| ObjectivesTo observe the therapeutical effect of Feng-Liao-Chang-Wei-Kang(FLCWK)guanule on rat model of diarrhea-predominant irritable bowel syndrome(IBS-D),and explore its underlying mechanism through contraction of isolated colonic longitudinal smooth muscle strips(CLSMSs)and chloride secretion across isolated colon mucosa.MethodsThis study includes four experiments:1.The study of therapeutical effect of FLCWK guanule on rat model of IBS-D.2.The study of influence and its underlying mechanism of FLCWK guanule on contraction of CLSMSs isolated from normal rat.3.The study of influence and its underlying mechanism of FLCWK guanule on contraction of CLSMSs isolated from rat model of IBS-D.4.The study of influence and its underlying mechanism of FLCWK guanule on chloride secretion across isolated colon mucosa of rat model of IBS-D.Detailed methods are as follows:Experiment one:one-day old specific pathogen free(SPF)Sprague-Dawley(SD)male rats were randomly divides into six groups as follows:normal group,IBS-D group,FLCWK granule high-dose(FLCWK-H)group,FLCWK granule medium-dose(FLCWK-M)group,FLCWK granule low-dose(FLCWK-L)group,positive control drug group(pinaverium group).Treat normal group with no handling and other groups with maternal separation from postnatal days 2 to 21 plus restraint stress from days 50 to 59 to establish rat model of IBS-D.At postnatal days 60,after the modeling,model evaluation was performed.model-evaluation indexes was based on feces condition(fecal pellet number,fecal water content)and visceral sensitivity(change of abdominal withdrawal reflex(AWR)score and ratio of external oblique muscle electromyographic area under the curve(AUC(%))induced by colorectal distension).After successful modeling,each group were given intervention measure for 14 consecutive days:FLCWK-H,FLCWK-M,FLCWK-L group was respectively given 0.47 g,0.95 g,1.89 g raw drug every 100 grams weight of rats each day,pinaverium group was given 2.1 mg every 100 grams weight of rats each day.One milliliter medicine was given through administration by gavage every 100 grams weight of rats.The normal group and IBS-D group was given the same amount of saline solution.After intragastric administration,therapeutical effect of FLCWK granule was evaluated by feces condition(fecal pellet number,fecal water content)and visceral sensitivity(change of abdominal withdrawal reflex(AWR)score and AUC(%)).Experiment two:Colonic longitudinal smooth muscle strips(CLSMSs)were isolated from SPF SD male rats(Weight:250-300 g).Krebs solution with mixed gas(95%02 and 5%C02)was nutrient solution of CLSMSs.Different dose of FLCWK solution was prepared by dry extract of FLCWK without excipients and Krebs solution.Via tissue perfusion system in vitro:1.with normal saline as control,influence of FLCWK solution on contraction of CLSMSs in basal conditions was observed;2.with normal saline as control,influence of FLCWK solution on contraction of CLSMSs induced by different stimuli was observed;3.with self-control methods,the mechanism of FLCWK solution which inhibited contraction of CLSMSs induced by different stimuli was discussed.Experiment three:According to modeling and administration methods in experiment one,one day old SPF SD male rats were randomly divides into three groups as follows:normal group,IBS-D group,FLCWK group.After administration by gavage,CLSMSs were isolated from rats from each group.Via tissue perfusion system in vitro and comparison between groups:1.influence of FLCWK solution on contraction of CLSMSs in basal conditions was observed;2.influence of FLCWK solution on contraction of CLSMSs induced by acetylcholine(Ach,0.1mmol/L)was observed;3.mechanism of FLCWK solution which inhibited contraction of CLSMSs induced by Ach was discussed.Experiment four:According to modeling,administration,grouping methods in experiment three,isolated colonic mucosal specimens with serosal muscular and submucous layers removed were prepared.Via short-circuit current(Isc)technique in vitro and comparison between groups:1.influence of FLCWK solution on basal volta potential difference(VPD),basal Isc and basal transepithelial electrical resistance(TEER)was observed.2.influence of FLCWK solution on change of ISC(ΔISC)induced by forskolin(1μmol/L)was observed.3.influence of FLCWK solution on AIsc induced by forskolin after substitution of chloridion was observed.4.mechanism of FLCWK solution which inhibit chloride secretion across isolated colon mucosa was discussed.ResultsExperiment one:1.Rat model evaluation of IBS-D:(1)defecation frequency and loose stools were increased;(2)visceral sensitivity was increased:AUC(%)and AWR score was higher than normal group(under colorectal distention of 40,60 mmHg(P<0.01),80 mmHg(P<0.05));2.therapeutical effect of FLCWK granule on rat model of IBS-D:(1 improvement of fecal conditions:①decrease of loose stools:compared with IBS-D group,fecal water content of FLCWK-H,FLCWK-M was lower(P<0.01).②decrease of defecation frequency:compared with IBS-D group,fecal pellet numbers of FLCWK-H,FLCWK-M ware fewer(P<0.01)(2)improvement of visceral hypersensitivity:① decrease of AUR(%):compared with IBS-D group,AUR(%)of FLCWK-H,FLCWK-M group was lower(under colorectal distention of 40,80 mmHg(P<0.01),60 mmHg:FLCWK-H(P<0.01),FLCWK-M(P<0.05));② decrease of AWR score:compared with IBS-D group,AWR score of FLCWK-H,FLCWK-M group was lower(under colorectal distention of 40,60,80 mmHg(P<0.05)).Experiment two:FLCWK solution can inhibit basal tension(%)and amplitude(%)of contraction of CLSMSs:from the cumulative dose of FLCWK solution reached 30 μg/ml,basal tension(%)and amplitude(%)was concentration-dependently decreased:(1)basal tension(%):when the cumulative dose of FLCWK solution reached 1000,3000 μg/ml(P<0.05);10000μg/ml(P<0.01)(2)basal amplitude(%):when the cumulative dose of FLCWK solution reached 1000μg/ml and above(P<0.01).2.FLCWK solution can inhibit the contraction of CLSMSs induced by different stimuli:from the cumulative dose of FLCWK solution reached 100μg/ml,tension(%)induced by different stimuli was concentration-dependently decreased:(1)Ach:when the cumulative dose of FLCWK solution reached 1000 μg/ml(P<0.05);3000,10000μg/ml(P<0.01).(2)KCl(20 mmol/L):when the cumulative dose of FLCWK solution reached 1000μg/ml and above(P<0.05).(3)exhausting the Ca2+ of internal calcium store and influx of extarcellular calcium:when the cumulative dose of FLCWK solution reached 1000 μg/ml and above(P<0.05).3.The inhibitory effect of FLCWK solution on contraction induced by Ach was partially blocked by inhibitors:(1)methylene blue(MB,an inhibitor of guanosine monophosphate cyclase,10 μmol/L):from the cumulative dose of FLCWK solution reached 300 μg/ml,Ach-induced amplitude(%)of contraction of CLSMSs was concentration-dependently decreased,but higher than control:when the cumulative dose of FLCWK solution reached 1000 μg/ml and above(P<0.05);(2)NG-nitro-L-arginine methyl ester(L-NAME,an inhibitor of nitric oxide synthase,100 μmol/L):from the cumulative dose of FLCWK solution reached 1000 μg/ml,Ach-induced amplitude(%)of contraction of CLSMSs was concentration-dependently decreased,but higher than control:when the cumulative dose of FLCWK solution reached 1000 μg/ml(P<0.05);3000,10000μg/ml(P<0.01),(3)4-aminopyridine(4-AP,a selective blocker of voltage-activated K+ channel,50 μmol/L)from the cumulative dose of FLCWK solution reached 3 μg/ml,Ach-induced amplitude(%)of contraction of CLSMSs was concentration-dependently decreased,but higher than control:when the cumulative dose of FLCWK solution reached 1000 μg/ml and above(P<0.05).Experiment three:1.FLCWK solution can inhibit contraction of CLSMSs in basal conditions:compared with IBS-D group,basal tension of FLCWK group was lower(P<0.01);2.FLCWK solution can inhibit contraction of CLSMSs induced by Ach:compared with IBS-D group,Ach-induced tension of FLCWK group was lower(P<0.05);3.after incubation of nifedipine(10 μmol/L,L-typed voltage-dependent Ca2+ channel blocker),nitro oxide synthase inhibitor L-NAME(100μmol/L),3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride(TMB-8,an intracellular Ca2+ antagonist,20 μmol/L),4-AP(50 μmol/L),MB(10μmol/L)respectively,there were no statistical significance of Ach-induced tension between three groups(P>0.05).Experiment four:1.There were no statistical significance of basal VPD,basal Isc and basal TEER(P>0.05),however FLCWK group showed a downward trend.2.FLCWK solution could inhibit chloride secretion induced by forskolin:compared with IBS-D group,forskolin-inducedΔisc of FLCWK group was lower(P<0.05).3.after substitution of chloride ion,the inhibitory effect of FLCWK solution on chloride secretion induced by forskolin was partially blocked:there were no statistical significance of ΔIsc induced by forskolin between three groups(P>0.05).4.the inhibitory effect of FLCWK solution on chloride secretion induced by forskolin was partially blocked by inhibitors:after incubation of diphenylamine-2-carboxylate(DPC,1 mmol/L,added on apical membrane),cystic fibrosis transmembrane conductance regulator(CTFR)inhibitor glibenclamide(1 mmol/L,added on apical membrane),Na-K-Cl cotransporter(NKCC)inhibitor bumetanide(100 mmol/L,added on basolateral membrane),there were no statistical significance of ΔIsc induced by forskolin between three groups(P>0.05).Conclusions1.FLCWK granule could effectively relieve diarrhea symptom and improve visceral hypersensitivity of rat model of IBS-D;2.FLCWK granule could concentration-dependently inhibit contraction of CLSMSs of normal rat and rat model of IBS-D,and its antispasmodic mechanism may be associated with blocking of calcium channel,voltage-gated potassium channel and nitric oxide(NO)-cyclic guanosine monophosphate(cGMP)-protein kinase G(PKG)pathway;3.FLCWK granule could inhibit chloride secretion across isolated colon mucosa of rat model of IBS-D and its anti-diarrhea mechanism may be associated with blocking apical chloride channel(including CFTR)and basolateral NKCC. |
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