| Backgrounds and Objective Hypertension is a chronic disease characterized by high blood pressure(BP).Group data provided by guidelines for prevention and treatment of hypertension Third Edition(2016 Revised Edition)show that about 1/3 of adults suffer from hypertension in China.Therefore,understanding of the pathogenesis of hypertension is particularly important for the prevention and treatment aganist hypertension.It has been documented that inflammation plays an important role in the development of neurogenic hypertension.The lipopolysaccharide(LPS)-induced systemic inflammation leads to a significant increase in blood pressure(BP).In the RAG-1-/-mice lacking T and B cells,hypertension and abnormal vascular function induced by angiotensin II(Ang II)infusion or deoxycorticosterone acetate-salt are significantly attenuated.It is reported that the low-grade inflammation has been detected in peripheral cardiovascular system in patients and animals with hypertension,suggesting that inflammation is involved in the processing of essential hypertension.The immune inflammatory response in the central nervous system,also called neuroinflammation,is characterized by an increase in production of pro-inflammatory cytokines,which is required to activation of microglia.It is reported that microinjections of the pro-inflammatory cytokine interleukin-1?(IL-1?)or tumor necrosis factor-?(TNF-?)into the cardiovascular center paraventricular nucleus(PVN)lead to an increase in BP and renal sympathetic nerve activity(RSNA).Preactivated microglia in the brain has prolonged the pressor response induced by intracerebroventricular injection of Ang II.The rostral ventrolateral medulla(RVLM)has been recognized as a pivotal region for maintaining basal BP and sympathetic tone.The previous evidence indicates that the systemic LPS-induced neuroinflammation in the RVLM is involved in high BP and sympathetic hyperactivity.Therefore,it would be significant to investigate the role of neuroinflammation in the central regulation of cardiovascular function in hypertension,and is helpful for researching for a new strategy against hypertension.Previous evidence suggests that the phosphoinositide-3 kinase(PI3K)pathway in the RVLM is involved in regulating BP and sympathetic outflow.It has been demonstrated that the PI3 K signaling in the RVLM is upregulated in hypertension,and its inactivation leads to reduction in reactive oxygen species(ROS)production and cardiovascular function.Moreover,we recently report that gene knockdown of the PI3 K subunit p110? in the RVLM exerts the centrally antihypertensive action.Interestingly,the PI3 K signaling is suggested to be involved in several key events in the inflammatory response to damage and infection.Therefore,the major aim of this study was to determine the role of the RVLM PI3 K signaling in the neuroinflammation in hypertension.Overactivity in renin-angiotensin system(RAS)has been shown to play an important role in the pathogenesis of hypertension.It is reported that Ang II stimulates the activation of resting microglia and induces the production of pro-inflammatory cytokines in vivo and in vitro.Recent evidence indicates that blockade of Ang II receptor(AT1R)with candesartan decreases the brain inflammatory response induced by administration of LPS.Therefore,it is suggested that neuroinflammation induced by Ang II acts as a molecular signal for increased sympathetic outflow in hypertension.It has been demonstrated that angiotensin-converting enzyme 2(ACE2)plays an important role in compensatory mechanisms to oppose the overactive RAS,and catalyzes the conversion from Ang II and Ang-(1–9)to Ang-(1–7),which exerts physiological functions through combining with Mas receptor.The cardiovascular protective effects(e.g.vasodilatation and anti-hypertrophic cardiomyopathy)of ACE2/Ang-(1-7)/Mas axis are resulted from increased synthesis of nitric oxide and decreased generation of reactive oxygen species(ROS).Cardiovascular neural regulatory regions in the brain targeted ACE2 overexpression are reported to reduce BP and sympathetic outflow in hypertension and ameliorate the baroreflex function in chronic heart failure.Interestingly,it is reported that Ang-(1-7)has a direct anti-inflammatory effect on microglia in vitro.However,it is not clear whether the effect of RAS in the RVLM on neuroinflammation in hypertension is associated with PI3 K signaling.Hence,the present study was designed to determine: 1)if the PI3 K gene silencing in the RVLM inhibits neuroinflammation in hypertensive rats;2)if the PI3 K silencing in the RVLM attenuates the Ang II-induced high BP and neuroinflammation in WKY rats;3)if the PI3 K signaling is involved in the antihypertensive effect of ACE2 overexpression by reducing the level of neuroinflammation in the RVLM in hypertensive rats.Methods Male normotensive WKY rats(280g-320g)and spontaneously hypertensive rat(SHR)were used in this work.The WKY rats were performed by intracisternal infusion of lipopolysaccharide(LPS)or angiotensin II(Ang II)for inducing neuroinflammation.Elisa and immunofluorescence was used to measure the level of pro-inflammatory cytokines and microglial activation,respectively.Western Blot was performed to detect the protein expression of PI3 K signaling pathway.Gene silencing of PI3 K p110? subunit and overexpression of ACE2 were realized by injecting of related lentivirus into the RVLM.Two weeks after the lentivirus infection,effects of silencing of PI3 K p110? or overexpression ACE2 on neuroinflammation in LPS or Ang II infused WKY rats and SHR were determined by Elisa.Results 1.Gene silencing of the PI3 K signaling in the RVLM attenuated neuroinflammation in SHR GFP immunofluorescence staining for lentivirus transfer was expressed restrictedly in the RVLM.Furthermore,transfer efficacy of lentivirus containing the specific PI3 K p110? shRNA fragment injected into the RVLM was confirmed by a decrease in expression level of PI3 K p110? protein.It was also observed that the expression level of the RVLM PI3 K p110? was significantly upregulated in SHR compared with WKY rats.Compared with WKY,SHR showed a significant increase in the content of pro-inflammatory cytokines(IL-1?: 55.9 ± 18.2 vs.141.8 ± 7.54 pg/mg,P<0.05;IL-6: 309 ± 18.2 vs.598 ± 26.0 pg/mg,P<0.05;TNF-?: 56.3 ± 3.76 vs.98.0 ± 15.4 pg/mg,P<0.05),MAP(125 ± 2.50 vs.183 ± 8.05 mmHg),HR(363 ± 10.6 vs.441 ± 11.4 bpm),and basal RSNA(11.3 ± 0.514 vs.38.0 ± 2.03 % Max).Importantly,the levels of IL-1?,IL-6 and TNF-? in the RVLM and cardiovascular activity were significantly decreased in SHR 4 weeks after knockdown of PI3 K p110? in the RVLM.In additional,injection of lenti-PI3 K shRNA into the RVLM had no influence on BP and HR in WKY rats.2.Gene silencing of PI3 K signaling in the RVLM attenuated the Ang II-induced neuroinflammation and cardiovascular excitation Intracisternal infusion of Ang II(24 ?g/d,2 weeks)significantly increased approximately 2.5-fold in the expression level of p110? subunit of PI3 K,and caused an approximately 1.5-fold increase in AKT phosphorylation at Ser473 in the RVLM of WKY rats.In order to confirm the role of PI3 K in the neuroinflammation induced by Ang II,PI3 K shRNA lentivirus was injected into the RVLM before intracisternal infusion of Ang II in WKY rats.It was found that intracisternal infusion(one week)of Ang II in the WKY rats significantly enhanced cardiovascular activity(MAP: 148 ± 1.89 vs.116 ± 2.75 mmHg;HR: 430 ± 10.6 vs.367 ± 14.6 bpm;and basal RSNA: 24.6 ± 2.72 vs.10.2 ± 1.00 % Max),but also increased levels of pro-inflammatory cytokines in the RVLM(IL-1?: 188 ± 17.7 vs.126 ± 3.76 pg/mg;IL-6: 686 ± 35.5 vs.544 ± 5.39 pg/mg;and TNF-?: 94.5 ± 5.94 vs.66.0 ± 3.55 pg/mg)compared with infusion of a CSF.Interestingly,the Ang II-induced increases in levels of pro-inflammatory cytokines(IL-1?,IL-6,and TNF-?)in the RVLM and cardiovascular activity(BP and HR)were significantly attenuated after pretreatment with silencing of PI3 K in the RVLM.3.Overexpression of ACE2 attenuated neuroinflammation in the RVLM via inhibiting PI3 K signaling Transfer efficacy of lentivirus containing ACE2 gene(lenti-ACE2)injected into the RVLM was confirmed by an increase in ACE2 protein expression and the ratio of Ang-(1-7)to Ang II in the RVLM.In the GFP group,levels of MAP and HR were significantly(P<0.05)increased in conscious WKY rats after treatment with intracisternal infusion of LPS(2 weeks)compared with artificial cerebrospinal fluid(aCSF)infusion.The WKY rats treated with LPS infusion showed a significant increase in baseline RSNA compared with aCSF infusion.Furthermore,the LPS-induced increases in the levels of pro-inflammatory cytokines(IL-1?,IL-6,and TNF-?)in the RVLM and cardiovascular activity(BP and HR)were significantly attenuated in WKY rat after pretreatment with ACE2 overexpression in the RVLM.Moreover,intracisternal infusion of LPS(2 weeks)significantly upregulated the expression level of p110? subunit of PI3 K and AKT phosphorylation at Ser473 in the RVLM of WKY rats,which was prevented by overexpression of ACE2.In SHR,It was observed that SHR showed a significant decrease in BP,HR,and basal RSNA 4 weeks after ACE2 overexpression in the RVLM.Compared with WKY,SHR showed a significant increase in the content of pro-inflammatory cytokines(IL-1?: 92.9 ± 13.3 vs.136 ± 9.62 pg/mg,P<0.05;IL-6: 428 ± 83.8 vs.582 ± 51.5 pg/mg,P<0.05;TNF-?: 40.6 ± 4.0 vs.65.4 ± 11.1 pg/mg,P<0.05),all of which were attenuated by pretreatment with overexpression of ACE2 in the RVLM.Overexpression of ACE2 in the RVLM caused a significant decrease in the expression level of p110? subunit of PI3 K and AKT phosphorylation in SHR.However,overexpression of ACE2 in the RVLM in WKY rats had no influence on BP and the level of PI3 K signaling.Conclusion The current results suggest that the PI3 K signaling in the RVLM is involved in the pathogenesis of neuroinflammation in hypertension.Neuroinflammation and cardiovascular excitation induced by central infusion of Ang II is prevented by pretreatment with RVLM PI3 K knockdown.It is also suggested that inactivation of the PI3 K signaling mediates the anti-neuroinflammation effect of ACE2 overexpression in the RVLM in hypertensive rats. |
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