| BackgroundReflux esophagitis(RE)present a condition of that backflow of gastric/duodenal contents into esophagus to induce mucosal injury,accompanied with the symptoms of regurgitation and heartburn.Some patients even exhibit external symptoms of esophagus like pharyngalgia and cough because of irritant refluxates to the throat and lung.In Asia countries,nearly 30%of gastroesophageal reflux disease(GERD)patients classified as RE.Nowadays,the pathophysiology of RE is unclear,mainstream opinion agree that the unbalance of anti-reflux defense mechanism and toxic refluxates is the mainly cause.With the deepening of research,irritant refluxates-induced inflammatory cascade reaction in esophageal epithelial cells is mainly attributed to cytokine release.CaSR mediated NLRP3 inflammation activation may play a role in the process of IL-1β maturation and secretion.CaSR can express in the basal cells of epithelium,maintain the calcium homeostasis through activating PLC/IP3 pathway,promoting intracellular calcium mobilization,finally inducing NLRP3 inflammation activation and cytokine release.However,it is unclear whether CaSR mediated NLRP3 inflammation activation and cytokine release participate in the inflammatory injury that induced by cytokine released from refluxates irritated esophageal epithelial cells.It is of importance to clarify this signal in the inflammatory injury of esophageal epithelium.Nowadays,oral medication of proton pump inhibitor(PPI)for eight weeks are primary.management of RE,while efficacy are not satisfying.Chinese herbal compounds possess advantage through treatment based on syndrome differentiation.Our previous animal experiment results had implied that Tojapride could effectively ameliorate inflammatory lesion in esophageal mucosa of RE rats suffered from live-gastric disharmony syndrome.However,whether the probable molecule mechanism underlying this effects of Tojapride is based on CaSR mediated NLRP3 inflammation activation,the questions need to be further studied.Part one In VivoObjectiveTo investigate the effects of Tojapride on the treatment of RE rats with live-gastric disharmony syndrome,and to further investigate the underlying mechanism of Tojapride on improving inflammatory lesion of esophageal epithelium via CaSR mediated NLRP3 inflammation activation pathway.Methods1.120 SD rats were randomly divided into sham and operation group,the later one suffered from esophagogastroduodenal anastomosis(EGDA).According the survival amount,randomly divided the rats into RE model(RE),RE combined with syndrome(RES),Toj apride-L(TL),Tojapride-M(TM),Tojapride-H(TH),Omeprazole(O)and Omeprazole mixed with Tojapride(OT)group.After raising for two weeks,RE combined with syndrome group and all of medication group were accepted tail clamp stimulation for one week.During three weeks of gastric administration,the daily body weight,amount of food-intake and drinking were recorded.Before the last day of gastric administration,autonomous activity capacity was tested through open-field ground.Then the pylorus ligation was made,and after two hours of the last gastric administration,gastric contents and esophagus were collected.For gastric contents,pH value was tested by pH-meter,contents of total bile acid,pepsin and pepsinogen were tested by Elisa.For the sample of esophagus,mRNA of CaSR、NLRP3、NLRC4、NLRP1A and AIM were measured by Real-time PCR;the expression of CaSR、NLRP3、Caspase-1、ASC and IL-1β was measured by Western blot;the distribution of CaSR、NLRP3 in esophageal epithelium were observed through enzyme linked immmunohistochemistry and the mean optical density value were counted;the co-localization of NLRP3-ASC was observed in esophageal epithelium through fluorescence immunohistochemistry.Results1.General situation of ratsUnanticipated death of rats post operatively achieved 46,survival rate was 57.5%,while the survival rate was 100%in sham group.2.Weight and food-intakeCompared with sham group,weight and food-intake in RE group was significantly increased(P<0.05),while weight in RES group was decreased(P<0.01).In addition,weight and food-intake in O group was significantly lower than other medication group(P<0.01).3.Water-intakeCompared with sham group,only RE group was significantly decreased(P<0.05).Compared with RES group,TH group was significantly increased(P<0.01),while TM(P<0.05)and O group(P<0.01)was significantly decreased.4.Autonomous activity capacityCompared with sham group,central area distance,total distance in RES group were significantly decreased(P<0.01),central area duration in RES group was decreased(P<0.01).Compared with RE group,central area distance(P<0.05)and frequence(P<0.01)in RES group were significantly decreased.Compared with RES group,central area distance,central area duration and frequence were significantly increased(P<0.01,P<0.05).5.Gastric fluid analysisCompared with sham group,pH value and total bile acid in both RE and RES group were significantly increased(P<0.01,P<0.05),the ratio of pepsin and pepsinogen in RES group was increased(P<0.05).Compared with RES group,pH value and total bile acid in both TH and O group were significantly decreased(P<0.01,P<0.05),while the ratio of pepsin and pepsinogen in TM,O and OT group were significantly increased(P<0.01,P<0.05).6.HE stainingThe morphology of esophageal epithelium was normal,a large amount of inflammatory cells immerged into epithelium was observed in RE group,mucosal hyperplasia and papillary elongation was observed in RES group,accompanied with rupture of basal layer.In the all medication group,the mucosa injury was ameliorated in different degree.7.Real-time PCRCompared with sham group,CaSR mRNA was elevated in RES group(P<0.05),and NLRP3 mRNA was elevated in both RE and RES group(P<0.05).8.Western blotCompared with sham group,the expression of CaSR、NLRP3、ASC and IL-1β in both RE group and RES group were significantly increase(P<0.01),the expression of Caspase-1 in RES group was increased(P<0.01).Compared with RES group,the expression of CaSR and NLRP3 in RE group were significantly decreased(P<0.01),the expression of CaSR、ASC and IL-1β in the all medication group were significantly decreased(P<0.01),the expression of NLRP3 in TM,TN and OT group were decreased(P<0.01,P<0.05),the expression of Caspase-1 in TM and TH groupwere decreased(P<0.01).9.Enzyme-linked immunohistochemistryPositive stain of CaSR and NLRP3 can be found in the basal layer and part ofspinal layer adjacent to basement.Compared with sham group,the mean optical density value of CaSR and NLRP3 in RES group were significantly increased(P<0.01).Compared with RES group,the mean optical density value of CaSR in all medication group was significantly decreased(P<0.01),the mean optical density value of NLRP3 in TL,TM,TH and OT group was significantly decreased(P<0.01).10.Fluorescence immunohistochemistryHomogeneous staining of NLRP3 and ASC,which in the cytoplasm,wereobserved in the whole esophageal epithelium.RE group present continuous yellow particals derived from the superposition of red and green fluorescence along the basal layer;RES group present strongly positive staining of NLRP3 and ASC in the breakage of esophageal epithelium,which with a large amount of yellow particles and sheets.All of medication groups present decreased yellow staining which derived from co-localization in esophageal epithelium.Conclusion 1.The rats suffered from EGDA and tail clamp stimulation can be made as anoptimum animal model used to study RE combined with liver-gastric disharmony syndrome.Tojapride can effectively ameliorate the pathological change in esophageal epithelium of model rats,restore the inflammatory lesion.2.The expression of CaSR、NLRP3、ASC and IL-1βwas elevated in model rats,Tojapride can inhibit those protein expression,especially the TH group.Part Two In VitroTo investigate the influence of Tojapride on the acidic bile salts impaired HET-1A cells,and to further explore the underlying molecule mechanism of this effect via CaSR mediated NLRP3 inflammation activation pathway.Methods1.Using acidic medium with various pH value(pH2、pH3、pH4、pH5 and pH6),and bile salts mixture with various concentration(200μmol/L、400μmol/L and 800μmol/L)to irritate HET-1A cell,three times per day(8:30AM,11:30AM and 14:30PM),12 minutes every time.The cell viability in first,third,fifth and seventh day were detected through CCK8.2.Grouping:①Normal group,LPS stimulated group,acid stimulated group,bile salts stimulated group and acidic bile salts stimulated group.②Normal group,acidic bile salts stimulated group,Tojapride-L,Tojapride-M,Tojapride-H and MCC950 group.The cell viability of the cells incubated with various concentration of Tojapride serum were detected through CCK8;the transepithelial electrical resistance(TEER)was measured by Ussing Chamber;the rate of LDH release was detected by LDH detection kit;the contents of ILβ、IL-18 release were detected by Elisa.3.The expression of CaSR and NLRP3 were detected by Western blot.CaSR was blocked by Calhe231,which is a CaSR specific inhibitor,and the expression of CaSR and NLRP3 in the cells stimulated with acidic bile salts were also detected by Western blot.The activity of Caspase-1 was detected by YVAD-AFC substrate coloration;mitochondrial membrane potential(△Ψm)was detected by JC-1;oxidative stress level of mitochondrial was detected by MitoSOX fluorescence probe.Result1.Establishment of acidic bile salts induced HET-1A cell injury①Compared with normal group,the cell viability of acidic stimulated cells were significantly decreased(P<0.01).Within the stimulated groups,the cell viability in pH5,pH6 group were higher than pH2,pH3,pH4 group(P<0.01).In time-effect relationship,the cell viability in normal group was gradually increase and peaked at the seventh day;the cells viability in pH2,pH3 and pH4 group were lower than pH5,pH6 group(P<0.01),until the fifth day,the cell viability were sharply decreased.②Compared with normal group,the cell viability of bile salts mixture stimulated cells were significantly decreased(P<0.01).Within the stimulated groups,the cell viability in 800mol/L group were significantly lower than 200μmol/L,400μmol/L group.In time-effect relationship,the cell viability in 200μmol/L,400μmol/L group has no statistical difference with normal group,untile the fifth day,the cell viability were sharply decreased.2.Selection of Tojapride serum concentration2.1 Blank serumThe cell viability of 5%,10%and 15%serum in the medium were higher than 20%,25%serum(P<0.01),and the cell viability of 5%serum in the medium was higher than 10%,15%serum(P<0.01).2.2 Low dose of Tojapride serumThe cell viability of 5%,10%drug serum in the medium were higher than 15%,20%and 25%drug serum(P<0.01).2.3 Medial dose of Tojapride serumThe cell viability of 5%,10%drug serum in the medium were higher than 15%,20%and 25%drug serum(P<0.01).2.4 High dose of Tojapride serumThe cell viability of 5%,10%drug serum in the medium were higher than 20%and 25%drug serum(P<0.01),15%drug serum was higher than 25%drug serum(P<0.05).3.TEER①Compared with normal group,the TEER of all stimulated groups were significantly decreased(P<0.01),and acidic bile salts stimulated group was lower than bile salts stimulated group(P<0.05).②Compared with normal group,TEER in the rest groups were all decreased(P<0.05).Compared with acidic bile salts stimulated group,the TEER of positive control and Tojaprode-L group were significantly increased(P<0.05).4.LDH release rate①Compared with normal group,the LDH release rate of all stimulated group were significantly increased(P<0.01),and LPS,acidic bile salts and acid stimulated group were higher than bile salts group(P<0.01).②Compared with normal group,the LDH release rate in the rest groups were significantly increased(P<0.01).Compared with acidic bile salts stimulated group,the LDH release rate of all medication group were significantly decreased(P<0.01,P<0.05)5.IL-1β、IL-18 release①Compared with normal group,IL-1β、IL-18 release in all stimulated group were significantly increased(P<0.01).②Compared with normal group,IL-1β、IL-18 release in the rest groups were significantly increased(P<0.01).Compared with acidic bile salts stimulated group,IL-1β、IL-18 release in all medication group were significantly decreased(P<0.01,P<0.05).6.Western blot①Compared with normal group,the expression of CaSR,NLRP3 in LPS,bile salts and acidic bile salts stimulated group were significantly increased(P<0.01,P<0.05).Within the stimulated groups,the expression of NLRP3 in acid,bile salts and acidic bile salts stimulated group were lower than LPS stimulated group(P<0.01,P<0.05).②Compared with normal group,the expression of CaSR,NLRP3 in acidic bile salts stimulated group were significantly increased(P<0.05).Pre-treated with Calhex231,the expression of CaSR,NLRP3 were both decreased in those stimulated cells(P<0.05).③Compared with normal group,the expression of CaSR,NLRP3 in acidic bile salts stimulated group were significantly increased(P<0.05).Compared with acidic bile salts stimulated group,the expression of CaSR in Tojapride-L was significantly decreased(P<0.05),the expression of NLRP3 in positive control and Tojapride-L groups were significantly decreased(P<0.01,P<0.05).Within the medication groups,the expression of CaSR in Tojapride-M and Tojapride-H were higher than Tojapride-L.7.Caspase-1 activityCompared with normal group,Caspase-1 activity of acidic bile salts stimulated group was significantly increased(P<0.01).Compared with acidic bile salts stimulated group.Caspase-1 activity in positive control,Tojapride-L and Tojapride-H were significantly decreased(P<0.01,P<0.05).8.ATmCompared with normal group,△Ψm of acidic bile salts stimulated group and all medication group were significantly decreased(P<0.01).Compared with acidic bile salts stimulated group,△Ψfm in positive control and Tojapride-L were significantly increased(P<0.05).9.Oxidative stress levelCompared with normal group,superoxide level in acidic bile salts stimulated group and all medication group were significantly decreased(P<0.01).Compared with acidic bile salts stimulated group,superoxide level in the all medication groups were significantly increased(P<0.01,P<0.05),especially for Tojapride-L group(P<0.01).Conclusion1.Acidic bile salts which involve acidic medium with pH5 and 400μmol/L bile salts mixture and interrupted stimulate the HET-1A cells can establish optimum cell injury model.2,LPS,acid,bile salts and acidic bile salts can effectively decrease TEER of HET-1A monolayer cell,increase the release of LDH and cytokines IL-1β IL-18.The toxic effect of acid,bile salts and acidc bile salts were parallel.Tojapride serum can antagonize acidic bile salts induced cell injury,especially for the low dose ofTojapride.3.Tojapride serum can effectively inhibit acidic bile salts induced elevated NLRP3 expression,Caspase-1 activity,lower △Ψm and mitochondrial oxidative stress,especially for the low dose of Tojapride. |
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