| Background:Chronic and excessive exposure to psychological stress can lead to hypertension development. The speedy life and fierce competition in modern society result in a growing occurrence of stress-induced hypertension, which is inflicting younger and younger people. Orexin (OXA), first discovered in1998as a new hypothalamic peptide, is recognized for their diverse functions in appetite, metabolism, and sleep-wakefulness regulation. OXA immunoreactive neurons in the lateral hypothalamus project to rVLM,which may participate in stress and cardiovascular regulation. The mechanism of acupuncture’s lowering BP of SIHR is unclear.Objectives:Our research is to investigate the expression of OXA immunoreactive neurons in LH-PFA and its selective receptor,OX1R, in rVLM of stress-induced hypertensive rats(SIHR) and another group of SIHR with the treatment of electroacupuncture(EA) in Zusanli acupoint(ST-36); to investigate the co-expression of OX1R and nNOS or iNOS in rVLM and the influence of the inhibitors of NOS and sGC on the cardiovascular effects of OXA in rVLM and test the content of amino acid transmitters in the rVLM. The present study was undertaken to further strengthen the hypothesis that OXA has the centrel control of cardiovascular activity and may take apart in the mechanism of stress-induced hypertension and depressor effects of acupuncture by adjust the NOS/NO systerm and regulate the release ratio of related amino acid in rVLM.Methods:The experiment was performed on male SD rats, the SIHR model established by stimuli of electric foot shock combined with buzzer noise for successive14days, and the SIHR+EA model which was anther group of SIHR with the treatment of electroacupuncture(EA) in Zusanli acupoint(ST-36) for5successive days. Immunohistochemistry (IHC) and Western blot analysis were used to detect the expression of OXA immunoreactive neurons in LH-PFA and OX1R in rVLM of SIHR and SIHR+EA. Confocal laser microscopy together with the double-label immunofluorescence was applied to determine the colocalization and close contact between OX1R and nNOS or iNOS or Neurofiliment. Intracerebroventricular injection of OXA was carried out to test the changes of BP and HR, and20min later the concentration of NOx, the by-product of NO in the rVLM, was measured to see the relationship between OXA and NO. By means of microinjection, we tested the central cardiovascular effects of OXA and the influence of the inhibitor of OX1R, nNOS, iNOS and sGC on the effects. In addition, Micro-dialysis combined with high performance liquid charomatography(HPLC) was employed to test the amino acid transmitters in rVLM of SIHR.Results:(1) Consecutive stress for14days induced a significant increase in SBP and HR, from110±5.4mmHg of basal level to142±7.6mmHg,372±19beats/min to420±23beats/min respectively, which means that the SIHR model was established successfully (p<0.01, n=20). Meanwhile, the raise of body weight of SIHR (270±14.6g) was decreased compared with control rats (340±16.8g)(p<0.01, n=20).(2) After the treatment of EA for5days, the SBP and HR decreased and body weight increased. On day14, SBP, HR and body weight were115±9mmHg,385±20beats/min,297±13.3g respectively, which means EA was effective.(3) Using IHC, we observed that the OXA immunoreactive neurons in SIHR were signigicantly up-regulated in LH-PFA, which was suggested by increased relative optical density (ROD) and the increased number of the positive neurons (p<0.01, n=6). EA treatment lead to the decrease of OXA immunoreactive neurons (p<0.05, n=6).(4) Western blot analysis showed that the expression of OX1R in rVLM of SIHRs was significantly increased, compared with control rats (p<0.01, n=4). EA treatment led to the decreased expression of OX1R in rVLM, which, however, had no significant statistical difference.(5) Confocal laser microscopy showed OX1R was co-expressed in cell membrane and cytoplasm with nNOS, iNOS or Neurofiliment in rVLM respectively.OX1R was co-expressed in a greater way with nNOS than with iNOS.(6) Intracerebroventricular injection of low dose (0.01nmol) of OXA induced no significant changes of SBP and HR while middle and high dose (0.1nmol and1nmol) increased SBP and HR dose-dependently. Intracerebroventricular injection of SB-408124alone, the selective inhibitor of OX1R, had no influence on SBP and HR, but it could block the cardiovascular effects of OXA.(7)20mins after intracerebroventricular injection of OXA or SB-408124or combined together, we tested the concentration of NOx in rVLM. The greater dose increase of OXA resulted in the lower concentration of NOx. The i.c.v. SB-408124alone had no such effects, but it could reverse the effects of OXA.(8) Unilateral microinjection of OXA (10pmol) into rVLM induced no significant changes of SBP and HR in normotensive rats. However, application of50or100pmol OXA in rVLM led to dose-dependent elevated SBP and HR. The hypertensive effects of100pmol OXA in rVLM was completely eliminated by SB-408124,7-Ni (a selective nNOS inhibitor) or Methylene Blue (a selective sGC inhibitor), which was microinjected in the same site5min in advance. But AG (a selectice iNOS inhibitor) had no influence on the effects of OXA on SBP and HR in the same manner. Unilateral microinjection of OXA (10,50,100pmol) into rVLM enhanced cardiovascular response in a dose-dependent manner, and the response was much more obvious in SIHR than in normotensive rats. It is worth noting that microinjection of SB-408124in rVLM alone could decrease the BP and HR of SIHR. SB-408124,7-Ni, or Methylene Blue, microinjected5min prior to OXA, could abolish the cardiovascular effects of OXA in rVLM of SIHR but AG had no such effect. The cardiovascular effects of OXA in rVLM in SIHRs+EA were basically the same as in normotensive rats.(9) During the first and second periods of10min after the microinjection of OXA into rVLM, the level of excitatory amino acid (EAA), Glu, increased while the inhibitory amino acid (IAA), Tau and GABA, decreased in the cerebrospinal fluid in rVLM. Compared with microinjection of OXA alone,7-Ni and Methylene Blue microinjected5min before OXA could abolish the effects OXA, leading to the decrease of Glu and an little increase of Tau but no change of GABA, while AG produced no signicicant change on amino acid transmitters in cerebrospinal fluid in rVLM.Conclusions:(1) OXA immunoreactive neurons in LH-PFA and OX1R in rVLM of the stress induced hypertensive rats were significantly up-regulated.(2) SB-408124, the selective OX1R antagonist, could decrease the BP and HR of SIHRs and abolish the cardiovascular response of exogenously-administrated OXA.(3) The enhanced BP and HR caused by microinjected OXA might be caused by the decrease of NO concentration in rVLM.(4) OXA in rVLM initiated an nNOS-NO-sGC-Glu cascade or iNOS-NO-GABA cascade, which may participate in central mechanisem of stress induced hypertension.(5) Acupuncture at Zusanli acupoint in SIHR could lower BP and HR, and decrease OXA immunoreactive neurons in LH-PFA and OX1R in rVLM. One of the mechansims of EA lowering BP and HR of SIHR. might be that OXA in rVLM was reduced to control nNOS/NO and iNOS/NO, then to regulate the ratio of excitatory/inhibitory amino acid neurotransmitters, and finally reduce the over-excited sympathetic outflow of SIHR, which leads to the depressor ofj hypertension. |
PDF Full Text Request