LPS和雨蛙肽诱导大鼠离体胰腺组织损伤并抑制HSP60蛋白的变化

LPS and cerulein induce isolated rat pancreatic tissue injury and inhibit expression of HSP60 protein

目的:观察脂多糖(LPS)、雨蛙肽(cerulein)刺激对大鼠离体胰腺组织热休克蛋白60(HSP60)表达的影响。方法:分离培养大鼠离体胰腺组织,用低、高浓度cerulein(Cer;10-11mol/L,10-5mol/L)或LPS(10 mg/L,20 mg/L)刺激,以生理盐水(NS)作为对照,在刺激前和刺激后1、4h测定胰腺组织的活力(MTT法)、胰蛋白酶原活性肽(TAP)水平(ELISA测定),培养上清液中白介素6(IL-6)水平(ELISA测定);并用real-time PCR和Westernblotting分别测定胰腺组织HSP60 mRNA和蛋白的表达。结果:低、高浓度的cerulein和LPS刺激胰腺组织后1 h,组织的活力与NS对照组比较稍有下降(P>0.05),刺激4h后,组织活力明显降低(P<0.05);组织中TAP水平在高浓度cerulein和低、高浓度的LPS刺激下明显升高;其培养上清液中IL-6水平在刺激后1 h变化不明显,4 h时升高,尤其以高浓度的cerulein或LPS作用显著(P<0.05)。胰腺组织HSP60 mRNA和蛋白的表达量随LPS刺激时间的延长和浓度的升高而降低;而低、高浓度的cerulein刺激HSP60 mRNA的表达增加,但蛋白的表达降低,以高浓度、长时间刺激的作用为明显(P<0.05)。结论:LPS及cerulein对离体胰腺组织具有损伤作用,并呈一定时间及浓度依赖性;同时,胰腺组织HSP60蛋白表达降低,提示HSP60细胞保护作用的减弱可能参与该损伤过程。

AIM ： To investigate the effects of lipopolysaccharides （LPS） or cerulein on the expression of HSP60 in isolated rat pancreatic tissues. METHODS： The tissue of rat pancreas was isolated by surgical operation and prepared into tissue snips. The isolated pancreatic tissue was cultured, and stimulated with low - and high - concentrations of cerulein （Cer, 10-11 mol/L, 10-5 tool/L） or lipopolysaccharides （LPS, 10 mg/L, 20 rag/L）. Normal saline （NS） was used as control reagent. Before stimulation and 1 h or g h after stimulation, the following parameters for evaluating injury were detected： the viability and the level of trypsinogen activation peptide （TAP） in the pancreatic tissues, and the level of interleukin 6 （ IL - 6） in the culture supernatants. Meanwhile, real - time PCR and Western blotting were used to determine the HSP60 expression at mRNA and protein levels respectively. RESULTS： Under the stimulation with LPS or cernlein, the viability of the pancreatic tissues decreased slightly at 1 h and became much lower with prolonged treatment for g h （ P 〈 0. 05 ）. The TAP level in the pancreatic tissues increased obviously at both 1 h and 4 h （ P 〈 0. 05 ） after treatment, except LPS at lower dose. The IL -6 level in the culture supernatant showed no significant change at 1 h stimulation, then increased remarkably at 4 h after stimulation, especially under the conditions of stimulating with LPS or cerulein at higher doses （P 〈 0. 05）. The expression of HSP60 at mRNA and protein levels decreased sharply with the increase in the concentration of LPS and the prolonged stimulation time; In cerulein stimulation group, the expression of HSP60 mRNA showed an obvious increase （P 〈 0. 01 ）, whereas HSP60 protein was reduced remarkably, especially stimulated with cerulein at higher dose and with longer stimulation time （ P 〈 0.05 ）. CONCLUSION： LPS or cernlein induce injuries in isolated pancreatic tissues in dose and time dependent manners. Meanwhile, the protein expression of HSP60 is reduced significantly, indicating that the decrease in the cellular protection of HSP60 may involve in the injury of pancreatic tissues.