The Study Of The Protective Effect Of Carbachol On Intestinal Epithelial Cell In Rats With Gut Ischemia-reperfusion And Oxidant Stress Injury

Objective: 1. To investigate the protective effect of carbachol on IEC against ischemia-reperfusion injury in vivo. 2. To study the protective effect of carbachol on IEC under oxidant injury and its mechanism in vitro.Method: In the study of investigating the protective effect of carbachol on IEC against I/R injury in vivo, a jejunal sac was formed in Wistar rat. The SMA was occluded for 45 minutes followed by 240 minutes of reperfusion. Animals were randomized into three groups: sham operation, I/R + saline injection (I/R+NS)group and I/R +carbachol injection (I/R+Ca)group. Immediately after occluded of SMA blood flow, either carbachol (0.1mg/kg) or same amount of 0.9% saline was injected into the jejunal sac. The pathological injury and Chiu's scores were observed. The activity of DAO and content of TNF-a and MDA and expression of TNF-a mRNA in intestinal mucosa tissue were determined. Mucosal blood flow was measured by laser Doppler. The apoptosis index of IEC was determined with TUNEL staining. Expressions of caspase-3 and bcl-2 in IEC were assessed by immunohistochemistry method. All determinations were done at 0min, 30min, 60min, 120min and 240min after reperfusion. In the study of the protective effects of carbachol on IEC with oxidant stress injury, IEC were cultured with H₂O₂ in vitro, to simulate the oxidative injured by OH Cultured IEC were divided into six groups: control group, H₂O₂ group, carbachol group, a-Bgt (an antagonist of a7 subunit of cholinergic N receptor) group, FITC-a-Bgt group and carbachol+FITC-a-Bgt group. In H₂O₂ group, IEC were cultured with H₂O₂(2.5mmol/L); In carbachol group, IEC were cultured with carbachol(100μmol/L) 30min in advance, then H₂O₂(2.5mmol/L) was given; In a-Bgt group, IEC were cultured with carbachol(100μmol/L) and a-Bgt (30μg/ml) 30min in advance, then H₂O₂(2.5mmol/L) was added; In FITC-a-BGT group, IEC were cultured with FITC-a-Bgt (30μg/ml); In carbachol+FITC-a-Bgt group, IEC were cultured with carbachol (100μmol/L) 30min in advance, then FITC-a-Bgt (30μg/ml) was added; In control group , IEC were cultured with H-DMEM. The IEC viability was observed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazol-ium bromide (MTT) assay. The level of lactate dehydrogenase (LDH) in the culture media, the content of TNF-a, IL-6, MDA and NO in IEC were measured. The expressions of TNF-a mRNA and IL-6 mRNA were determined by RT-QPCR assay. Apoptosis rate of IEC was evaluated by Annexin-V/PI and flow cytometry. Fluorescence intensity was observed with fluorescence microscope. Expression of NF-κB P⁶⁵ was examed by immunohistochemistrymethod.Results: In the study of investigating the protective effect of caracole on IEC against I/R injury in vivo, The pathological changes were alleviated and Chiu's scores was lower in I/R+Ca group than in I/R+NS group (P < 0.01). In I/R+Ca group the activity of DAO in intestinal mucosa and mucosal blood flow increased(P < 0.01); meanwhile the content of TNF-a, MDA and expression of TNF-a mRNA in intestinal mucosa was decreased dramatically compared with those in I/R+NS group(P < 0.01); Apoptosis index of IEC and expressions of caspase-3 were significantly decreased(P < 0.01) , while the expressions of bcl-2 increased dramatically in I/R+Ca group compared with those in I/R+NS group(P < 0.01). In the study of the protective effect of carbachol on IEC under oxidant stress injury, the IEC viability was obviously higher than that of H₂O₂ group(P < 0.01), LDH of caracole group were lower than that of H₂O₂ groups < 0.01). The content of MDA,TNF-a, NO, expressions of TNF-a mRNA, IL-6 mRNA in IEC and apoptosis rate of IEC in I/R+Ca group were obviously lower than those in H₂O₂ group(P < 0.01). In carbachol+FITC-a-Bgt group, the fluorescence intensity was significantly weakened compared with that in FITC-a-Bgt group. There were a few expressions of NF-κB P⁶⁵ in nucleus. In H₂O₂ group, expression of NF-κB P⁶⁵ in nucleus was increased. Expression of NF-κB P⁶⁵ in nucleus ofI/R+Ca group was lower than that of in H₂O₂ group.Conclusion: 1. Carbachol can protect IEC from I/R injury in vivo by decreasing TNF-a production in IEC after I/R, increasing blood flow in intestinal mucosa after I/R, and inhibiting apoptosis of IEC after I/R. 2. Carbachol can protect IEC from oxidative stress injury in vitro by decreasing the production of TNF-a, IL-6 and NO in IEC after oxidative injury. 3. Carbachol protect IEC through a7 subunit of cholinergic N receptor and inhibiting transferring of NF-κB P⁶⁵ from cytoplasm to nucleus of IEC after oxidative stress injury.