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Proinflammatory Effects and Mechanisms of Extracellular HSP60 in Cardiac Myocytes

Author: LiuXueMei
Tutor: YuanWenJun；LinLi
School: Ningxia Medical University
Course: Physiology
Keywords: Heat shock protein 60 Myocardial cells Inflammatory cytokines toll-like receptor
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract

Heat shock protein 60 (heat shock protein 60, HSP60) located within the cell under physiological conditions, is indispensable to maintain the normal function of cells and cellular stress response has a protective effect. In cells subjected to ischemia and other stimulating factors, HSP60 can \Studies have shown that extracellular HSP60 can activate immune cell membrane toll like receptors (toll-like recepors, TLRs) -4 and cause inflammation. Myocardial cells, the TLRs distribution, but whether extracellular HSP60 may activate cardiac myocyte TLRs and direct cause of the inflammatory response is not clear. The subject in the digestion and separation of adult rat cardiomyocytes cultured rat H9C2 cardiac cell model to study the role of exogenous H9C2 to stimulate the expression of inflammatory cytokines, and to explore its mechanism of action. Objective: To identify the receptor and its signaling pathway mediated the extracellular HSP60 direct stimulation of the inflammatory cytokine expression, and explore the myocardial cells TLRs. Under pathophysiological conditions such as myocardial ischemia, HSP60 abnormal exocytosis, endogenous factor may become HSP60 cause aseptic inflammation, this research will help the understanding of the ischemic myocardium aseptic inflammation reaction mechanism clues. Methods: 1, by ligation adult male SD rats left anterior descending artery (left anterior descending coronary artery, LAD), cardiac ischemia model. Take the sham or coronary ligation-induced myocardial ischemia in the rat heart, the left ventricle of the isolated heart Langendorff system retrograde perfusion, collagenase digestion of isolated cardiac myocytes. To given the HSP60 stimulus, by real-time quantitative PCR assay TNF, IL-6 mRNA expression amount TLR2/TLR4 change; ELISA assay in the cell culture supernatant of inflammatory cytokines TNF, IL-6 content; colorimetric detection LDH activity in the cell culture supernatant myocardial cell injury. 2, according to the conventional culture H9C2 rat myocardial cells Department of given HSP60 stimulus, by real-time quantitative PCR assay TNF-а, IL-6, TLR2, of TLR4 expression levels change; ELISA assay inflammation in the cell culture supernatant factor TNF-а, IL-6 content; western blot assay TLR2 and TLR4 receptor protein expression. Application of TLR2/TLR4 specific antibody or transfected with siRNA to block the TLR2/TLR4, observe how it affects HSP60 proinflammatory effects. And HSP60 detected by immunofluorescence whether caused P65 occurrence nuclear translocation, in order to determine the HSP60 whether activation of the proinflammatory transcription factor NF-kB. Results the outside of endogenous HSP60 incubation enzymatically isolated adult rat cardiac myocytes, inflammatory cytokines TNF-a and IL-6 expression and release were significantly up-regulated. Given HSP60 1ug/ml incubated for 3h sham group myocardial cell TNF-а and expression levels of IL-6mRNA were raised to 1.76 ± 0.13 and 1.89 ± 0.09-fold; LAD ligation group myocardial cells TNF-а and the expression of IL-6mRNA respectively the raised 3.07 ± 0.11 and 3.35 ± 0.18-fold. Given HSP60 5ug/ml incubated for 3h sham group myocardial cell TNF-а and expression levels of IL-6mRNA were raised to 2.04 ± 0.12 and 2.46 ± 0.13-fold; TNF-а and the amount of expression of IL-6mRNA LAD ligation group were raised by 3.97 ± 0.13 and 4.28 ± 0.17; expression of HSP60 caused TNF-а and IL-6mRNA,, raised were statistically significant. , HSP60 and cause a significant increase in TNF-а and IL-6 release. HSP60 1ug/ml incubated for 3h, TNF-а and IL-6 release in the sham group were 84.5 ± 6.8 and 72.7 ± 3.7 pg / ml, significantly higher than the non-administered group (63.1 ± 5.2 and 53.6 ± 4.5pg/ml P lt; 0.05); LAD ligation group TNF-а and IL-6 release were 98.6 ± 7.9 and 90.6 ± 7.4pg/ml, significantly high non-administered group (90.8 ± 6.5 and 78.6 ± 4.4pg/ml P lt; 0.05). Sham group TNF-а and IL-6 release further increased when given high doses of HSP60 (5ug/ml), 112.7 ± 6.2 and 95.7 ± 4.7pg/ml LAD ligation group TNF-а and IL-6 The release also further increased, respectively, 137.6 ± 8.0 and 132.4 ± 6.8pg/ml. 2 derived HSP60 incubated cultured H9C2 myocardial cells, cytokines TNF-а and IL-6 expression and release outside also significantly increased. The HSP60 1ug/ml treated inflammatory cytokines TNF-а and IL-6mRNA expression levels were increased 1.42 ± 0.06 and 1.69 ± 0.11-fold; of HSP60 5ug/ml treated TNF-а and IL-6mRNA expression levels were raised to 1.87 ± 0.13 and 2.25 ± 0.12-fold compared with the non-administration groups were statistically significant (P lt; 0.05). The TLR2 and TLR4mRNA expression significantly up-regulated, there is a statistically significant difference. Western blot analysis showed that treatment of TLR4 protein expression in HSP60 3h significantly elevated best is still high, 24h, 12h reached. In addition, the results of LDH activity was detected in the cell culture supernatant LDH released in 24h HSP60 treatment showed an increasing trend, there is a significant difference (P lt; 0.05). Blocking TLR4 receptor TLR4-specific antibodies, the H9C2 cell culture supernatant of TNF and IL-6 release were 25.8 ± 4.9 and 33.9 ± 4.5pg/ml, both significantly lower than HSP60 release of the treatment group (54.1 ± 4.7 and 60.7 ± 4.1pg/ml P lt; 0.05). The results suggest that TLR4 participate in mediated proinflammatory effect of HSP60. 4, observed in cultured H9C2 myocardial cells, cell immunofluorescence method inflammatory transcription factor NF-кB subunit P65 exogenous HSP60 translocation from the cytoplasm into the nucleus (NF-кB TLRs receptor downstream key signal molecules). Showed that HSP60 activation of NF-кB. Conclusion 1, extracellular HSP60 can directly induce myocardial cells to produce inflammatory cytokines TNF-a and IL-6. 2, HSP60 caused upregulation of TLR2 and TLR4 receptors. 3, TLR4 antibody can effectively inhibit HSP60 proinflammatory effect, suggesting that TLR4 involved in mediating the proinflammatory effect of HSP60. TLR4 may be used as the receptor and HSP60 combination, and thus the role of HSP60 mediated directly; may also be raised by its expression of HSP60 indirectly mediated role. 4, HSP60 cause myocardial cell P65 nuclear translocation, indicating that HSP60 activation of the inflammatory transcription factor NF-кB, prompted TLR4/NF-кB pathway is involved in mediated HSP60 proinflammatory effect.

Proinflammatory Effects and Mechanisms of Extracellular HSP60 in Cardiac Myocytes

Abstract

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