The Role Of RhoA/ROCK Pathway And Angiotensin 1-7 In Long-term Facilitation Of Renal Sympathetic Nerve Activity And Rat Hypertension Induced By Chronic Intermittent Hypoxia

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Establishment of hypertensive model induced by chronic intermittent hypoxiaObjective:Our aim is to explore a kind of hypertensive animal model which simulate the features of the pathological and physiological symptom of obstructive sleep apean(OSA)and therefore we can evaluate the effect.Methods:The model of chronic intermittent hypoxia(CIH)was established by sealed chambers which were used to generate a hypoxic environment.Pure nitrogen and compressed air were distributed into each chamber through timed solenoid valves and computer system.Using 90s cycles,pure nitrogen was infused into each chamber for the first 30s until the minimum oxygen concentration reached 5%.Compressed air was infused for the remaining 60s subsequently to allow the oxygen concentration in the chambers to gradually return to 21%.Hypoxia exposure experiments were performed for 8h a day for 21 days and RA group was in normoxic state.16 SD rats were divided into two groups:normoxia(RA)and chronic intermittent hypoxia(CIH)groups.RA group was exposed to normoxia and CIH group was exposure to chronic intermittent hypoxia.Systolic blood pressure(SBP)was measured at the starting of the experiment and every week.Renal sympathetic nerve activity(RSNA)was measured at the end of the experiment.Serum noradrenaline were detected.Results:1.Compared with RA group,CIH rats showed a significant SBP elevation beginning in the second week,and SBP gradually increased throughout the experimental period in response to CIH.2.CIH resulted in a significant increase in RSNA(long-term facilitation)compared with room air exposure3.CIH resulted in a marked increase in the NE level in serum.Conclusion:1.CIH induced hypertension in SD rat.2.CIH Lead to long-term facilitation of RSNAPart ? Role of RhoA/ROCK pathway in long term facilitation of renal sympathetic nerve activity and hypertension induced by chronic intermittent hypoxiaBackground:OSA,which is a risk factor for secondary hypertension,is characterized by temporary upper airway obstruction during sleep.Although many mechanism have been proposed to explain the OSA related hypertension,much more work remains to be done to support the hypothesis.Objective:Our aim is to explore the role and significance of oxidative stress and RhoA/ROCKpathway in CIH rats.Methods:Forty male SD rats were divided into five groups:RA(room air),CIH-V(CIH+vertical,ip),CIH-G(CIH+GKT137831,ip,40mg/kg/d),CIH-N(CIH+N-ace tylcyst-eine,ip,300mg/kg/d),CIH-Y(CIH+Y27632,ip,1 Omg/kg/d).Hypoxia exposure experiments were performed for 8h and RA group was in normoxic state.Systolic blood pressure(SBP)was measured at the starting of the experiment and every week.Renal sympathetic nerve activity(RSNA)was recorded at the end of the experiment.Blood samples and renal tissues were harvest for the molecular biological detection.TUNEL assay was used to determine the apoptosis of the kidney.Results:1.The blood pressure became increase from the second week and continued to rise until the third week.CIH caused elevation of RSNA and oxidative stress and Nox4 expression in SD rats.2.Administration of Y27632 inhibited the increase of blood pressure and RSNA in CIH-Y group.Intervene of GKT137831 and NAC reduced the development of hypertension in CIH-G and CIH-N group.In addition,the level of oxidative stress and overactivity of RSNA were attenuated.The expression of RhoA/ROCK was inhibited by the administration of the two antioxidants.Conclusions:1.CIH induce hypertension and overactivity of RSNA.RhoA/ROCK pathway may involve in the physiopathologic mechanism of hypertension induced by CIH.2.CIH caused enhanced oxidative stress in vivo.Antioxidants may relieve hypertension induced by CIH by the inhibition of RhoA/ROCK pathway.CIH induced elevation of BP is at least partially mediated via the Nox4 induced ROS/RhoA/ROCK pathway.3.CIH caused increased apoptosis in renal tissue.Inhibition of oxidative stress and RhoA/ROCK pathway may relieve renal injury induced by CIH.Part ? The role of Ang(1-7)in long-term facilitation of renal sympathetic nerve activity and hypertension induced by chronic intermittent hypoxiaObjective:Activation of(renin-angiotensin system,RAS)was found clinically in OSA patient.The activation of RAS system is the important reason for the increased blood pressure,and one of the important mechanisms of cardiovascular disease result from OSA.angiotensin-converting enzyme 2(ACE2)-angiotensin(1-7)(Ang(1-7))-mas axis is a new pathway which may be served as a counterbalance to Ang?.There's limited data about Ang(1-7),and the results are controversial.Our aim is to explore the relationship between hypertension induced by CIH and renal injury,and the role of angiotensin 1-7(Ang(1-7))on the long term facilitationof renal sympathetic nerve activity and renal injury in CIH rats.Methods:Male Sprague-Dawley rats were randomly divided into four groups:CIH(CIH exposure without any treatment),norm(normoxia without any treatment),CIHAng(CIH exposure+Ang(1-7)infusion at 400 ngkg-1min-1,for 28 days)or NormAng(normoxia exposure+Ang(1-7)infusion at 400 ngkg-1min-1for 28 days).Systolic blood pressure(SBP)was measured at the starting of the experiment and every week.Renal sympathetic nerve activity(RSNA)was recorded at the end of the experiment.CTGF and TGF-b were detected by immunohistochemistry and western blotting.The concentration of SOD,MDA and CAT were also determined by chemoluminescence.3-nitrotyrosine,interleukin-6,hypoxia-inducible factor-1 and tumor necrosis factor-a were determined by immunohistochemistry and ELISA.hypoxia-inducible factor-1 was determined by immunohistochemistry and immunoblotting.TUNEL assay and cleaved caspase 3 and 12 were also determined for the apoptosis.Results:1.CIH rats displayed a significant increase in SBP compared with the Norm and NormAng groups from the end of the second week to the end of the experiment.By the end of the treatment period with Ang(1-7),SBP was restored,reaching values statistically different from those obtained in the CIH group.2.The RSNA baseline of the CIH group was significantly higher than that of the CIHAng group,which was higher compared with the Norm and NormAng groups.3.Immunohistochemistry showed that the nontreated CIH group presented an increased renal expression of IL-6,TNF-a,and HIF-a compared with the Norm group.Chronic treatment with Ang(1-7)reduced the expression of inflammators in the CIHAng kidney.Immunostaining results were confirmed by immunoblotting analysis(HIF-la)and ELISA(IL-6and TNF-?).4.Both CAT and SOD renal enzymatic activities were decreased and MDA was increased in the CIH group.After chronic treatment with Ang(1-7),a significant decline of oxidative stress levels were observed.Immunohistochemistry showed that the content of 3-NT significantly increased in CIH group(the main location was in thecytoplasm).The intervention of Ang(1-7)reduced the content of 3-NT but higher than the Norm and NormAng groups.5.Immunohistochemistry and immunoblotting showed that the CIHAng group chronically treated with Ang(1-7)showed a significant reduction of fibrosis,for the abundance of CTGF and TGF-? declined compared with CIH group but higher than norm and normAng group.ELISA showed that The Ang? level was lower in the CIHAng group compared with CIH groupbut higher than the Norm and NormAng groups.6.The ratio of TUNEL-positive cells in the CIHAng group was lower than in the CIH group but higher than in the Norm and NormAng groups.Conclusions:1.Hypoxia can result in the elevation of blood pressure and overactivation of renal sympathetic nerve.The administration of Ang(1-7)attenuated the development of hypertension and overactivation of RSNA.2.CIH increased the level of inflammation,oxidative stress and fibrosis,these factors may stimulate RSNA and injure the regulatory function of RSNA in blood pressure with the long-term facilitation of RSNA.The intervention of Ang(1-7)attenuated inflammation,oxidative stress and fibrosis.We hypothesis that the anti-hypertensive role of Ang(1-7)may due to the attenuation of RSNA.In addition,Ang(1-7)may reduce the extent of inflammation,oxidative stress and fibrosis,which may stimulate RSNA and raise blood pressure.3.CIH induced more apoptosis and renal injury,however,the infusion of Ang(1-7)improved kidney injury in rats.Ang(1-7)may represent a potential strategy in CIH induced hypertension and related target organ damage.