| Acute pancreatitis(AP)refers to a disease in which pancreatic enzymes are activated in the pancreas due to a variety of causes,which cause the pancreatic tissue to digest,edema,hemorrhage and even necrosis.It is one of the main reasons for hospitalization due to gastrointestinal diseases.Among them,20% will progress to severe acute pancreatitis(SAP),and the risk of death caused by local or systemic complications and invasive intervention is as high as 30%.Persistent organ failure is the main determinant of the severity of SAP.Since the intestine is close to the pancreas,it is susceptible to inflammation and is closely related to the progression of the disease.Severe intestinal injury can easily cause a "second blow" and cause SAP to worsen.Therefore,the control of SAP-related intestinal injury may be a key goal for effective treatment of SAP.Guanylate cyclase C(GC-C)is a transmembrane receptor located on the membrane of intestinal epithelial cells.It is activated by endogenous ligands to cause intracellular cyclic guanosinc monophosphate(c GMP)increase and exert biological effects.GC-C plays an important role in regulating water and electrolyte balance,maintaining the integrity of the intestinal barrier,controlling inflammation,relieving abdominal pain and regulating intestinal microecology,and has recently been regarded as a potential therapeutic target for intestinal diseases.Linaclotide,a GC-C agonist,is an endogenous ligand analog synthesized from amino acids.It has the characteristics of high affinity,weakened PH dependence and local action on the intestine,and has made many progresses in the treatment of digestive diseases.At present,it is not clear whether GC-C changes in SAP-related intestinal injury and its mechanism of action,and whether Linaclotide has a mitigating effect on SAP-related intestinal injury,and there is a lack of relevant research.Based on this,this project intends to observe the change rule and role of GC-C in SAP-related intestinal injury and explore whether activation of GCC through Linaclotide can effectively alleviate the severity of SAP-related intestinal injury,and hope that this can provides a theoretical basis for new targets of SAP treatment.Part 1: Experimental study of GC-C’s changing law and function in SAP-related intestinal injury.Objective: To explore the change rule and role of GC-C in SAP-related intestinal injury.Methods: 36 adult male SD rats were divided into SO group(n=18,only turning the pancreas several times after opening the abdomen)and SAP group(n=18,5% sodium taurocholate(0.1m L/100 g)was retrogradely pumped into the pancreaticobiliary duct after laparotomy to prepare the SAP model).Each group was randomly divided into 12 h group,24 h group and 48 h group according to the medication time(n=6).Rats were killed in batches at12 h,24 h,and 48 h after modeling,and serum,pancreas and colon tissues were collected.HE staining was used to evaluate the pathological changes of the pancreas and colon;the concentration of AMY,DAO,D-Lac and TNF-α in serum was determined by ELISA;The expression of GC-C and Claudin-1 in colon tissue was detected by RT-q PCR,western blotting and immunohistochemical staining.The time point with the lowest expression of GC-C was selected as the approximate time point of the most serious intestinal injury,that is,as the intervention time point for follow-up experiments.Results: The expression of GC-C in the colon of the SAP group was significantly reduced,and the expression was the lowest at 12 h after modeling(P<0.05),but GC-C gradually increased over time.Claudin-1 showed a similar trend with GC-C in the colon.Compared with the SO group,the SAP group’s colon showed varying degrees of mucosal discontinuity,interstitial edema and inflammatory cell infiltration,and pathological scores increased(P<0.05);serum AMY,DAO,D-Lac and TNF-α both were significantly increased(P<0.05);but the expression of GC-C and Claudin-1 in the colon decreased.Conclusion: GC-C showed a trend of first decline and then rise in SAP-related intestinal injury,and its expression was lowest in the SAP-12 h group,so 12 h after modeling was used as the follow-up intervention time point.The expression of Claudin-1 in the intestine is similar to GC-C,suggesting that GC-C may maintain the integrity of the intestinal barrier function by effecting the expression of tight junction proteins.Part 2: Experimental study on the best times of Linaclotide intervention in SAPrelated intestinal injury.Objective: Clarify the number of times that Linaclotide has the best effect after administration.Methods: 30 adult male SD rats were randomly divided into SO group,SAP group and Linaclotide group.According to Linaclotide’s half-life of 6 h,it was again divided into one intervention group(L1),two intervention group(L2)and three intervention group(L3)(n=6).The modeling method of SO group and SAP group was the same as before;Linaclotide group was given intragastric Linaclotide solution(10 μg/kg/d)immediately after SAP induction.The L1 group was given an intragastric administration immediately after the model was made,the L2 group was given an intragastric administration at 0 h and 6 h after the model was made,and the L3 group was given an intragastric administration at 0 h,4 h,and 8 h after the model was made.Rats were sacrificed 12 h after modeling,colon tissues were collected,and the expression of GC-C in the colon was detected by RT-q PCR and western blotting.Results: The expression of GC-C in the SAP group was significantly lower than that in the SO group and Linaclotide group.Compared with the SAP group,the expression of GC-C in the Linaclotide group was increased,and the expression in the L2 group was significantly higher than that in the L1 and L3 groups(P<0.05).Conclusion: The expression of GC-C increased most significantly in the L2 group,indicating that Linaclotide has the best effect of interfering with intestinal injury in SAP rats for 2 times.Part 3: Experimental study of Linaclotide to relieve SAP-related intestinal injury.Objective: To explore the curative effect evaluation and mechanism of Linaclotide in relieving the inflammatory response and tissue damage in SAP-related intestinal injury.Methods: 18 male SD rats were randomly divided into SO group,SAP group and Linaclotide group(n=6).The method of modeling rats in each group was the same as before.Rats were sacrificed 12 h after modeling to collect serum and colon tissue.HE staining was used to evaluate colonic pathological changes;ELISA method was used to measure serum DAO,D-Lac,TNF-α and endotoxin concentrations;RT-q PCR was used to detect the expression of TNF-α and GC-C m RNA in colon tissue.The expressions of GC-C,Claudin-1and JAM-A in colon tissue were detected by western blotting,immunohistochemical staining and transmission electron microscopy.Results: Compared with the SO group,the colon tissues in the SAP group and Linaclotide group showed different degrees of damage,and the pathological scores were increased(P<0.05);serum DAO,D-Lac,TNF-α and endotoxin were all significantly increased High(P<0.05);the expression of TNF-α in the colon increased while the expression of GC-C,Claudin-1,and JAM-A decreased(P<0.05).Compared with the SAP group,the Linaclotide group’s colonic pathology score decreased(P<0.05),the serum levels of the above factors decreased(P<0.05),and the expression levels of GC-C,Claudin-1,and JAM-A in the colon tissue increased(P<0.05),the expression of TNF-α decreased(P<0.05).Conclusion: Linaclotide,a GC-C agonist,not only inhibited the systemic inflammatory response,but also reduced the levels of colon injury factors and pro-inflammatory factors in SAP rats,and up-regulated the expression of GC-C,Claudin-1 and JAM-A in colon tissue,and improved the intestinal barrier function,thereby alleviating the SAP-related intestinal injury. |
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