Reg蛋白与重症急性胰腺炎肠黏膜损伤的关系及其作用机制

Relationship between Reg proteins and intestinal mucosa barrier damage in rats with severe acute pancreatitis

目的:研究胰岛再生源蛋白(regenerating isletderived protein,Reg)Ⅰ、Ⅲ在大鼠重症急性胰腺炎(severe acute pancreatitis,SAP)小肠中表达,评价RegⅠ、Ⅲ水平与肠黏膜屏障损伤的关系,并初步探其作用机制.方法:72只SD大鼠随机分为对照组(N)、重症急性胰腺组(S)、10 mg/kg吡咯烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate,PDTC)预处理组(P)各为12 h及24 h两组.S组腹腔注射20%L-精氨酸2.5 g/kg大鼠体质量2次,中间间隔1 h;N组于腹腔注射等体积0.9%氯化钠;P组:于造模前1 h腹腔注射PDTC 10 mg/kg预处理.HE染色观察胰腺、小肠的病理变化,ELISA方法检测血清中白介素22(interleukin22,IL-22)、肿瘤坏死因子α(tumor necrosis f a c t o r-α,T N F-α)及肠型脂肪酸结合蛋白(intestinal fatty acid binding protein,I-FABP)水平,RT-PCR测定小肠组织中RegⅠ、ⅢmRNA表达含量,Western blot检测小肠组织中核转录因子κB(nuclear-factorκB,NF-κB)p65及RegⅠ、Ⅲ蛋白水平.结果:(1)SAP组在胰腺评分(S12 h 8.92±1.130,S24 h 11.31±1.609)、肠道评分(S12 h3.79±0.689,S24 h 4.33±0.354)、IL-22(S12h 712.46 ng/mL±81.549 ng/mL,S24 h 751.02ng/mL±104.054 ng/mL)、TNF-α(S12 h 138.08ng/mL±20.369 ng/mL,S24 h 159.43 ng/mL±24.46 ng/mL)、I-FABP(S12 h 338.04 IU/mL±61.876 IU/mL,S24 h 395.26 IU/mL±58.547IU/mL)、RegⅠ蛋白(S12 h 0.45±0.047,S24 h0.56±0.033)、RegⅢ蛋白(S12 h 0.70±0.084,S24 h 0.92±0.163)、NF-κB p65蛋白(S12 h0.51±0.065,S24 h 0.60±0.066)水平较对照组均有明显升高(P<0.05);(2)应用PDTC预处理后各指标较SAP组均有表达降低(P<0.05),但仍高于对照组(P<0.05);(3)RegⅠ、Ⅲ蛋白表达与肠黏膜病理评评分、IL-22、I-FABP、TNF-α及NF-κBp65表达呈正相关.结论:SAP时大鼠小肠组织RegⅠ、Ⅲ表达上调,与肠黏膜损伤及NF-κB通路活性相关.

AIM： To detect the expression of regenerating islet-derived proteins（Reg）Ⅰand Ⅲ in the intestinal mucosa of rats with severe acute pancreatitis（SAP）, and to evaluate the relationship between the levels of RegⅠ and Ⅲ and intestinal mucosal barrier damage.METHODS： Seventy-two adult SD rats were randomly divided into three groups： a normal control（N） group, an SAP（S） group, and a pyrrolidine dithiocarbamate（PDTC, 10 mg/kg） pretreatment（P） group. Each group wasfurther divided into two subgroups for testing at different time points（12 and 24 h）, with 12 rats in each subgroup. The rats in the S group were given 20% L-arginine（L-Arg, 2.5 g/kg） by intraperitoneal injection twice at one-hour interval to induce SAP. The N group was given equal volume of normal saline. The P group was given PDTC 10 mg/kg by intraperitoneal injection 1 h before the first injection of L-Arg. All rats were killed 12 h or 24 h after L-Arg injection to collect blood, pancreatic and intestinal tissue samples. The pathological changes in pancreatic and intestinal tissues were observed and graded under an optical microscope. ELISA was used to detect the levels of serum interleukin 22（IL-22）, tumor necrosis factor-α（TNF-α） and intestinal fatty acid binding protein（I-FABP）. The expression of RegⅠ and Ⅲ mRNAs in intestinal tissue was evaluated by RT-PCR. The levels of RegⅠ, Ⅲ and nuclearfactor κB（NF-κB） proteins in intestinal tissue were detected by Western blot.RESULTS： In the SAP group, the scores of pancreatic changes（12 h： 8.92 ± 1.130; 24 h： 11.31 ± 1.609） and intestinal mucosal changes（12 h： 3.79 ± 0.689, 24 h： 4.33 ± 0.354）, and the levels of IL-22（12 h： 712.46 ng/mL ± 81.549 ng/mL, 24 h： 751.02 ng/mL ± 104.054 ng/mL）, TNF-α（12 h： 138.08 ng/mL ± 20.369 ng/mL, 24 h： 159.43 ng/mL ± 24.46 ng/mL）, I-FABP（12 h： 338.04 IU/mL ± 61.876 IU/mL, 24 h： 395.26 IU/mL ± 58.547 IU/mL）, intestinal NF-κB p65（12 h： 0.51 ± 0.065, 24 h： 0.60 ± 0.066）, RegⅠprotein（12 h： 0.45 ± 0.047, 24 h： 0.56 ± 0.033）, and Reg Ⅲ protein（12 h： 0.70 ± 0.084, 24 h： 0.92 ± 0.163） were significantly higher（P 0.05） than those in the control group. Compared with the S group, pretreatment with different doses of PDTC significantly decreased the above parameters（P 0.05）, although the levels of these parameters were still significantly higher than those in the N group（P 0.05）. There were positive correlations among RegⅠ and Ⅲ protein expression, intestinal mucosal pathological score, IL-22,I-FABP, TNF-α, and NF-κB p65 expression.CONCLUSION： RegⅠ and Ⅲ protein expression is upregulated in SAP, which is possibly associated with intestinal mucosa damage and NF-κB signaling pathway activation.