Nitric Oxide Donor Protects Against Acetic Acid-induced Gastric Ulcer In Rats

Part ?The protective role of exogenous NO against acetic acid-induced gastric ulcer in ratsObjectivesVagal afferents are extensively distributed in the GI tract to control and coordinate gastrointestinal functions. Its effect in controlling the gastrointestinal inflammation has been well recognized, but the underlying mechanism especially that of the modulation of the afferent discharge has not been clearly understood. It has been demonstrated that NO excited vagus afferents in GI tract. There is no convictive evidence to suggest that NO-induced vagal afferent firing evokes protective effect during inflammation. So, we hypothesize that NO participate in the vagal anti-inflammatory reflex to modulate inflammatory responses.MethodsWe examined the actions of NO on an acetic acid-induced gastric ulcer in a rat model.In order to investigate if vagus nerve participated in the protective effect of NO, we tested the effect of subdiaphragmatic vagotomy on the protective effect of SNP (i.p.).We used a TRPV1 antagonist capsazepine (CZP) to explore the underlying mechanism. We sacrificed the rats at the 4th day. The extent of the ulcer areas were measured (UA, mm2), the histologic samples were stained with HE, and the inflammatory degree of the gastric lesions was also measured by myeloperoxidase(MPO) activity.ResultsAcetic acid induced characteristic necrotic lesions in the stomach, including loss of folds, edema, discoloration, white fur coating and mucosal necrosis in acetic acid control group. The UA was 1.16 ±0.12 mm2. Mice treated with SNP (i.p.) for four days exhibited considerably less inflammatory change and more intact fold around ulcer tissue. The UA was reduced to 0.74 ± 0.12 mm2 (n = 6,P < 0.05 vs acetic acid control group). The protective effective of SNP was abolished by vagotomy or CZP(i.p.).The protective effect of SNP was confirmed histologically. Acetic acid induced severe damage of gastric mucosa, which was characterized by submucosa destruction,hemorrhagic injury and inflammatory cell infiltration. Compared with the acetic acid vehicle mice, SNP-treated mice exhibited much more intact histological structure of the mucosa. The destruction and leucocytes infiltration in mice were much less severe,while vagotomy or CZP abolished SNP's effect.The MPO activity in acetic acid group was increased to 1.79±0.37 U/g (n=6),higher than that of the NS control group (0.23±0.05 U/g, n= 6,P< 0.05). The MPO activity was reduced in acetic acid + SNP group (0.65±0.10 U/g,n=6,P<0.05 vs acetic acid control group). The effect of SNP was reversed by vagotomy or CZP.ConclusionPeritoneal injection of SNP decreased the ulcer area, inflammatory cell infiltration and MPO degree in acetic acid-induced gastric ulcer in rat. This effect was abolished by vagotomy and a TRPV1 antagonist. These results indicate that NO might play a beneficial role in the gastric ulcer healing process via the inflammation reflex. Our findings reveal a new mechanism for vagal afferent activation, and a new potential anti-inflammatory target.Part IIThe role of exogenous NO on mesenteric afferent discharge of ratsObjectivesNO is believed to mediate the cellular effects via two signaling pathways, the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway and cGMP-independent S-nitrosylation. The importance of S-nitrosylation modification towards protein thiols is increasingly recognized by researchers since it exerted a large amount of NO's ubiquitous influence on cellular signal transduction. An increasing number of ion channels have been identified as targets for S-nitrosylation.It has been proved that NO excited vagus afferents in GI tract indirectly, but there is no convictive evidence to suggest that NO activates spontaneous mesenteric afferent discharge via S-nitrosylation. Abundant expression of TRPV1 was detected on vagal afferent neurons in the gastrointestinal tract. As a target of S-nitrosylation,TRPV1 was also proved to be involved in the inflammatory response. So, we hypothesize that NO has direct S-nitrosylation effect on mesenteric afferents, involving TRPV1.MethodsWe recorded the mesenteric afferent nerve firing of male rats, mice and male TRPV1-dificient mice. To identify the subtype of the mesenteric afferent nerve that was activated by SNP, the mechanical sensitivity of the spontaneous afferent spikes was assessed by ramp distension. Subdiaphragmatic vagotomy, CZP, a selective inhibitor of guanylate cyclase 1H- [1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one(ODQ), and N-ethylmaleimide (NEM), a protein thiol protective agent were used to testify the underlying mechanisms.ResultsEffect of NO donor on spontaneous discharge of mesenteric afferent multi-unit activityWhen added to the serosal side of the gut segment for 2 min, SNP (4 mM) elicited significant increase in afferent multiunit firing rates. The mean discharge above baseline was increased from 1.3 ± 1.2 imp/s to 48.5 ± 7.3 imp/s (P < 0.05, n=6). The afferent nerve activation effect of SNP acted in a dose-dependent manner, and the half-maximum effective dose (ED50) was 0.16mM.Effect of NO donor on spontaneous discharge of different subunits of mesenteric afferent fibersBased on their spike waveform, 59 single units were discriminated in the multiunit recordings of 5 rats. According to their response to distension, 17 units was identified as the low thresholds (LT, maximal discharge rates at < 20 mmHg), 22 as the wide dynamic range (WDR, increased monotonically over the entire range of 60 mmHg),and 20 as the high thresholds (HT, activation threshold > 20 mmHg). SNP activated the spontaneous afferent firing of LT units (P < 0.05 vs vehicle), while did not influence that of the HT units. The mesenteric afferent nerve bundle contains both vagal and spinal fibers, and most of LT are vagal afferent fiber, while most of HT is spinal afferent fibers.Vagotomy eliminated the excitatory effects of SNP on spontaneous afferent dischargeIn vagotomy group, the administration of SNP did not influence the spontaneous discharge of mesenteric afferent nerve discharge. The mean discharge above baseline was decreased from 26.9 ± 5.2 imp/s (sham-operation group) to 2.6 ± 0.4 imp/s(vagotomy group, P < 0.01, n=6).Involvement of S-nitrosylation and TRPV1 in the SNP-induced activation of the mesenteric afferent nervePretreatment with NEM for 15 min completely eliminated the excitatory effect of SNP on the mesenteric afferent nerve. The mean discharge above baseline was decreased from 23.4 ± 5.2 imp/s to 0.2±5.7 imp/s (P < 0.05,n=6). However,the presence of ODQ (10 ?M,10 min) did not exert any influence on the excitatory effect of SNP (P = 0.52, n=6).Pre-treatment of CZP (50 ?M) diminished the excitatory effect of SNP on mesenteric afferent discharge. The mean frequency of the afferent discharge was decreased from 25.8 ± 5.6 imp/s to 8.4 ± 4.1 imp/s (P < 0.05, n= 6). SNP also markedly enhanced the excitatory effect of capsaicin (0.3 ?M), the agonist of TRPV1, on the mesenteric afferent discharge. The mean afferent discharge was increased from 8.2 ± 1.8 imp/s to 20.9 ± 2.7 imp/s (P < 0.05 vs capsaicin control group, n=6). Administration of SNP failed to increase the nerve firing of TRPV1-/- mice (P < 0.001, n = 6).ConclusionSNP increased the jejunal mesenteric afferent discharge via vagus nerve.S-nitrosylation and TRPV1 may involve in these processes.Part ?The role of exogenous NO on electrical properties of NG/DRGneurons of ratsObjectivesWe provided evidence that SNP enhanced the spontaneous discharge of vagus afferent nerve by S-nitrosylation, while TRPV1 might participate in this process. To further determine whether exogenous NO selectively activates vagus afferents, and whether TRPV1 was the key target of NO S-nitrosylation, we used NG neurons and DRG neurons to testify the effect of SNP.MethodsBoth the NG neurons and T7-L5 DRG neurons were excised, digested, and plated onto dishes. Whole-cell patch-clamp recordings were carried out to record the membrane potential and current of the neurons in rats.ResultsThe resting membrane potential of NG neurons was -57.4 ± 1.3 mV (n = 43). The membrane was depolarized following SNP (0.16 mM) administration, and partially recovered after washing the cells with Krebs saline. Pretreatment of CZP (50 ?M) or NEM (5 mM) significantly diminished this effect, while ODQ did not influence it.Pretreatment of SNP (0.16 mM) increased the amplitude of capsaicin-induced inward current (Icap) significantly,from -609.4±88.0 pA to -1224.3±202.1 pA (P< 0.05, n=7). This effect of SNP was abolished by NEM (5 mM), but was not influenced by ODQ(10?M).Contrast to the depolarization of the NG neurons, the membrane of DRG neurons was hyperpolarized following SNP (0.0016 to 0.16mM) administration (P < 0.05, n = 7).ConclusionSNP can activate TRPV1 on NG neurons via S-nitrosylation, increase its sensitivity to capsaicin. NO elicited a contrary effect on sympathetic and vagal afferents. Our findings reveal a new mechanism for vagal afferent activation.