Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues
Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues
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RNAi 下调 HSP60 表达会增加脂多糖和雨蛙蛋白诱导的大鼠离体胰腺组织损伤
DOI：
10.1007/s12192-010-0207-9
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发表时间：
2010-11-01
影响因子：
3.8
通讯作者：
Chen, Chang-Jie
中科院分区：
文献类型：
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作者：
Li, Yong-Yu;Lu, Shuai;Chen, Chang-Jie
本研究旨在通过应用热休克蛋白60（HSP 60）小干扰RNA（siRNA）抑制HSP 60的表达，探讨HSP 60在胰腺组织中的作用。分离大鼠胰腺，制备胰腺组织切片，培养，并用低浓度和高浓度雨蛙肽（10（-11）和10（-5）mol/L）或脂多糖（LPS，10和20 μg/mL）刺激。在刺激前、刺激后1 h和4 h，测定组织碎片中的活力和胰蛋白酶原激活肽（TAP）水平，并测定培养上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素6（IL-6）的水平。采用实时PCR和蛋白质印迹法检测HSP 60 mRNA和蛋白的表达。在施用siRNA以抑制分离的组织中的HSP 60表达后，测量并比较这些损伤参数。对照组胰腺组织经雨蛙肽或LPS刺激后，存活率不同程度降低，组织和/或培养上清中TAP、TNF-α和IL-6水平显著升高（p < 0.05）。低浓度蛙皮素或LPS刺激1h后，HSP 60 mRNA和蛋白表达适度升高，高浓度毒素刺激后，HSP 60 mRNA和蛋白表达下降。特别是两种毒物刺激4 h后，HSP 60蛋白表达显著降低（p < 0.05）。相反，与对照组相比，其中应用HSP 60 siRNA的组织片段在用相同剂量和相同持续时间的毒物刺激后显示出低得多的组织活力（p < 0.01）和组织或培养上清液中较高水平的TNF-α、IL-6和TAP（p < 0.01）（p < 0.05）。结果表明，雨蛙肽和脂多糖均能引起离体胰腺组织损伤，但其损伤效应与刺激时间和毒物浓度有关。HSP 60 siRNA可降低HSP 60的表达，减轻蛙皮素或LPS对胰腺组织的损伤，提示HSP 60对胰腺组织具有保护作用。
The objective of this study was to investigate the function of heat shock protein 60 (HSP60) on pancreatic tissues by applying HSP60 small interfering RNA (siRNA) to reduce HSP60 expression. Rat pancreas was isolated and pancreatic tissue snips were prepared, cultured, and stimulated with low and high concentrations of cerulein (10(-11) and 10(-5) mol/L) or lipopolysaccharide (LPS, 10 and 20 μg/mL). Before the stimulation and 1 and 4 h after the stimulation, the viability and the level of trypsinogen activation peptide (TAP) in the tissue fragments were determined and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the culture supernatants were measured. Real-time PCR and Western blotting were used to evaluate the HSP60 mRNA and protein expression. After the administration of siRNA to inhibit HSP60 expression in the isolated tissues, these injury parameters were measured and compared. The pancreatic tissues in the control (mock-interfering) group showed a decreased viability to varying degrees after being stimulated with cerulein or LPS, and the levels of TAP, TNF-α, and IL-6 increased significantly (p < 0.05) in the tissues and/or in the culture supernatant. The expressions of HSP60 mRNA and protein were raised moderately after stimulating 1 h with low concentrations of cerulein or LPS, but decreased with high concentrations of the toxicants. In particular, the expression of HSP60 protein was reduced significantly (p < 0.05) when the tissues were stimulated by the two toxicants for 4 h. In contrast, the tissue fragments in which HSP60 siRNA was applied showed much lower tissue viability (p < 0.01) and higher levels of TNF-a, IL-6, and TAP (p < 0.01) in the tissues or culture supernatant after stimulating with the toxicants at the same dose and for the same time duration as compared with those of the control groups (p < 0.05). The results indicated that both cerulein and LPS can induce injuries on isolated pancreatic tissues, but the induction effects are dependent on the duration of the stimulation and on the concentrations of the toxicants. HSP60 siRNA reduces HSP60 expression and worsens the cerulein- or LPS-induced injuries on isolated pancreatic tissues, suggesting that HSP60 has a protective effect on pancreatic tissues against these toxicants.