The Level Change Of Growth Factors VEGF, GDF-9 And GDF-15 On The Intestinal Mucosal Barrier In Rats With Severe Acute Pancreatitis

Severe acute pancreatitis(SAP) is a common clinical acute abdomen with an acute onset, complex complications. Bacterial translocation through the damaged intestinal mucosa plays a critical role in the secondary infection. Studies found that, intestinal bacteria and endotoxin were easy to translocate because of the intestinal mucosa barrier dysfunction and the increased intestinal permeability in SAP, which makes sepsis, induce and aggravate the systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction syndrome(MODS),this is the main reason that lead to the death of acute pancreatitis,therefore,the sound barrier function of intestinal mucosa in acute pancreatitis is very important. Studying on the influence factors of the intestinal mucosal barrier function and reducing the intestinal barrier function injury are very helpful for the treatment of pancreatitis. In recent years, the roles of growth factors in the development of SAP attract more and more attentions.Growth factor is a kind of polypeptide that can combine with cell specific membrane receptor with high affinity, which effect the regulation of cell growth and other cell functions. There are many kinds of growth factors, such as epidermal growth factor [transforming growth factor(TGF alpha), epidermal growth factor(EGF)], insulin like growth factors(IGF-I, IGF-II), vascular endothelial cell factor(VEGF)], transforming growth factor beta [(TGF- beta family, activin, bone morphogenetic protein(BMP), growth differentiation factor(GDF)] etc. Searchers pay more and more attentions to the effect of VEGF and GDF in acute pancreatitis, VEGF can increase vascular permeability, promote vascular endothelial cell proliferation, especially,plays the major role in stimulating the growth and pathological angiogenesis.GDF plays a role in apoptosis and anti-tumor in a variety of pathological and physiological conditions, while its effect on the intestinal mucosal barrier of severe acute pancreatitis is unknown.Obeject: This experiment aimed to determine the VEGF, GDF-9and GDF-15 protein and m RNA in the expression of SAP in rats intestine,analyse the level change of growth factors VEGF, GDF-9 and GDF-15 on the intestinal mucosal barrier in rats with severe acute pancreatitis; preliminary study on the meaning of growth factors VEGF, GDF-9 and GDF-15 on the intestinal mucosal barrier in rats with severe acute pancreatitis; through analysis the changes of intestinal VEGF, GDF-9、GDF-15 in the application of NOD signaling pathway inhibitor two methyl benzoyl hydroxyl benzophenone(Caspase-1 inhibitor) after pretreatment.Methods: 60 SD rats were randomly divided into sham operation group(N), acute pancreatitis(SAP) group and acute pancreatitis+Caspase-1 inhibitor(SC) group, each group was divided into 6h and 12 h subgroups, each group involved 10 rats, the method of the pancreatic duct retrograde injection of 5% sodium taurocholate was used to make animal model. And then pancreatic, blood, ileum tissue were got after 6 h and 12 h modeling, respectively.Pathological changes of pancreas; intestinal were observed through HE dyeing, plasma levels of D-lactic acid were detected using the method of ELISA detection for the evaluation of intestinal permeability;VEGF, GDF-9, GDF-15 protein and m RNA expression among the three groups were compared through Western-blot and RT-PCR.Results:1 Pathological evaluation of pancreatic tissue: HE staining, pancreatic tissue and cell morphology approximately normal in rats of group N 6 h, with occasional edema cells in rats of group N 12 h; SAP group showed pancreatic tissue inflammatory reactions, hyperemia and edema, hemorrhage and necrosis, it is more obvious in 12 h group than in 6 h group; the pancreatic lesions of SC group were lighter when compared with SAP group lesions, less inflammatory cell infiltration, hemorrhage and necrosis; Pathology score shows that, the scores of group SAP and group SC were higher(P<0.01) when compared with N group score, the score of group SC was lower when compared with SAP group score, there had significant difference in 6 h subgroup and 12 h subgroup(P<0.01),pancreatic pathological score of 12 h subgroup was significantly higher than 6 h subgroup in SAP group(P< 0.01),the score of 12 h subgroup was significantly higher than 6 h subgroup in SC group(P < 0.05), while no significant difference was observed in N group when compared between 6 h and12 h time points(P > 0.05).2 Intestinal pathology score: in group N, intestinal mucosal structure normal, it has no inflammatory cells infiltration; SAP group showed villous desquamation, epithelial cell layer necrosis and shedding, it has inflammatory cells infiltration; SC group showed less inflammatory cell infiltration, epithelial and villous structure is occasionally not complete; the score of SAP group and SC group was significantly higher when compared with the N group(P < 0.01); the score of SC group was lower when compared with SAP group(P < 0.01); no significant difference was showed between the two time points in N group and SC group(P > 0.05), while statistically significant difference was observed between the two time points in SAP group,the score of 12 h subgroup was significantly higher than 6 h subgroup in SAP group(P< 0.01).3 The level of plasma D-lactat:no significant difference was displayed between the two time points in N group(P > 0.05), plasma D-lactate levels in SAP group or SC group were significantly higher than that in N group at each time point(P < 0.01),while that in SC group is significantly lower when compared with SAP group at each time point(P< 0.01);however, plasma D- lactate levels in12 h subgroups were higher than 6 h subgroups in both SAP group and SC group(P < 0.05).4 The expression of intestinal VEGF: Expression of VEGF protein through Western blot detection, the VEGF protein expressions in SAP group and SC group were significantly higher when compared with that in N group at two time points(P < 0.01), while that in SC group were significantly lower than that in SAP group at two time points(P < 0.01).There were differences between two time points in SAP and SC group(P < 0.05),the expression of 12 h subgroup was significantly higher than 6 h subgroup(P< 0.01), while not in N group(P > 0.05) in terms of VEGF expression.The VEGFm RNA expression through RT-PCR detection, the VEGFm RNA expressions in SAP group group were significantly higher when compared with that in N group at two time points(P< 0.01), while that in SC group were significantly lower than that in SAP group at two time points( P< 0.05),while when compared with N group were significantly higher than 6h subgroup(P < 0.05) and 12 h subgroup(P < 0.01).There were differences between two time points in SC or SAP group,the expression of 12 h subgroup was significantly lower than 6h subgroup(P < 0.05),while not in N group(P > 0.05) in terms of VEGF m RNA expressions.5 The expression of intestinal GDF-15: Expression of GDF-15 protein through Western blot detection,the GDF-15 protein expressions in SAP group and SC group were significantly higher when compared with that in N group at two time points(P < 0.01),There were no differences between two in SAP and SC group, While,there were differences between two time points in SAP and SC group,the expression of 12 h subgroup was significantly lower than 6h subgroup(P < 0.01), while not in N group(P > 0.05) in terms of GDF-15 expression. The GDF-15 m RNA expression through RT-PCR detection, the GDF-15 m RNA expressions in SAP group and SC group were significantly higher when compared with that in N group at two time points(P < 0.01),There were no differences between two in SAP and SC group, While,there were differences between two time points in SAP group,the expression of 12 h subgroup was significantly lower than 6 h subgroup(P < 0.05), while not in N and SC group(P > 0.05) in terms of GDF-15 expression.6 The expression of intestinal GDF-9:Expression of GDF-9 protein through Western blot detection,the GDF-9 protein expressions in SAP group or SC group were significantly higher when compared with that in N group at 6 h and 12 h(P < 0.01),GDF-9 protein expressions at in SC group were significant lower than that in SAP group(P < 0.05),There was difference between two time points in SC and SAP group(P < 0.05) while not in N group(P > 0.05) in terms of GDF-9 expressions. The GDF-9m RNA expression through RT-PCR detection, the GDF-9m RNA expressions in SAP group or SC group were significantly higher when compared with that in N group at 6h and 12 h(P < 0.01), GDF-9m RNA expressions at in SC group were significant lower than that in SAP group(P < 0.05),There was difference between two time points in SC and SAP group(P < 0.01) while not in N group(P > 0.05) in terms of GDF-9m RNA expressions.7 The expression of intestinal VEGF and the level of plasma D-lactat expression were positively correlated.Conclusions:1 VEGF, GDF-15、GDF-9 showed high expression in rat intestinal tissue in SAP, which act as regulation factor for intestinal mucosal barrier.2 NOD signaling pathway inhibitor two methyl benzoyl hydroxyl benzophenone(Caspase-1 inhibitor) can inhibit the NOD pathway activity, reduce pancreatic and intestinal injury, reduce the expression of VEGF and GDF-9 in the intestinal mucosal. Does not affect the expression of GDF- 15.