Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues.
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.
Li
Yong-Yu
YY
Institute of Digestive Diseases, Department of Pathophysiology, Tongji University School of Medicine, 1239 Si Ping Road, Shanghai, 200092, China. liyongyu@tongji.edu.cn
Lu
Shuai
S
Li
Kun
K
Feng
Jia-Yan
JY
Li
Yan-Na
YN
Gao
Zhi-Rong
ZR
Chen
Chang-Jie
CJ
D000818:Animals; D002478:Cells, Cultured; D002108:Ceruletide / Q000633:toxicity*; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity*; D009842:Oligopeptides / Q000378:metabolism; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D018834:Chaperonin 60; D015850:Interleukin-6; D008070:Lipopolysaccharides; D009842:Oligopeptides; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C080396:trypsinogen activation peptide; D002108:Ceruletide