of AMPK.(6)In Ang ?-treated A7R5 cells,exogenous FNDC5 inhibited Ang ? induced up-regulation of ROS production,the protein expression of NOX2,NLRP3 and IL-1?production,which can be eliminated by the inhibitor EX527.(7)In Ang ?-treated A7R5 cells,exogenous FNDC5 inhibited Ang ? induced up-regulation of ROS production,the protein expression of NOX2,NLRP3 and IL-1?production,and these effects can be eliminated by the integrin receptor inhibitor GLPG0187.Moreover,FNDC5 promoted the phosphorylation of AMPK?,SIRT1activity and protein expression,which can be inhibited by the inhibitor GLPG0187.(8)Ang ?(100 n M)treated mouse primary VSMCs for 24 h,but it had no significant effect on the expression of FNDC5 m RNA and protein in mouse primary VSMCs.Exogenous FNDC5 inhibited Ang ?-induced ROS production,up-regulation of NOX2,NLRP3 protein expression and IL-1?production.At the same time,the inhibitory effects of Ang ? on AMPK?phosphorylation,SIRT1 activity and protein expression were also eliminated by exogenous FNDC5.ConclusionFNDC5 gene knockout aggravates Ang ?-induced vascular oxidative stress and activation of NLRP3 inflammasome.Exogenous FNDC5 inhibits Ang ?-induced NLRP3 inflammasome activation and oxidative stress through integrin receptor-mediated AMPK phosphorylation and increased SIRT1 enzyme activity.Part 2 MiR-31-5p promotes vascular smooth muscle cell migration and oxidative stress by inhibiting FNDC5 expressionBackgroundHypertension is an important factor of global mortality,and affects more than 25%population of the world.The renin-angiotensin system(RAS)plays an important role in the course of hypertension and end-organ damage caused by hypertension.Ang ? is an acute vasoconstrictor,directly induced vasoconstriction and excessive proliferation,migration,inflammation,oxidative stress of VSMCs,and is closely related to hypertension induced by Ang ?.Micro RNA(miRNA)is a type of endogenous non-coding small RNA that inhibits the transcription of target gene by binding to the 3'UTR domain.Studies have shown that miR-31-5p promotes the proliferation,migration,and invasion of colorectal cancer cells by inhibiting the expression of membrane-associated proteins in colorectal cancer tissues.However,the roles of FNDC5 in Ang ?-induced VSMCs migration and oxidative stress and the regulation of miR-31-5p on FNDC5expression are still unclear.2.ObjectiveThe purpose of this experiment was to explore the role and molecular mechanisms of miR-31-5p in regulating FNDC5 expression,VSMCs migration and oxidative stress.3.Methods8-week-old male C57/BL6J(WT)mice and FNDC5^-/-mice were from theLaboratory Animal Center of Nanjing Medical University.Spontaneously hypertensive rats(SHR)and Wistar-Kyoto(WKY)rats were from Beijing Vital Lihua Laboratory Animal Technology Co.,Ltd.Enzymatic digestion method was used to extract primary VSMCs of SHR and WKY rats,male wild-type(WT)and FNDC5^-/-mice.Mouse primary VSMCs,rat primary VSMCs and vascular smooth muscle cell line(A7R5)were cultured in a cell incubator at 37oC and 5%CO2.Western Blot was used to detect the protein expression level of NOX2,NOX4.RT-PCR was used to detect the m RNA expression level of FNDC5,miR-31-5p.DHE staining was used to detect the level of reactive oxygen species in VSMCs.Boyden chamber experiment and wound healing experiment were used to detect the migration ability of VSMCs.Double luciferase reporter assay was used to detect the binding site of FNDC5 gene to miR-31-5p.4.Results(1)MiR-31-5p mimic promoted the migration of primary VSMCs in SHR and WKY rats,and this effect was more pronounced in SHR primary VSMCs.MiR-31-5p inhibitor only inhibited the migration of SHR primary VSMCs,but had no significant effect on the migration of primary VSMCs of WKY rats.(2)MiR-31-5p mimic inhibited the luciferase molecular activity of wild-type(WT-FNDC5)plasmid,but has no inhibitory effect in mutant FNDC5(mut FNDC5)plasmid;miR-31-5p expression in SHR primary VSMCs is much higher than that in WKY rat VSMCs;miR-31-5p mimic simultaneously inhibited the expression of FNDC5 in WKY,SHR primary VSMCs and A7R5 cells.Mmoreover,miR-31-5p mimic also inhibited the expression of FNDC5.(3)Exogenous FNDC5 were added to the supernatant of rat primary VSMCs.Exogenous FNDC5 inhibited the migration of SHR primary VSMCs but had no effect on WKY rat primary VSMCs.(4)Mouse primary VSMCs were treated Ang ?(100 nmol/L)for 24 hours to induce cell migration in vitro.Compared with wild-type mouse primary VSMCs,Ang ?-induced cell migration promotion was more significant in FNDC5^-/-mouse primary VSMCs.(5)The production of ROS in primary SHR VSMCs,the expression of NOX2 and NOX4 protein were higher than those in WKY rat primary VSMCs.And exogenous FNDC5 significant inhibited the production of ROS and expression of NOX2 protein in primary SHR VSMCs but had no significant effects on the expression of NOX4protein in WKY and SHR primary VSMCs.(6)Ang ? treatment significantly increased ROS production and NOX2 protein expression in mouse VSMCs,and this effect was more significant in FNDC5^-/-mice.But Ang ? treatment had no effect on the expression of NOX4 in VSMCs of WT and FNDC5^-/-mice.(7)MiR-31-5p inhibitor had no effect on ROS production and protein expression of NOX2 in VSMCs of WKY rats,but significantly inhibited the production of ROS and the protein expression of NOX2 in SHR primary VSMCs,but had no effect on the expression of NOX4 in WKY and SHR primary VSMCs.(8)MiR-31-5p mimic,MiR-31-5p mimic+exogenous FNDC5 had no significant effect on ROS production,NOX2 protein expression in VSMCs of WKY rats.However,miR-31-5p mimic significantly increased ROS production and protein expression of NOX2 in SHR primary VSMCs,while exogenous FNDC5 suppressed this phenomenon.However,miR-31-5p mimic,miR-31-5p mimic+exogenous FNDC5 did not significantly affect the protein expression of NOX4 in both WKY and SHR primary VSMCs.5.ConclusionMiR-31-5p aggravates the migration and oxidative stress of SHR primary VSMCs by inhibiting the expression of FNDC5.Exogenous FNDC5 inhibits SHR primary VSMCs migration and oxidative stress.FNDC5 gene knockout aggravates Ang ?-induced migration and oxidative stress in mouse primary VSMCs.