Effect Of Hydrogen Sulfide On Visceral Sensitivity And DSS-induced Colitis In Mice And The Underlying Mechanism

In the gastrointestinal(GI)system,visceral pain is the main symptom of many functional gastrointestinal diseases,which seriously affects the daily work and quality of life of patients.Inflammatory bowel disease(IBD)is a group of chronic and recurrent inflammatory conditions with diarrhea and abdominal pain as the main clinical manifestations.The pathogenesis of both visceral pain and IBD is not yet well defined and effective treatment of visceral pain and IBD remains a huge challenge.Hydrogen sulfide(H2S)is the third gas signal molecule found after nitric oxide(NO)and carbon monoxide(CO)which can regulate a number of cellular activity and organ functions under both physiological and pathological conditions.Numerous studies have found that H2S is closely related to the function of the GI system.In GI system,it is produced throughout the digestive tract by resident microbes and generated endogenously by the action of three endogenous producing enzymes.H2S in GI system has been proved to regulate intestinal motility.secretion,inflammation,and visceral pain.H2S plays a complex role in visceral pain and intestinal inflammation,which is manifested as a dual role of pro-and anti-nociceptive actions,as well as pro-and anti-inflammatory actions.Therefore,further study of the effect and mechanism of H2S on visceral sensitivity and inflammation can provide new insights for the treatment of visceral pain and IBD,and provide new targets for anti-inflammatory and analgesic drugs.Part ⅠEffect of exogenous hydrogen sulfide on mesenteric afferent sensitivity in mice and the underlying mechanismObjectives:Hydrogen sulfide(H2S)has been increasingly recognized as a key gasotransmitter regulating a number of cellular activity and organ functions under both physiological and pathological conditions.The effect of H2S on visceral nociception is elusive.The coplex effects of H2S on visceral sensitivity have been shown in the conflicting reports obtained from different experimental protocols and diverse species.So the purpose of this study was to explore the effect of exogenous H2S on colonic afferent sensitivity in mice to reveal the mechanism underlying the effect of H2S in mesenteric afferent nerve levels.Methods:The effects of sodium hydrosulfide(NaHS),the exogenous donor of H2S,on colonic afferent sensitivity of male C57BL6/J mice and Wistar rats were measured by recording the mesenteric afferent nerve discharge in vitro.The change of spontaneous activity,chemosensitivity and mechanosensitivity of colonic mesenteric afferent nerves caused by NaHS were recorded.Non-specific K+ channel blocker TEA,non-specific NOS inhibitor L-NMMA,the specific nNOS inhibitor NPLA,N-type Ca2+ channel blocker co-conotoxin and P-type Ca2+ channel blocker ω-agatoxin IVA were used to study the underlying mechanism of the effects of NaHS.Results:1.Effect of H2S donor on spontaneous discharge of colonic mesenteric afferent activity in mice.After the nerve activity was stabilized,NaHS(0.01 mM,0.1 mM,0.5 mM,1 mM,and 2 mM)was added to the serosal side of the colonic segment.Serosal application of NaHS induced a significant decrease in colonic afferent firing rate in a dose-response manner in mice.The average firing rate above baseline at 5 min after NaHS(0.5 mM)application was decreased from 0.5 ± 0.2 imp/s(vehicle control,n= 7)to-9.8 ± 1.2 imp/s(NaHS,P<0.001,n=10).2.The role of intraluminal pressure in NaHS-induced inhibition of the mesenteric afferent nerve.The intraluminal pressure at 5 min after NaHS(0.5 mM)administration was reduced by 0.5 ± 0.1 mmHg,and that of vehicle group was reduced by-0.3 ± 0.3 mmHg(P<0.05,n = 6).We cannot exclude the possibility that the decrease of spontaneous discharge caused by NaHS was due to the changes of intraluminal pressure.So we used the nonspecific K+ channel blocker TEA(10 mM)to attenuate the decrease in intraluminal pressure caused by NaHS.After pretreatment with TEA(10 mM)for 10 min,the change of intraluminal pressure caused by NaHS was increased from-0.5 ±0.1 mmHg(NaHS group,n = 6)to-0.1 ± 0.04 mmHg(TEA + NaHS group,P<0.05,n = 6).However,TEA(10 mM)did not block the inhibitory effect of NaHS on colonic mesenteric afferent activity(P>0.05.n = 6).These results indicated that the inhibitory effect of NaHS on colonic afferent nerves was not mediated by K+ channels,nor is it secondary to the decreased colonic motility caused by NaHS.3.Effect of H2S donor on mesenteric afferent discharge to bradykinin.The afferent response to bradykinin was markedly inhibited by prior NaHS application.The peak of firing rate above baseline caused by bradykinin was decreased from 37.3 ± 2.52 imp/s(vehicle control,n=8)to 20.1 ± 3.1 imp/s(NaHS 0.5 mM,P<0.01,n = 6).4.Effect of H2S donor on mesenteric afferent discharge to colonic ramp distention.To further examine the effect of NaHS on the mechanosensitivity of colonic mesenteric afferent nerves,colonic ramp distention was preformed which induced a pressure-dependent increase in afferent nerve discharge.Compared with the vehicle group,the colonic segments treated with NaHS(0.5 mM)exhibited significantly reduced mechasensitivity to high pressure distention and no apparent change in that to low pressure distention.The AUC above baseline discharge during distention from 20 to 60 mmHg was120.33 ± 7.59 imp/s in vehicle group(n = 10)and 75.43 ±7.44 imp/s in NaHS-treated group(P<0.001,n = 9).5.Effect of NO on NaHS-induced inhibition of the mesenteric afferent nerve.To testify our hypothesis that NO maybe play a vital role in NaHS-induced inhibition of afferent nerve excitability,non-specific NOS inhibitor L-NMMA and specific nNOS inhibitor NPLA were used to explore whether the action of NaHS would be affected.5.1 Effect of L-NMMA and NPLA on NaHS-induced inhibition of spontaneous discharge.The mean value of spontaneous discharge above baseline at 5 min after NaHS administration was increased from-9.73 ± 0.94 imp/s(vehicle + NaHS group,n =8)to-2.63 ± 0.63 imp/s(L-NMMA + NaHS group,P<0.001,n= 7)and-2.86 ±1.13 imp/s(NPLA + NaHS group,P<0.001,n = 6)respectively.5.2 Effect of L-NMMA and NPLA on NaHS-induced inhibition of mesenteric afferent discharge to bradykinin.The peak of firing rate above baseline induced by bradykinin was increased from 20.1 ± 3.2 imp/s(vehicle + NaHS group)to 36.8 ± 5 imp/s(L-NMMA + NaHS group,P<0.05,n= 6)and 44.48 ± 7.34 imp/s(NPLA + NaHS group,P<0.05,n=6)respectively.Nevertheless,neither L-NMMA nor NPLA applied alone affected the afferent excitation to bradykinin(P>0.05,n = 6).5.3 Effect of L-NMMA and NPLA on NaHS-induced inhibition of mesenteric afferent discharge to colonic ramp distention.The AUC above baseline discharge during distention from 20 to 60 mmHg was increased from 70.85 ± 6.65 imp/s(vehicle + NaHS group,n= 8)to 127.85 ±17.02 imp/s(L-NMMA + NaHS group,P<0.05,n= 6)and 112.26 ± 11.29 imp/s(NPLA + NaHS group,P<0.05,n = 6).6.Effect of Ca2+ channel blockers on NaHS-induced inhibition of the mesenteric afferent nerve.Previous studies have shown that the activity of nNOS in the ENS can be stimulated by intracellular Ca2+,which rely heavily on N-type or P-type Ca2+ channels.Therefore,we used ω-conotoxin GVIA(an N-type calcium channel blocker)and co-agatoxin IVA(a P-type calcium channel blocker)in the recording of mesenteric afferent discharge.6.1 Effect of Ca2+ channel blockers on NaHS-induced inhibition of spontaneous discharge.At 5 min after NaHS application,the mean spontaneous firing rate above baseline was markedly increased from-9.6 ± 1.07 imp/s(vehicle + NaHS group,n = 7)to-2.02 ± 1.3 imp/s(ω-conotoxin + NaHS group,P<0.001,n= 6).However,P-type Ca2+ channel blocker ω-agatoxin IVA(0.1 μM)did not diminish the inhibitory effect of NaHS on colonic afferent activity(P>0.05,n = 5).Therefore,in the subsequent experiments we only used ω-conotoxin GVIA(1 μM).6.2 Effect of ω-conotoxin GVIA on NaHS-induced inhibition of inhibition of mesenteric afferent discharge to bradykinin.The peak of discharge frequency above baseline induced by bradykinin was increased from 20.19 ± 3.15 imp/s(vehicle + NaHS group,n = 5)to 59.29 ± 5.74imp/s(ω-conotoxin + NaHS group,P<0.001,n = 6).Besides,co-conotoxin GVIA applied alone did not alter the bradykinin-induced afferent excitation(P>0.05,n = 6).6.3 Effect of co-conotoxin GVIA on NaHS-induced inhibition of mesenteric afferent discharge to colonic ramp distention.The AUC above baseline discharge during high pressure distention was increased from 70.85 ± 6.65 imp/s(vehicle + NaHS group,n = 8)to 121.53 ±12.12 imp/s(ω-conotoxin + NaHS group,P<0.05,n = 6).7.Effect of H2S donor on spontaneous discharge of colonic mesenteric afferent activity in rats.In order to verify the effect of NaHS on the mesenteric afferent nerve activity in the rat colon,we added NaHS(0.5 mM,1 mM and 3 mM)to the serosal side of the colonic segment and found NaHS also showed inhibition on colonic afferent firing rate in a dose-response manner in rats.The low concentration of 0.5 mM NaHS had no significant effect on afferent discharge,while 1 mM and 2 mM NaHS significantly reduced the frequency of mesenteric afferent nerve discharge.Conclusions:NaHS reduced the colonic afferent sensitivity in nNOS-dependent way,including spontaneous activity,chemosensitivity and mechanosensitivity.Our findings demonstrated the antinociceptive effect of H2S in the periphery.Part ⅡThe role of cystathionine β-synthase/H2S pathway in colonic mesenteric afferent sensitivity of miceObjectives:Endogenous H2S and its producing enzymes have been found to play different roles in different pain models.We have found that exogenous H2S down-regulated the colonic afferent sensitivity in the healthy mice.However,whether endogenous H2S could exert the same effect and whether endogenous H2S has a tonic effect on afferent nerve sensitivity remains unclear.Besides,Cbs+/-mice exhibit dysfunctions in multiple organs,but the effects on visceral pain have not been clearly studied.Therefore,the purpose of this study was to explore the role of CBS/H2S pathway in colonic mesenteric afferent sensitivity using Cbs+/-mice.Methods:We compared the mesenteric afferent nerve activity in WT and Cbs+/-mice by recording the mesenteric afferent nerve discharge in vitro and studied the effect of NaHS and CBS on visceral nociception induced by colorectal distension.Results:1.The spontaneous activity of the mesenteric afferent nerves of Cbs+/-mice.There was no significant change in the spontaneous activity of the mesenteric afferent nerves of Cbs+/-mice compared to WT littermates.The average firing rate was 16.15± 1.65 imp/s in the WT mice(n= 10)and 18.51 ± 0.88 imp/s in the Cbs +/-mice(P>0.05,n = 6).2.The afferent response to bradykinin in Cbs+/-mice.There was no significant change in the afferent response to bradykinin of the colonic mesenteric afferent nerves of Cbs+/-mice compared to WT littermates.The peak of firing rate above baseline caused by bradykinin was 31.74 ± 3.18 imp/s(Cbs+/-,n=6)and 38.04 ± 2.62 imp/s(WT,P>0.05,n = 6).3.The afferent response to colonic distention in Cbs+/-mice.Compared with WT littermates,Cbs+/-mice were found to exert increased afferent response to high pressure distention.The AUC above baseline discharge during distention from 20 to 60 mmHg was 71.47 ± 9.21 imp/s in WT mice(n= 6)and 106.28 ± 9.59 imp/s in Cbs+/-mice(P<0.05,n=7).4.The visceral pain response to CRD in Cbs+/-mice.The pain response to the second CRD procedure without any drug stimuli was not significantly different from that to the first CRD in both WT and Cbs+/-mice(P>0.05,n = 6).Compared with WT mice,Cbs+/-mice showed an enhanced hyperalgesic response to CRD(P<0.001,n = 7).5.The effect of NaHS on CRD-induced nociception in WT and Cbs+/-mice.Intraperitoneal administration of NaHS(60 μmol kg-1)in Cbs+/-mice evoked a remarkable reduction of the AWR score at all volumes of distension applied except distension with 0.04 ml of water(P<0.01,n = 6).However,administration of NaHS(60 μmol kg-1 i.p.)in WT mice reduced the CRD-induced AWR only at distensions with 0.12 ml and 0.16 ml of water(P<0.05,n = 6).Conclusions:Cbs+/-mice showed increased afferent sensitivity to colonic distention and enhanced visceral hyperalgesia induced by CRD.Our results provide additional evidence that endogenous H2S also exerts analgesic effect on the visceral nociception and provide new targets for analgesic drugs.Part ⅢThe role of cystathionine β-synthase/H2S pathway in DSS-induced colitis in miceObjectives:Cumulative studies have shown that H2S plays an important role as an inflammatory mediator in inflammatory bowel disease(IBD).The exact role of CBS/H2S system in colitis is not well understood due to the lack of specific CBS inhibitors.Hence,the aim of this study was to explore the role of CBS/H2S pathway in dextran sodium sulfate(DSS)-induced colitis in Cbs+/-mice.Methods:Colitis was induced by administration of 2.5%DSS for 7 days in Cbs+/-mice and wild-type littermates(WT).The degree of inflammation of each mouse was assessed by disease activity index(DAI),histological score of colitis tissue,colonic mucosal permeability,JAM-1 and Occludin immunohistochemistry,PAS staining,and the expression of pro-inflammatory factors.Results:1.Cbs+/-mice are more susceptible to DSS-induced colitis.In the DSS-induced colitis mice,the body weight began to decrease on the 3-4 days after the administration of DSS.The following symptoms gradually appeared:reduced diet,apathy,decreased activity,increased stool frequency,diarrhea,and bloody stools.1.1 Body weight and colon lengthThere was no apparent change in body weight and colon length between healthy WT and Cbs+/-mice(P>0.05,n = 5-8).Compared with WT colitis group,the Cbs+/-colitis group had a greater weight loss and a shorter colon length.On the 7th day of DSS,the body weight of the Cbs+/-mice was decreased by 23.84%±1.19,while that of WT mice was decreased by 16.10%±2.11(P<0.001,n =8-11).The colon length of the Cbs+/-group was 5.9 ± 0.22 cm,and the colon length of the WT group was 5.11 ± 0.1 cm(P<0.001,n = 10-12).1.2 Disease activity index(DAI)Compared with WT colitis group,the Cbs+/-colitis group lost more weight,and the symptoms of diarrhea and bloody stool were more serious.From the fourth day,the DAI score of Cbs+/-colitis group was significantly higher than that of WT colitis group(P<0.05,n = 9-11).1.3 Histological scoreOn the 7th day of DSS,the histological score is 5.17 ± 0.4 in Cbs+/-colitis group and 3.67 ± 0.21 in WT colitis group(P<0.05,n = 6).The histological score in Cbs+/-group was higher than that in WT group2.Increased inflammatory cytokine expression in Cbs+/-colitis mice.The mRNA expression levels of proinflammatory factors IL-1β,IL-6 and TNFa in Cbs+/-colitis group were significantly higher than those in WT group(P<0.05,n =7-10).There was no significant difference in the expression levels of inflammatory cytokine between WT and Cbs+/-control groups(P>0.05,n = 5-8).3.Increased colonic mucosal permeability in Cbs+/-colitis mice.The FITC-dextran mucosal permeability test showed that FITC fluorescence value in the serum of the Cbs+/-colitis group was higher than that of the WT colitis group,indicating that the mucosal permeability of Cbs+/-colitis group was increased compared to WT colitis group(P<0.05,n = 6-7).The colonic mucosal permeability of the Cbs+/-control group was similar to that of the WT control group(P>0.05,n =6-8).4.More severe destruction of colonic mucosal barrier in Cbs+/-colitis mice.We further detected the colonic epithelial cell barrier by JAM-1 and occludin immunohistochemistry.The expression of these two proteins were not significantly different in WT and Cbs+/-control group,but was significantly reduced in the colonic epithelial cells of the Cbs+/-colitis mice compared to the WT colitis group.We also detected the mucus barrier of colonic epithelium by PAS staining.Following DSS administration,the number of colonic mucosal glycogen was significantly reduced,but there was no significant difference in glycogen expression between the Cbs+/-colitis group and the WT colitis group.Conclusions:DSS-induced colitis in Cbs+/-mice showed more severe symptoms of colotis,which may be associated with the up-regulation of pro-inflammatory cytokines and the aggravation of colonic mucosal barrier disruption.Our findings suggest that the CBS/H2S pathway may play an endogenous protective role in DSS-induced colitis.