| Objective: Roux-en-Y gastric bypass is one of the current clinical metabolic surgery,which has a excellent and lasting effect on weight and diabetes control,but its mechanism has not been unified.Studies have shown that gram-negative bacteria in the intestinal produce lipopolysaccharide(LPS),through the intestinal mucosal barrier to produce low-grade systemic inflammatory,leading to obesity,diabetes,hyperlipidemia,hypertension and other metabolic diseases.In this study,the Roux-en-Y gastric bypass model of obese diabetic rats was established to observe the changes of portal vein circulation LPS and inflammatory index and the changes of terminal ileum mucosal mechanical barrier after gastric bypass,and to explore the possible mechanism of gastric bypass.Methods:(1)Obese diabetic rats model: 50 6-week-old Sprague Dawley(SD)rats were reared on high-fat feed for 12 weeks,and a low-dose of streptozotocin(STZ)was injected intraperitoneally at 30 mg/kg.,random blood glucose≥ 16.7mmol/L as the diabetes rats.(2)Surgery pre-experiment: 20 rats in the practice group completed the Roux-en-Y gastric bypass study,observed the effects of anesthesia,operation time,intraoperative bleeding,perioperative fluid replacement and feeding methods on rat survival.Draw the surgical learning curve and summarized the modeling experience.(3)Experimental grouping:Model rats were randomly divided into Bypass group(n=10),Sham group;(n=10),high-fat control group(HFD group;n =5).Bypass group underwent Roux-en-Y gastric Bypass,Sham group underwent exploratory laparotomy and incision and suture of stomach and intestinal wall,the operation time was the same,and ordinary feed was used for postoperative feeding,while high-fat feed was used for continuous feeding in the HFD group.Normal control group(n=5),the rats were fed with ordinary feed continuously,and their body weight and fasting blood glucose were measured to establish the baseline level.(4)General observation items: the baseline level of fasting blood glucose and body weight of the rats were measured before surgery and twice a week after surgery,the curves were drawn for statistical analysis.(5)Portal vein serum indicators and liver histological examination: Intraperitoneal anesthesia with 1% pentobarbital was used preoperative and 12 weeks after surgery.After laparotomy,1.5ml of fasting portal venous blood was extracted from ligamentum hepatoduodenale in each group by fine needle.LPS,lipopolysaccharide binding protein(LBP)and high sensitive C reaction protein(hs-CRP)were determined by ELISA.To evaluate the effect of gastric bypass on portal endotoxemia in obese diabetic rats.Before the rats were sacrificed,liver tissue was taken and HE staining was used to evaluate the improvement of hepatic steatosis.(6)Histological examination of rat ileal mucosa: IHC staining was used to observe the distribution of Occludin and Claudin-1 in the terminal ileum mucosa.WB was used to detect the expression level of Occludin and Claudin-1 proteins in intestinal tissues,and qPCR wasused to detect the relative mRNA content of Occludin and Claudin-1.Transmission electron microscopy(TEM)was used to observe the tight junction between intestinal epithelial cell and organelles,and to observe the effect of gastric bypass on the intestinal mucosal epithelial structure and mechanical barrier.Results:(1)50 high-fat rats were fed for 12 weeks with body weight(651.9±33.0)g,while the Normal group was(500±52.0)g.The weight of the experimental group was 30.4% higher than the Normal group,with a significant statistically difference(P<0.001).Forty-seven SD rats successfully induced were given low-dose STZ 30mg/kg intraperitoneal injection after 12 hours of abstinence from drinking and fasting,and four of them were given a secondary intraperitoneal injection of STZ 20mg/kg.The modeling success rate was 94%.(2)20 rats completed surgical practice and nursing mode exploration,and the survival rate was 35%.The main factors affecting survival included:anaesthesia accident(3 cases),intraoperative bleeding(2 cases),postoperative circulatory failure(6 cases)and anastomotic leakage(2 cases).The overall complication rate was 60%.The main difficulty of modeling is the prevention of intraoperative bleeding and postoperative care,through continuous practice,shorten the operation time,reduce the dosage of anesthesia and the postoperative care is continuously improved.Adopting continuous water supply,early liquid diet and restrictive solid intake,increase the survival rate of rats.In the experimental stage,the overall survival rate reached 90%.(3)There was no significant difference in the preoperative weight and fasting blood glucosebetween the three obese diabetes groups(P>0.05).Both the Bypass group and the Sham group showed a decrease in postoperative body weight,the Sham group increased slowly after 2 weeks,which was consistent with the HFD group.In the Bypass group,the blood glucose level decreased significantly one week after the operation,and basically returned to normal at the fourth week,then showing a persistent and stable level.The blood glucose of the Sham group and the HFD group was maintained at a relatively higher level.Compared with the Sham group,the blood glucose and weight changes of the Bypass group were statistically significant from 2 weeks after the operation(P<0.01).(4)The preoperative portal vein LPS[(1.23±0.46)vs(0.40±0.11)]EU/ml,LBP[(39.48±5.69)vs(19.89±3.27)]ng/ml,and hs-CRP [(5.11±1.29)vs(1.81±0.49)]ng/ml were significantly higher in obese diabetic rats than the Normal group(P<0.001).After 12 weeks,Bypass group showed significant difference in LBP[(25.71±3.56)vs(37.03±1.69)]ng/ml compared with Sham group(P<0.001),hs-CRP[(2.60 ±0.53)vs(3.49±1.28)] ng/ml,LPS[(0.57 ± 0.12)vs(0.64±0.21)] EU/ml,the concentrations were lower than those in the Sham group,but the difference was not statistically significant(P >0.05).The inflammation indexes of the Bypass group and the Sham group decreased to varying degrees,but compared with the Normal group,the Sham group was still significantly increased(P <0.05).Comparing the Bypass group with the HFD group,LPS [(0.57± 0.12)vs(1.24 ± 0.25)] EU/ml,LBP [(25.72±3.56)vs(41.24±7.14)]ng/ml,hs-CRP[(2.59±0.53)vs(5.87±1.01)]ng/ml,concentrationreduction-n had statistical significance(P<0.001).(5)Fatty-induced obese rats have more severe fatty livers,HE staining of the HFD group shows that a large number of hepatocytes have vacuole degeneration,swelling deformation obviously,cytoplasmic dissolve or disappear,net-like or nearly transparent,hepatocyte steatosis.Microscopic morphology of liver tissues in the Bypass group and the Sham group showed significant remission,among which the Bypass group was the highest,and the fatty liver was also significantly improved in the Sham group after long-term ordinary feed.(6)The expression levels of Occludin and Claudin-1 in terminal ileum mucosa were detected by IHC and WB,and the relative contents of Occludin and Claudin-1 mRNA were detected by qPCR.The results showed that the expression levels of Occludin and Claudin-1 were significantly increased in the HFD group(P<0.05),but there was no significant difference between other three groups(P>0.05).TEM observation of terminal ileum mucosa epithelium,all groups were observed the tight junction structure,we found that the HFD group and the Sham group has widespread mitochondria vacuoles,HFD group is more serious,but Bypass group and Normal group can not observe this change,suggesting that intestinal mucosal mitochondria function of obese diabetic rats was impaired.Conclusion:(1)The method of high-fat diet feeding combined with low-dose STZ to construct the model of obese diabetic rats is reliable.(2)Roux-en-Y gastric bypass can significantly improve metabolic inflammation in obese diabetic SD rats,and can treat metabolic diseases such as obesity,diabetes and fatty liver.(3)Mitochondrial function in intestinal mucosa ofobese diabetic rats was impaired,Roux-en-Y gastric bypass may reduce the level of LPS by affecting the intestinal mucosal epithelial barrier function through energy metabolism of the mucosa. |
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