| Endotoxemia is a major consequence of infectious diseases triggered by Gram-positive and/ or -negative organisms, which can proliferate and or release endotoxin and lead to tissue injury or even multiple organ dysfunction syndrome. Until now, sepsis is still a leading cause of death in intensive care unit, however, unlike other major epidemic illnesses, treatment for sepsis is nonspecific, limited primarily to support of organ function and administration of intravenous fluids, antibiotics, and oxygen. Therefore, studies on the pathogenesis of endotoxemia, provide theoretical basis for the treatment and treatment strategies is very important. Lipopolysaccharide (LPS) is a large lipid and polysaccharide structure which is the major component of the outer cell wall of Gram-negative bacteria. After binding to its innate immunity pattern recognition Toll-like receptor 4 (TLR4), LPS can trigger the release of many inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-1,IL-6,and IL-8. So far LPS/TLR4/mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB)/TNF-α pathway is still thought to be the classical signal pathway for production of TNF-a induced by LPS. However, in recent years, LPS was found to be able to transactive epithelial growth factor receptor (EGFR). EGFR belongs to tyrosine kinase receptor family, which is expressed in a variety of cells and plays an important role in cellular proliferation, differentiarion and tumor growth. In some chronic airway diseases, LPS-induced airway inflammation increases the expression of inflammatory cytokines such as IL-1, IL-6 and this effect depends on the activation of EGFR. Meanwhile, Kuper C, et al found that in renal collecting duct cells, LPS induced EGFR activation via TLR4/TNF-α-converting enzyme (TACE), and finally resulted in induction of cyclooxygenase (COX)-2 expression. All these studies suggested that EGFR activation may play a critical role in LPS induces endotoxemia. In endotoemia or sepsis, excessive released inflammatory mediators render septic patients at high risk of developing multi-organ failure, which is associated with high mortality. TNF-a has been shown to be a major factor responsible for myocardial depression during endotoxemia and cardiomyocytes are the major local source of TNF-a.β-arrestins play important role as adaptor proteins involved in G protein-coupled receptor (GPCR) desensitization and also implicated in regulation of TLR signaling and proinflammatory gene expression. Some studies found that the membrane-associated GPCR/β-arrestins/c-Src signaling complex induced the transactivation of EGFR, and further activated downstream signaling cascades such as PI3K/Akt and Ras/Raf/MAPK/ERK pathways resulting in increased proliferation and migration.So far, there is no study especially focusing on the effect of EGFR on the production of TNF-α induced by LPS in cardiomyocytes or myocardium. Similarly, there is no study about that, whether β-arrestin 2, which plays an important regulating role in inflammation,was also involved in the transactivation of EGFR in endotoxemia or sepsis. In the present study, in cultured neonatal cardiomyocytes,wild type C57BL/6 mouse and β-arrestin 2 knockout C57BL/6 mouse, both in the levels of cell and in vivo, to explicit the effect of transactivation of EGFR on the production of TNF-a of myocardium or cardiomyocytes during endotoximia, and whether p-arrestin 2 also involved in the LPS transaction of EGFR.Materials and methods1. Animal preparation and model preparationSPF wild type C57BL/6 male mice,6-8 weeks, weight 22 to 25 g, bought from laboratory animal center of southern medical university.β-arrestin 2 knockout C57BL/6 mouse were brought from the Jackson laboratory animal center. Before and after the experiment, mice were raised in the south of the hospital laboratory animal center (room temperature 25 to 27℃, humidity 50% to 70%),5 in a cage, free food and water intake,8:00 in the morning and evening light turn on, avoiding glare and noise stimulation. We conducted the endotoxin model mice by intraperitoneal injection of LPS (5 mg/kg).2. Preparation of Neonatal Mouse CardiomyocytesNeonatal hearts fronm C57BL6 mice born within 24h were minced in a nominally Ca2+ and Mg2+ free D-Hanks balance solution. Cardiac myocytes were dispersed by the addition of Liberase TH with a final concentration of 22.5μg/mL in D-Hanks solution and incubated in 37℃water bath for 10 min. After being mixed by pipette for about 2-4 min, we collected the supernatant into a 15 ml clean tube, then adding new fresh digestion buffer and incubation in 37℃ water bath again. The cell suspension was centrifuged at 800 rpm for 5 min to obtain a cell pellet and the debris of heart was redigested one more time by Liberase TH and collected in the same tube, Cells were then suspended in M199 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution and preplated for 45-60 min to remove noncardiomyocytes. Then the cell suspension was filtered through a polypropylene macroinvolved porous filter (mesh opening 105μm, Spectra/Mesh, Spectrum Medical Industries). The cardiomyocytes were plated at a density of 5×105 cells/ml in M199 supplemented with 10% FBS and 1% penicillin-streptomycin solution on 24-well plates precoated with 1% gelatin. Cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2. A confluent monolayer of spontaneously beating cells was formed within 2 days.3. The experimental process3.1 The effect of PD168393 and erlotinib on LPS-induced EGFR phosphorylationFirst, we cultured neonatal mouse cardiomyocytes, divided into 6 groups:Control group:added saline.PD168398 group:added PD168393 (50μM) into the cardiomyocytes.Erlotinib group:added erlotinib (20μM) into the cardiomyocytes.LPS group:added LPS (4μg/ml) into the cardiomyocytes.LPS+PD168398 group:pretreated with PD168393 (50μM) 30 min before LPS (4μg/ml);LPS+Erlotinib group:pretreated with erlotinib (20μM) 30 min before LPS (4μg/ml);0.5 hour after LPS treatment, harvested the cardiomyocytes, Phospho-EGFR and total EGFR were determined by western blot analysis at 0.5 hour after LPS treatment.Then we analysed the results in vivo.16 wild type C57BL/6 mice were divided into four groups:Control group:saline i.p.Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days;LPS group:LPS (5mg/kg) i.p.LPS+Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days Before LPS (5mg/kg) i.p;Phospho-EGFR and total EGFR in the myocardium were determined by western blot analysis at 1 hour after LPS treatment.3.2 The inhibitory effect of EGFR inhibitor on the production of cardiac derived TNF-a after LPS treatmentFirst, we cultured neonatal mouse cardiomyocytes, divided into 9 groups:Control group:added saline.Erlotinib 20μM group:added erlotinib (20μM) into the cardiomyocytes.PD168398 50 μM group:added PD168393 (50μM) into the cardiomyocytes.LPS group:added LPS (4μg/ml) into the cardiomyocytes.LPS+ PD1683982 μM group:pretreated with PD168393 (2μ) 30 min before LPS (4pg/ml);LPS+ PD168398 10μgroup:pretreated with PD168393 (10μM) 30 min before LPS (4μg/ml);LPS+ PD168398 50 μM group:pretreated with PD168393 (50μM) 30 min before LPS (4μ/ml);LPS+ Erlotinib 10μM group:pretreated with erlotinib (10μM) 30 min before LPS (4μg/ml);LPS+ Erlotinib 20μM group:pretreated with erlotinib (20p.M) 30 min before LPS(4μg/ml);TNF-a protein and mRNA in the cultured medium were determined by ELISA and real-time RT-PCR at 4 hours after LPS treatment.Then we analysed the results in vivo.16 wild type C57BL/6 mice were divided into four groups:Control group:saline i.p.Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days;LPS group:LPS (5mg/kg) i.p.LPS+ Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days Before LPS (5mg/kg) i.p;TNF-aprotein in the myocardium was measured by ELISA at 6 hour after LPS (5mg/kg) treatment.3.3 EGFR is required in the activation of p38 and ERK1/2 in LPS treated cardiomuocytes.First, we cultured neonatal mouse cardiomyocytes, divided into 6 groups:Control group:added saline.Erlotinib group:added erlotinib (20μ) into the cardiomyocytes.PD168398 group:added PD168393 (50μM) into the cardiomyocytes.LPS group:added LPS (4μg/ml) into the cardiomyocytes.LPS+ PD168398 group:pretreated with PD168393 (50μM) 30 min before LPS (4μg/ml);LPS+ Erlotinib group:pretreated with erlotinib (20μM) 30 min before LPS (4μg/ml);Phospho-p38 or ERK1/2 and total p38 or ERK1/2 were determined by western blot analysis at 2 hours after LPS treatment.Then we analysed the results in vivo.16 wild type C57BL/6 mice were divided into four groups:Control group:saline i.p.Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days;LPS group:LPS (5mg/kg) i.p.LPS+ Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days before LPS (5mg/kg) i.p;Phospho-p38 or ERK1/2 and total p38 or ERK1/2 in the myocardium were determined by western blot analysis at 2 hours after LPS treatment.3.4 The inhibitory effect of β-arrestin2 on the production of TNF-a in the myocardium after LPS treatmentWe employed β-arrestin 2 knock out mice, and divided into 6 groups;Control group:saline i.p;β-arrestin 2+/- group:saline i.p;β-arrestin 2-/- group:saline i.p;LPS group:wild type C57BL/6, LPS (5mg/kg) i.p;LPS+β-arrestin 2+/- group:LPS (5mg/kg) i.p;LPS+β-arrestin 2--/- group:LPS (5mg/kg) i.p;TNF-aprotein and mRNAin the myocardium was determined by ELISA and RT-PCR at 6 hours after LPS treatment.3.5 β-arrestin2 is required in the activation of EGFR, p38 and ERK1/2 in the myocardium of endotoxemic miceβ-arrestin2 and EGFR could affect the production of TNF-α, we hypothesis whether β-arrestin2 is required in the activation of EGFR, p38 and ERK1/2 in the myocardium of endotoxemic mice.Control group:saline i.p;β-arrestin 2+/- group:saline i.p;β-arrestin 2-/- group:saline i.p;LPS group:wild type C57BL/6, LPS (5mg/kg) i.p;LPS+β-arrestin 2+/- group:LPS (5mg/kg) i.p;LPS+β-arrestin 2-/--group:LPS (5mg/kg) i.p;Phospho-EGFR and total EGFR were determined by western blot analysis at 1 hour after LPS treatment, Phospho-p38 or ERK1/2 and total p38 or ERK1/2 were determined by western blot analysis at 2 hours after LPS treatment.3.6 Measurement of left ventricle pump function with cardiac ultrasound during endotoxemia16 wild type C57BL/6 mice were divided into four groups:Control group:saline i.p.Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days;LPS group:LPS (20mg/kg) i.p.LPS+ Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days beforeLPS (20mg/kg) i.p;Changes of cardiac output (CO), ejection fraction (EF), fractionalshortening (FS) and stroke volume (SV) in left ventricle were measured with cardiac ultrasound 6 hours after LPS treatment.3.7 Effects of erlotinib on survival of mice treated with LPS16 wild type C57BL/6 mice were divided into four groups:Control group:saline i.p.Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days;LPS group:LPS (20mg/kg) i.p.LPS+ Erlotinib group:pretreated with erlotinib (45mg/kg) orally for 3 days beforeLPS (20mg/kg) i.p;After LPS administration, survival of mice was monitored at every 6 hour for 72 hour.Result1. PD168393 and Erlotinib effectively inhibited the phosphorylation of EGFR induced by LPSWe found in cultured neonatal cardiomyocytes of C57BL/6 mice, EGFR was obviously transactivated 0.5 h after LPS (4μg/ml) treatment and this kind of trasactivation could be effectively inhibited by EGFR irreversible inhibitor PD168393 (10 μM) or reversible inhibitor erlotinib (15μM). To further verify this result in vivo,16 wild type C57BL/6 mice were divided into four groups:control group, Erlotinib group, LPS group and LPS+Erlotinib group. As shown in Fig. 1B, EGFR in the myocardium was transactivated by LPS and this effect was partly inhibited by Erlotinib pretreatment. All these results indicated that both in vitro and vivo, LPS induced EGFR activation could be partly inhibited by EGFR selective inhibitor PD168393 or Erlotinib.2. Inhibiting the phosphorylation of EGFR decreased the production of TNF-a induced by LPSTo investigate the role of EGFR on LPS induced TNF-a expression, neonatal cardiomyocytes were pretreated with PD168393 or erlotinib 30 minites before LPS (4μg/ml) treatment. As we expected PD 168393 or Erlotinib obviously inhibited the production of TNF-a both in mRNA and protein level compared with LPS group. Meanwhile, as we increased the concentration of PD168393, the amount of TNF-a in the medium of cultured neonatal cardiomyocytes decreased correspondingly. To further verify this result in vitro, wild type C57BL/6 mice were treated with saline or LPS (5mg/kg, i.p.) with or without Erlotinib pretreatment. Compared with LPS group, the expression of TNF-a in the myocardium of LPS+Erlotinib group was effectively decreased.3. LPS trans-ativated EGFR regulated the phosphorylation of ERK1/2 and p38In cultured neonatal cardiomyocytes, p38 and ERK1/2 phosphorylation were measured 1.5 hours after LPS treatment with or without PD168393/Erlotinib pretreatment. We found that LPS promoted the phosphoralation of ERK1/2 and p38 and this effect could be inhibited by EGFR selective inhibitor PD168393 or Erlotinib. Then, we verified this result in vivo. Wild type C57BL/6 mice were divided into four groups:control group, Erlotinib group, LPS group, and LPS+Erlotinib group. Phosphorylation of ERK1//2 and p38 in the myocardium of LPS group obviously increased, compared with those of control and Erlotinib groups. Pretreatment of Erlotinib could partially decrease the phosphorylation of both ERK1/2 and p38 induced by LPS. These results demonstrated that both p38 and ERK1/2 were involved in the mechanism of how EGFR regulating the production of TNF-a after LPS treatment.4. β-arrestin 2 regulated the production of TNF-a in the myocardium of LPS induced endotoxemia(3-arrestin2 knockout mice heterozygote (β-arrestin2+/-) and homozygote (β-arrestin2-/-) were employed and treated with vehicle or LPS (5mg/kg). Six hours after LPS treatment, Both TNF-a mRNA and protein in the myocardium were significantly decreased in β-arrestin 2-/- compared with WT mice. These results indicated that β-arrestin 2 was a regulator for the production of TNF-a in the myocardium of LPS induced endotoxemia.5.β-arrestin 2 was required for the transactivation of EGFR induced by LPSSince both β-arrestin 2 and EGFR could negatively regulate the production of TNF-a in the myocardium of LPS-induced endotoxemia, we just wondered whether β-arrestin 2 was involved in the transactivation of EGFR induced by LPS. Therefore we measured the phosphorylation of EGFR in β-arrestin 2 knockout and wild type mice treated with or without LPS. In wild type mice, LPS effectively induced the phosphorylation of EGFR. However, in β-arrestin 2 knockout mice, the phosphorylation state of EGFR in the myocardium showed no difference in saline and LPS group. Then we further measured the phosphorylation of ERK1/2 and p38 in wild type and P-arrestin 2 knockout mice with or without LPS treatment. As we expected, in β-arrestin 2 knockout mice, LPS lost the ability of transactivating ERK1/2 and p38. These results indicated that P-arrestin 2 was critical important for LPS transactivating EGFR and its downstream signal in LPS induced endotexemia.6. Inhibiting the phosphorylation of EGFR alleviated myocardial dysfunction dramaticallly in endotoxemic miceWe compared the hemodynamic changes of heart with cardiac ultrasound in all the four groups after 6 hours of LPS (20 mg/kg, i.p.) treatment. Although there was no significant change of heart rate in all the four groups, the cardiac output (CO), ejection fraction (EF), fractionalshortening (FS) and stroke volume (SV) of left ventricle were significantly reduced in endotoxemic mice compared with control and Erlotinib group. However all these changes induced by LPS could be obviously reversed by Erlotinib pretreatment. These data demonstrated that inhibiting the phosphorylation of EGFR effectively improved left ventricular pump function and ameliorated cardiac dysfunction induced by LPS in mice.7. Effects of EGFR activation on the survival rate of wild type C57BL/6 mice during endotoxemiaWe found that 8,24 h after LPS treatment,48% of saline-pretreated mice died, while 32% of Erlotinib-pretreated mice died and no deaths occurred in saline or Erlotinib control group. At 72 h, the LPS group was associated with a 72 h survival rate of about 16%. In contrast, LPS-injected mice pretreated with Erlotinib had a higher survival rate of 52%.Therefore, pretreatment with EGFR inhibitor, Erlotinib significantly improved survival during acute endotoxemia in mice (P< 0.01).ConclusionIn summary, our study demonstrated that in endotoxemia, LPS transactivated EGFR played a key role in regulating the production of cardiac derived TNF-a both in DNA and protein level. Inhibiting the phosphorylation of EGFR with PD168393 or erlotinib also decreased the phosphorylation level of p38 and ERK1/2 induced by LPS. In LPS treated β-arrestin 2 knockout mice, the phosphorylation of EGFR, p38 and ERK1/2 were decreased obviously compared with those in wild type mice indicating β-arrestin 2 involves in regulating the transactivation of EGFR by LPS. Inhibiting the activation of EGFR in the myocardium of endotoxemic mice with erlotinib, we found erlotinib effectively improved left ventricular pump function and ameliorated cardiac dysfunction and ultimately improving survival rate. All these results provided a new insight of the cross talk between tumorigenesis and sepsis and the production of TNF-a in cardiomyocytes and a potential new target for the treatment of cardiac failure in sepsis. |
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