Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues

Abstract

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.

Pre-inhalation of Hydrogen-rich Gases Protect Against Caerulein-induced Mouse Acute Pancreatitis While Enhance the Pancreatic Hsp60 Protein Expression

“…Studies have shown that the ordered synthesis, transport, modi cation and secretion of pancreatic enzymes are closely related to Hsp60 [3,13]. And there are reports to reveal the protective effects of Hsp60 on AP, that is, water immersion stress induced Hsp60 expression protects against pancreatitis in rats [14], and down-regulation of Hsp60 worsen the injuries of isolated rat pancreatic tissues [5]. However, few studies have observed the protective effect of Hap60 on AP in mice, and the mechanism of Hsp60 in the pathogenesis and protection of AP need further investigation [15].…”

“…Heat shock protein 60 (Hsp60), known as molecular chaperonin, is a member of heat shock protein family which functions in protein folding, transmembrane translocation, assembly, and disassembly. In pancreas, there is evidence that Hsp60 coexists with pancreatic zymogen granules along the exocrine secretion pathway in pancreatic acinar cells [3], and studies suggest that the decreased expression of Hsp60 in pancreas may correlate with the occurrence of AP in rat [4,5]. The protective effect of HSP60 on AP is to be discussed further.…”

Long-Lasting Effect of Infant Rats Endotoxemia on Heat Shock Protein 60 in the Pancreatic Acinar Cells: Involvement of Toll-Like Receptor 4

“…This expression of HSP60 in pancreatic tissues has been decreased with prolonged stimulation with LPS [64]. Ohashi et al [14] claimed that TLR4 mediates HSP60 signaling, as a putative endogenous ligand of the TLR4 complex.…”

HSP60 is a Ubiquitous Player in the Physiological and Pathogenic Interactions between the Chaperoning and the Immune Systems

“…HSP60 is typically a mitochondrial protein, which together with its co-chaperone HSP10 (also designated HSPE1 in humans, and Hsp10 or Cpn10 in prokaryotes), assists protein folding inside the organelle [12][13][14][15]. The presence and integrity of HSP60 in mitochondria are crucial for cell survival as demonstrated by inducing HSP60 knockdown with siRNA oligonucleotides, which leads to apoptosis [12][13][14][15][16][17][18], and by the occurrence of at least two serious human genetic chaperonopathies associated with mutations of the HSP60 gene [19,20].…”

“…Distinctive functional domains in chaperone molecules recognize a polypeptide in need of assistance, build a chaperoning complex, form a network with other chaperoning complexes, or interact with a protein-degrading machine like the ubiquitin-proteasome system. HSP/chaperones also play 'extrachaperoning' roles, namely non-canonical functions, such as participation in IS regulation, cell differentiation, gene expression, DNA replication, signal transduction, programmed cell death, cellular senescence, and carcinogenesis [12][13][14][15][16][17][18].…”