| Heat shock protein 60 (HSP60) locates inside of cells and maintains their normal functions under physiological condition.HSP60 plays a role in protecting cells in stress. However, when cells are in an ischemic stress, HSP60 is released from cells in an autocrine manner or by injured cells.Extracellular HSP60 is reported to activate toll-like receptor-4 (TLR4) of cell membrane, leading to inflammatory response. Besides immunocytes, TLRs also locate in cardiac myocytes. Whether the extracellular HSP60 directly activates TLRs leading to inflammatory response in cardiac myocytes remains unknown.Objective:Inflammatory effects and mechanisms of HSP60 were studied isolated rat cardiac myocytes and H9C2 cell line for the purpose of providing clues for understanding the mechanism of noninfectious inflammatory reaction caused by ischemic heart diseases.Methods:1. A myocardial ischemia model was established by ligating the left anterior decending coronary artery (LAD) of adult male SD rats. Single rat cardiac myocytes were acquired by perfusing the isolated heart of LAD-ligation rats and sham rats with collagen solution using a Langendorff apparatus. Cells were treated with HSP60 to determine the expression level of TNF-α, IL-6, TLR2, TLR4 mRNA by quantitative real-time PCR, the content of inflammatory factors in the supernatant was determined by ELISA. LDH activity was determined to analyze the extent of injury to cardiac myocytes.2. H9C2 cell line was incubated in a normal condition and treated with HSP60. Expression levels of TNF-α, IL-6, TLR2, TLR4 mRNA determined by quantitative real-time PCR, and the content of inflammatory factors in supernatant determined by ELISA, expression level of TLR2/TLR4 protein was deternmined by western blot. The inflammatory effect of HSP60 was studied after inhibiting TLR2/TLR4 receptors via TLR2/TLR4 blocking antibodies or RNAi. P65 determined by immunofluorescence assay to analyze whether the NF-КB was activated after HSP60 treatment.Results:1. Cytokine TNF-a and IL-6 were upregulated significantly after cardiac myocytes of adult rats were incubated with HSP60. After incubation with HSP60 at a concentration of 1ug/ml for 3h, the expression of TNF-αand IL-6 mRNA increased by 1.76±0.13 and 1.89±0.09 fold in sham operation rats, and by 3.07±0.11 and 3.35±0.18 fold in LAD-ligation rats respectively. After incubation with HSP60 at a concentration of 5ug/ml for 3h, the expression of TNF-αand IL-6 mRNA increased by 2.04±0.12 and 2.46±0.13 fold in sham operation rats, and by 3.97±0.13 and 4.28±0.17 fold in LAD-ligation rats respectively. After incubation with HSP60 at a concentration of 1ug/ml for 3h, the content of inflammatory factors TNF-αand IL-6 in the supernatant increase by 84.5±6.8 pg/ml and 72.7±3.7 pg/ml respectively versus 63.1±5.2 pg/ml and 53.6±4.5 pg/ml in the untreated controls (P<0.05); by 98.6±7.9 pg/ml and 90.6±7.4 pg/ml in LAD-ligation rats vs 90.8±6.5 pg/ml and 78.6±4.4 pg/ml in untreated control (P<0.05). After treatment with the concentration of 5 ug/ml for 3h, the content of inflammatory factors TNF-αand IL-6 in the supernatant increased progressively, increased by 112.7±6.2 pg/ml and 95.7±4.7 pg/ml the sham rats, and by 137.6±8.0 pg/ml and 132.4±6.8 pg/ml in the LAD-ligation rats respectively.2. After incubation with HSP60, the expression of cytokine TNF-αand IL-6 of H9C2 also increased significantly. With the concentration 1 ug/ml of HSP60, the expression level of TNF-αand IL-6 mRNA increase by 1.42±0.06 and 1.69±0.11 fold and with the concentration 5 ug/ml of HSP60 the expression level of TNF-αand IL-6 mRNA increase by 1.87±0.13 and 2.25±0.12 fold respectively compared with the control group (P<0.05).3. After TLR4 recptors were blocked with specific antibodiesn H9C2 cell line, the content of inflammatory factors TNF-αand IL-6 in the supernatant decreased significantly by 25.8±4.9 pg/ml and 33.9±4.5 pg/ml respectively ,versus 54.1±4.7 pg/ml and 60.7±4.1 pg/ml in the group treat with HSP60 (P<0.05), indicating that TLR4 participated in the inflammatory effect of HSP60.4. Treatment with HSP60 induced translocation of P65 (a subunit of NF-кB) from the cytosol to the nucleus, as detected by immunofluorescence assay. Indicating that HSP60 activated NF-кB.Conclusion:1. Extracellular HSP60 directly induced the synthesis of inflammatory factors TNF-a and IL-6 in cardiac mycytes.2. HSP60 upregulated TLR2/TLR4 receptors.3. The inflammatory effects of HSP60 could be inhibited by TLR4 blocking antibodies, indicating that TLR4 participated in the inflammatory effect of HSP60.We hypothesized that TLR4 binds with HSP60 as a receptor to mediate the inflammatory effects directly or mediate the effect by upregulating the receptor indirectly.4. Treatment with HSP60 induced translocation of P65 (a subunit of NF-кB) from the cytosol to the nucleus, indicating that NF-кB participated in the inflammatory effects of HSP60. |
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