Effects Of Angiotensin â…¡ Microinjected Into The Paraventricular Nucleus On Gastric Ischemia-reperfusion Injury In Rats

Currently,gastric ischemia-reperfusion(GI-R)injury is widely investigated for its pathogenic mechanisms,such as the increase in free oxygen radicals,endothelin levels, microvascular dysfunction,polymorphonuclear leukocyte infiltration,nitric oxide release, gastric acid secretion,and prostaglandin decrease during GI-R.However,little is known about the systemic regulation of GI-R injury,in particular about the roles of the relevant central nuclei. The hypothalamic paraventricular nucleus(PVN)regulates various activities of the stomach, such as secretion,electrical activation,and gastric motion.The PVN contains many types of neurons that secrete many kinds of neurotransmitters,including angiotensinⅡ(AngⅡ) immunoreactive cells,fibres and high densities of AngⅡAT1 receptors.AngⅡneurons and their receptors in the PVN contribute to the production of stress-induced gastric ulcers.More importantly,increases in central AngⅡhave been shown to increase sympathetic outflow.Our previous work showed that electrical stimulation of the PVN attenuates GI-R injury via the vagal and sympathetic efferent nerves.However,it remains unknown whether exogenous AngⅡin the PVN can regulate GI-R.injury and its possible peripheral mechanisms.Therefore,it needs to clarify whether the sympathetic and parasympathetic nervous systems mediate the effects of AngⅡmicroinjected into the PVN.Gastric mucosa is the tissue with a high renewal rate.Although gastric mucosa is vulnerable to injury,it is also restored quickly.The damage and repair in the gastric mucosa are physiologically common phenomenons,indicating that gastric mucosal cells have a potential ability to regulate the apoptosis and proliferation.The integrity of gastric mucosa depends upon the interplay between epithelial cell proliferation and apoptosis.The signal pathway of nuclear factor-κB(NF-κB),one family of transcription factors,tightly relates to cell apoptosis and proliferation.NF-κB is responsible for the regulation of numerous target genes involved in the inflammatory,immune reactions,cell proliferation and apoptosis.Furthermore,NF-κB is activated by ischemia-hypoxia,oxygen free radicals,tumor necrosis factor-or(TNF-α)and COX-2.When cells are subjected to stimuli,NF-κB translocates to the nucleus where it binds to specific response elements in the DNA,activates the transcription of genes including apoptotic genes(Bcl-2/Bax),COX-2,intercellular adhesion molecules,thereby promotes their expression,and finally lead to the injury of tissues and cells.Several genes and their proteins regulate epithelial cell apoptosis,such as the Bcl-2 family proteins.Both the family members,Bcl-2 and Bax,regulate apoptosis that can affect gastric mucosal injury.The ratio of Bcl-2/Bax is a key determiant to inhibit apoptosis.However, studies of GI-R injury are still limited to anti-injury mechanisms,and little is known for the mechanisms underlying anti-apoptosis or pro-proliferation.Therefore,in order to explore the role of AngⅡin the PVN in regulation of GI-R injury, the time- and dose-dependent effects of AngⅡmicroinjected into the PVN were observed in the present study.Then,we firstly examined the peripheral nervous regulation.We determined whether the parasympathetic or the sympathetic pathway is involved in the effects of PVN AngⅡon GI-R injury using pharmacological and surgical approaches.Secondly,we examined the molecular regulation in the gastric mucosa.We observed the change patterns of apoptosis and proliferation in gastric mucosal after GI-R.The critical role of NF-κB activation in regulating GI-R injury and the effects of PVN AngⅡwas investigated by detecting the alterations of NF-κB,IκB-αand p-IκB-α.The molecules relevant to apoptosis(Bcl-2,Bax and caspase-3) were also detected. PartⅠEffect of angiotensinⅡmicroinjected into the paraventricular nucleus on gastric ischemia-reperfusion injury in rats and the peripheral nervous mechanismOur previous study demonstrated that electrical stimulation of the PVN protects against GI-R injury,but it is still unknown whether AngⅡin the PVN plays a role in the development of GI-R injury.The purpose of this part was to investigate the effect of PVN AngⅡon GI-R injury and the regulation by autonomic nerves.GI-R injury was induced in rats by clamping the celiac artery for 30 min,and then reperfusing for 30 min,1 h,3 h,6 h or 24 h,respectively.A cannula was inserted into the unilateral PVN for microinjection of AngⅡ.The extent of gastric mucosal damage was determined by gross and histological methods.We found that microinjection of pharmacological doses of AngⅡ(3,30,and 300 ng)into the PVN dose-dependently inhibited GI-R injury,and that AngⅡ(30 ng)markedly attenuated GI-R injury at 1 h and 3 h after reperfusion.The effect of AngⅡwas prevented by pretreatment with the AngⅡAT1 receptor antagonist losartan(5μg)into the lateral cerebral ventricle.Furthermore,the protective effect of AngⅡon GI-R injury was abolished by propranolol(1 mg/kg,i.v.)or disconnection of the nerves innervating the adrenal glands,was augmented by sympathectomy or phentolamine(1 mg/kg,i.v.),and was not affected by subdiaphragmatic vagotomy or atropine(1 mg/kg,i.v.).In addition,AngⅡmicroinjected into the PVN increased the gastric blood flow after ischemia. These results indicate that the PVN is a responsive site for central AngⅡ-induced protection against GI-R injury.The central effects of AngⅡare mediated by AT1 receptors in the PVN,and the peripheral effects by a sympathetic-adrenal gland-β-adrenoceptor pathway.The attenuation of GI-R injury may result from increasing gastric blood flow. PartⅡThe role of nuclear factor-κB in the protective effect of angiotensinⅡmicroinjected into the paraventricular nucleus on gastric ischemiareperfusion injury in ratsThe PVN is one of the specific central nuclei regulating GI-R injury.The purpose of this part was to elucidate the local molecular mechanisms underlying the development of GI-R injury and the effects of PVN AngⅡon GI-R injury.The GI-R injury in Sprague-Dawley rats was induced by clamping the celiac artery and then reperfusing.A cannula was inserted into the unilateral PVN for microinjection of AngⅡ.The changes in the proliferation and apoptosis,the activation of NF-κB,and the expressions of Bcl-2,Bax and COX-2 mRNA in rat gastric mucosa were examined by Western blotting,immunohistochemistry or RT-PCR.The gastric mucosal malonaldehyde(MDA)content and superoxide dismutase(SOD)activity were examined as well.The results showed that:(1)GI-R significantly decreased the proliferation cells at 30 min and 1 h,then increased gradually at 3 h and recovered to normal level at 6 h after reperfusion.GI-R increased the apoptotic cells significantly at 30 min,1 h and 3 h and recovered to normal level at 6 h after reperfusion.The apoptosis rate in the cultured human gastric mucosal cells was detected by flow cytometer,and was significantly increased at 6 h,24 h and 48 h after reoxygen following 1-h hypoxia.(2)Microinjection of AngⅡ(30 ng)into the PVN significantly decreased mucosal cell apoptosis at 1 h after reperfusion.PVN AngⅡalso accelerated the mucosal cell proliferation after GI-R.(3)The expressions of NF-κB(p65)and phosphorylated IκB-αprotein were increased after 1-h GI-R,which were markedly reduced by microinjection of AngⅡinto the PVN.In contrast,the expression of IκB-αprotein was decreased after 1-h GI-R,which was reversed to the normal level by microinjection of AngⅡinto the PVN.The effects of AngⅡwere prevented by pretreatment with AngⅡAT1 receptor antagonist losartan(5μg)into the lateral cerebral ventricle.Pyrrolidine dithiocarbamate (PDTC,200 mg/kg),an inhibitor of NF-κB,inhibited GI-R-induced NF-κB(p65)activation similarly in the rats with or without microinjection of AngⅡinto the PVN.PDTC also attenuated gastric mucosal injury and apoptosis,and promoted cell proliferation.(4) Microinjection of AngⅡinto PVN significantly increased the protein expression of Bcl-2(an inhibitor of apoptosis)and did not alter that of Bax(a promoter of apoptosis),thereby resulting in a significant increase in the Bcl-2/Bax ratio.Microinjection of AngⅡinto the PVN markedly decreased the levels of caspase-3 and COX-2 mRNA after 1-h GI-R.Microinjection of AngⅡinto the PVN also increased the MDA content,but did not alter the SOD activity in gastric mucosa after GI-R.These results indicate that microinjection of AngⅡinto the PVN has a significant protective effect on GI-R injury by inhibiting gastric mucosal cell apoptosis and promoting proliferation.This protection may result from the increase in gastric blood flow, the decrease in oxygen-derived free radicals,and the inhibition of COX-2 expression,which in turn may lead to the inhibition of NF-κB activation,the increase in the expression of its downstream regulating factors Bcl-2 and Bcl-2/Bax ratio,and finally result in a significant protective effect on GI-R injury. Summary1.Microinjection of AngⅡ(3,30,and 300 ng)into the PVN dose-dependently inhibits GI-R injury;AngⅡ(30 ng)markedly attenuated GI-R injury at 1 h and 3 h after reperfusion,and also increased the gastric blood flow after ischemia.2.After AngⅡis microinjected into the PVN,it centrally stimulates AT1 receptor in the PVN,peripherally activates a sympathetic-adrenal gland-β-adrenoceptor pathway,and attenuates GI-R injury mediated by the central and peripheral ways.3.AngⅡmicroinjected into the PVN decreases GI-R-induced NF-κB activation, resulting in the inhibition of apoptosis and accelation of cell proliferation in gastric mucosa.4.AngⅡmicroinjected into the PVN protects gastric mucosa against GI-R injury through dilating gastric vessels,decreasing oxygen free radicals,inhibiting COX-2 expression;thereby increasing the activities of anti-apoptotic and proliferation-promoting factors via inhibition of NF-κB activation.