Mechanism Of Sympathetic Nerve Regulation Of Macrophage Polarization In Ventricular Arrhythmia

Part ⅠMechanism of sympathetic regulation of macrophagepolarization in ventricular arrhythmia after strokeObjective: To investigate the mechanism of sympathetic nerve regulation of macrophage polarization in ventricular arrhythmia after acute stroke.Methods: Canine acute stroke models were established and divided into three groups: sham operation group(n=6),acute stroke group(n=7)and stellate ganglion ablation group(n=7).Canine craniotomies for right acute middle cerebral artery occlusion were performed in the acute stroke group and stellate ganglion ablation group.In the stellate ganglion ablation group,left stellate ganglion ablation was performed simultaneously with right acute middle cerebral artery occlusion.Brain magnetic resonance imaging(MRI)was performed 24 hours postoperatively to confirm successful modeling.After 3 days,electrophysiological and neural activity of the heart was measured under anesthesia.After the canines were sacrificed,the concentration of norepinephrine in plasma and ventricle was detected by enzyme-linked immunosorbent assay(ELISA),the expression of monocyte chemoattractant protein-1(MCP-1),tumor necrosis factor-α(TNF-α)and nuclear factor kappa-B p65(NF-κB p65)in ventricle was detected by western blot.The expression of nerve growth factor(NGF)in ventricle was analyzed by immunohistochemistry.Immunofluorescence was used to detect pro-inflammatory polarization of macrophages in ventricular tissue.Results: Canines in the acute stroke group had an increased susceptibility to ventricular tachycardia,a lower ventricular fibrillation threshold(3.0 ± 0.8 vs 9.0 ±1.4 V,P < 0.01),increased stellate ganglion activity and norepinephrine concentration,increased number of M1 macrophages(42 ± 6 vs 14 ± 4 per mm2,P < 0.01),increased expression of NGF,inflammatory cytokines and NF-κB p65 compared with the sham group(all P < 0.01).Compared with the acute stroke group,canines in the stellate ganglion ablation group had a lower induction rate of ventricular arrhythmias,higher ventricular fibrillation threshold(7.1 ± 1.3 vs 3.0 ± 0.8V,P < 0.01),lower concentration of norepinephrine,decreased number of ventricular M1 macrophages(22 ± 4 vs 42 ± 6 per mm2,P < 0.01),decreased expression of NGF,inflammatory cytokines and NF-κB p65(all P < 0.01).Conclusions: Increased sympathetic activity after acute stroke promotes pro-inflammatory polarization of ventricular macrophages,upregulates expression of inflammatory cytokines,and increases susceptibility to ventricular arrhythmias by activating the NF-κB pathway.Ablation of stellate ganglion reduces sympathetic activity,inhibits pro-inflammatory polarization of ventricular macrophages,down-regulates expression of inflammatory cytokines,and reduces susceptibility to ventricular arrhythmias.Part Ⅱ Mechanism of sympathetic regulation of macrophagepolarization in methamphetamine-induced ventricular arrhythmiasObjective: To explore the mechanism of sympathetic regulation of macrophage polarization in methamphetamine-induced ventricular arrhythmias.Methods: Seventy-two mice were randomly divided into three groups with 24 mice in each group.Methamphetamine group and methamphetamine + dapagliflozin group received intraperitoneal injection of methamphetamine for 14 weeks,during which the dose was gradually increased(3mg/kg/week to 42mg/kg/week).Control mice received the same amount of normal saline injection.Methamphetamine +dapagliflozin group mice were given dapagliflozin 1 mg/kg/d by gavage daily.After14 weeks,the cardiac function of each group was measured by ultrasound.Lead electrocardiogram was recorded and blood was collected under anesthesia.After blood collection,some of the mice underwent in vitro perfusion to detect the susceptibility to ventricular arrhythmias.The other mice were killed and their hearts were harvested.Plasma and ventricular norepinephrine concentrations were detected by ELISA.CD68 and NGF immunofluorescence double-standard staining were used to detect the sympathetic nerve density and macrophage number in the ventricle.Hematoxylin-eosin(HE)staining,transmission electron microscopy and deoxyribonucleotide terminal transferase-mediated notch end labeling(TUNEL)staining were used to detect myocardial injury and apoptosis.The degree of myocardial fibrosis was detected by masson staining,Sirius red staining and smooth muscle actin α(α-SMA)protein immunohistochemistry.The expression of MCP-1,IL-1β,NF-κB p65,transforming growth factor β(TGF-β),α-SMA,gap junction protein(CX43),Bcl-2 associated protein(Bax),B-cell lymphoma 2(Bcl-2),cleaved caspase3 were detected by western blot.Interleukin-6(IL-6)expression was detected by immunofluorescence.Immunohistochemical staining of spl IT1 hirsute enhancer(HES1)and ice cut tissue staining of reactive oxygen species(ROS)to explore further signaling pathway mechanism;CX43 immunofluorescence staining was used to detect CX43 density.Results: Mice in the methamphetamine group showed a higher incidence of premature ventricular beats and a higher susceptibility to ventricular fibrillation than those in the control group.Meanwhile,compared with the control group,the methamphetamine group had higher norepinephrine concentrations in plasma and ventricles(plasma norepinephrine concentration /pg/ml: 567.6 ± 23.9 vs 326 ± 20.6,P< 0.01),increased sympathetic nerve density and the number of M1 macrophages,upregulated expression of inflammatory cytokines MCP-1,IL-1β,IL-6;enhanced apoptosis of myocardial cells(percentage of apoptotic cells: 7.4 ± 1.3 vs 1.3 ± 0.5,P< 0.01),increased degree of myocardial fibrosis(masson staining collagen fiber %:5.01 ± 1.90 vs 0.73 ± 0.35,P < 0.05),decreased cardiac function(left ventricular ejection fraction: 56.51 ± 6.01 vs 73.62 ± 1.31,P < 0.01),upregulated NF-κB p65 and HES1 expression.The incidence of premature ventricular beats and susceptibility to ventricular fibrillation in the methamphetamine + dapagliflozin group were lower than those in the methamphetamine group.In addition,compared with the methamphetamine group,the methamphetamine + dapagliflozin group had lower norepinephrine concentrations in the ventricles and plasma(plasma norepinephrine concentration/pg/ml: 440 ± 20.5 vs 567.6 ± 23.9,P < 0.01),decreased sympathetic nerve density and the number of M1 macrophages,downregulated expression of inflammatory cytokines MCP-1,IL-1β,IL-6;suppressed myocardial apoptosis(apoptotic cells % : 2.4 ± 0.8 vs 7.4 ± 1.3,P < 0.01),decreased myocardial fibrosis(masson staining collagen fiber % : 1.60 ± 0.46 vs 5.01 ± 1.90,P < 0.05),improved cardiac function(left ventricular ejection fraction: 56.51 ± 6.01 vs 70.99 ± 5.57,P <0.01)and inhibition of NF-κB p65 and HES1 expression.Conclusions: Increased sympathetic nerve activity induced by methamphetamine sympathetically promotes pro-inflammatory polarization of ventricular macrophages,further increases myocardial fibrosis and enhances susceptibility to ventricular arrhythmias,mediated at least in part by NF-κB pathway.Dapagliflozin decreased sympathetic nerve activity,inhibited ventricular macrophage pro-inflammatory polarization,improved myocardial fibrosis,and protected methamphetamine-induced ventricular electrical remodeling.Part Ⅲ Mechanism of norepinephrine promoting macrophagepolarization and inflammatory cytokine secretionObjective: To investigate the effect of norepinephrine on macrophage polarization and secretion of inflammatory cytokines and its specific mechanism.Methods: Macrophages were divided into control group,norepinephrine preconditioning group,lipopolysaccharide stimulation group and norepinephrine preconditioning + lipopolysaccharide stimulation group.The expression of i NOS,a marker of M1 macrophage,was detected by PCR.Western bolt and ELISA were used to detect the expression or supernatant concentration of IL-6,MCP-1 and TNF-α.The expression of IL-6 in each group was further verified by immunofluorescence staining.Western bolt was used to detect NF-κB p65,mitogen and stress-activated protein kinase1(MSK-1)and phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2)protein expression.Results: Norepinephrine promoted the polarization of macrophages induced by lipopolysaccharide(i NOS m RNA relative level: 5.09 ± 0.28 vs 3.98 ± 0.30,P < 0.01),but norepinephrine alone had no significant effect.Noradrenaline preconditioning promoted the synthesis of IL-6 and MCP-1 in LPS stimulated macrophages,and the secretion of TNF-α and IL-6 in LPS stimulated macrophages.Noradrenaline stimulation alone promoted the expression of IL-6(IL-6 concentration of cell supernatant /pg/ ml: 9.3 ± 2.5 vs 3.3 ± 1.5,P < 0.05),and had no significant effect on MCP-1 and TNF-α.The expressions of NF-κB p65,MSK-1 and p-ERK1/2 were significantly increased by noradrenaline pretreatment and single stimulation.Conclusions: Norepinephrine pretreatment can promote the polarization of macrophages to M1 type induced by lipopolysaccharide and increase the secretion of inflammatory cytokines.The pro-inflammatory effect of norepinephrine is related to the phosphorylation of NF-κB p65.