ABSTRACT
Background:Inflammation plays an important role in intestinal ischemia-reperfusion injury(IRI),and erythropoietin (EPO)has been reported to have anti-inflammatory activity. Aims:To explore the protective effect of EPO on intestinal IRI and its potential mechanism. Methods:Thirty-two healthy male Sprague-Dawley rats were randomly divided into four groups:sham operation group(sham group),IRI group,EPO group and 740Y-P group. Rats in IRI,EPO and 740Y-P groups were injected intraperitoneally with 0. 9% NaCl,EPO and EPO + 740Y-P,respectively,one hour before the establishment of intestinal IRI model by superior mesenteric artery clamping(45 min)-reperfusion. All rats were sacrificed one hour after reperfusion. Histopathological changes of small intestine were observed;expression of proteins in PI3K/ Akt and NF-κB signaling pathways was measured by Western blotting;expression and secretion of inflammatory cytokines, including interleukin-8(IL-8),tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein-1(MCP-1)were examined by real-time PCR and ELISA. Results:Compared with sham group,the damage score of small intestine,protein expressions of PI3K,p-Akt and p-NF-κB p65,as well as mRNA expressions and serum levels of IL-8,TNF-α and MCP-1 in IRI group were significantly increased(P < 0. 05). EPO pretreatment could ameliorate the histopathological changes of small intestine in IRI model rats,inhibit PI3K/ Akt and NF-κB signaling activation and down-regulate expression and secretion of inflammatory cytokines. When 740Y-P,a PI3K agonist,was used combinedly,the effect exerted by EPO was diminished. Conclusions:EPO pretreatment can protect against intestinal IRI by inhibiting the activation of PI3K/ Akt/NF-κB signaling and the subsequent inflammatory response.
ABSTRACT
Objective To explore the effect of biliary obstruction caused acute acalculous cholecystitis (AAC) on ultrastructure of gallbladder interstitial cells of Cajal (ICCs),and the possible mechanism of impaired contraction of gallbladder smooth muscle. Methods Total 60 healthy adult guinea pigs were in this study. The guinea pigs AAC model was induced by common bile duct ligation (BDL). The guinea pigs were divided into five groups equally,including sham control group (Sham),BDL for 12 hours (BDL-12),24 hours (BDL-24),48 hours (BDL-48) and 72 hours (BDL-72)groups. The gallbladder specimens were collected by the end of study. Gallbladder pathological changes were observed with HE staining under light microscope. Three muscle strips were collected of each gallbladder,fixed in constant temperature water bath with different concentration of eight peptide cholecystokinin agonist (CCK-8,1010 mmol/L,10-9 mmol/L,10-8 mmol/L,10-7 mmol/L and 10-6mmol/L),acetylcholine (Ach,10-8 mmol/L,107 mmol/L,10-6 mmol/L,10-5 mmol/L,10-4 mmol/L)and potassium chloride (KC1) (60 mmol/L). The contraction activity of gallbladder muscle strips was recorded by tonotransducer. The ultrastracture changes of gallbladder ICC in sham,BDL-12 and BDL-72 groups was examined by transmission electron microscopy. Results There was no obvious inflammation in Sham and BDL-12 groups. Compared with sham group,there were significant differences of biology score of gallbladder in BDL-48 and BDL-72 groups (P＜0. 05). After adding CCK-8,Ach and KC1,the contraction amplitude of gallbladder muscle increased in each group,and in dose-dependent manner. Compared with sham group,the effect value of each other groups decreased significantly (P＜0. 05). Compared with sham group,the morphology of ICC changed in BDL-12group,and more obvious in BDL-72 group. Conclusion Biliary obstruction can induce AAC. At the earlier stage of ACC,the impaired contraction of gallbladder smooth muscle present even without gallbladder inflammation occurrence. ICC may play an important role in impaired contraction.
ABSTRACT
BACKGROUND: Apoptosis plays a key role in intestinal mucosal ischemia-reperfusion injury and recovery; meanwhile, effect of shenfu injection on apoptosis of intestinal epithelial cells during intestinal mucosal ischemia-reperfusion injury should be studied further.OBJECTIVE: To investigate the relationship between the apoptosis of intestinal epithelium and characteristics of intestinal mucosal ischemia-reperfusion injury and recovery.DESIGN: Randomized controlled animal experiment.SETTING: Department of General Surgery, Xianning Central Hospital;Department of General Surgery, Renmin Hospital of Wuhan University.MATERIALS: The experiment was carried out in the Central Laboratory,Xianning Central Hospital from March to August 2005. Fifty-four healthy male SD rats weighting 200-250 g were provided by Animal Center of Medical School, Wuhan University.METHODS: The rats were divided randomly into 3 experimental groups:control group (n=6), ischemia-reperfusion group (n=24) and shenfu treatment group (n=24). ① Pentobarbital sodium solution (40 mg/kg) was administrated into the intraperitoneal cavity to induce anaesthesia. Through a midline abdominal incision, the mesenteric blood vessel of a 15-cm segment of mid-intestine was occluded for 60-minute with an atraumatic vascular forceps. The control group underwent the same procedure except for unblocking the mesenteric blood vessel. At the end of 60 minutes ischemia period the forceps was removed to allow reperfusion, the abdominal cavity was closed. ShenFu injection (8 mL/kg ·h, 20 mL/kg ·d, produced from Yaan Three-Nine Pharmaceuticals Co, No: 030302) was injected 30 minutes before occlusion in SF treatment group, same quantity of 0.9% natrii chloride was injected in control group and ischemia-regeneration group at the same time, and oxygen was inbreathed during the operation and ischemia-regeneration. ② Experimental intestinal canals were sampled for the following analysis when all groups were respectively performed sham ischemia for 1 hour, intestinal ischemia for 1 hour and reperfusion for 1, 24and 72 hours. Sections were observed in light microscope. Histological mucosal damage in each sample was evaluated as followed scoring system: 0score, normal muscosal villi and gland; 1 score, slight lesion near the tip of the villi; 2 scores, slight lesion of subepithelial gland; 3 scores, development of subepithelial (Gruenhagen) spaces near the tip of the villi with capillary congestion; 4 scores, extension of the subepithelial space with moderate epithelial lifting from the lamina propria; 5 scores, a few denuded villous tips; 6 scores, massive denuded villi; 7 scores, denuded villi with exposed lamina propria and obvious gland lesion; 8 scores, disintegrateon of the lamina propria; 9 scores, haemorrhage and ulceration. ③ The Tunel method (TdT mediated biotin-dUTP nick and labeling; TdT-Frag EL DNA fragmentation detection kit) was used. Inbrief, this method allowed the identification of apoptosis nuclei in tissue samples through DNA fragment and labeling. Apoptosis Index (AI) was set as the average number of apoptosis cells in per 100 cells by observing ten high power fields of adjacent villi and crypts. ④ The mitotic phase of crypt epithelial nucleus within intestinal mucosa was observed in intestinal sections stained with haematoxylin and eosin. The number of cells with nucleus mitotic phase was counted in ten adjacent mucosal crypts, which was taken as the index of mitotic activity of intestinal mucosal epithelial cell.MAIN OUTCOME MEASURES: Intestinal mucosal histopathological changes, apoptosis of intestinal mucosal epithelial cell and mitotic activity of intestinal mucosal crypt.RESULTS: All 54 rats were involved in the final analysis. ①) Scores of histopathological changes were (0.65 ±0.35) points in 1-hour ischemia group, (3.87±0.86) points in 1-hour reperfusion group and (0.65±0.35)points in 24-hour reperfusion group; which were lower than those in ischemia-reperfusion group [(7.11±1.01), (8.05±1.34), (1.53±0.48) points; P＜ 0.05]. ② Indexes of apoptosis were 17.24±7.05 in 1-hour ischemia group, 24.20±9.87 in 1-hour reperfusion group, 11.49±4.71 in 24-hour reperfusion group and 6.02±2.16 in 72-hour reperfusion; which were lower than those in ischemia-reperfusion group (51.09±13.76, 54.89±15.58,23.54±9.64, 12.47±5.52; P ＜ 0.05). Activities of mitosis were 10.37±2.03and 11.72±2.07 in 1-hour ischemia group and 1-hour reperfusion group,respectively; which was higher than those in ischemia-reperfusion group(8.24±1.69, 9.95±1.93; P ＜ 0.05).CONCLUSION: Shenfu injection can significantly attenuate apoptosis of intestinal epithelium, increase crypt mitotic activity, and promote intestinal epithelium regeneration or repair.