| BackgroundMyocardium ischemia/reperfusion injury remains the major cause of cardiac dysfunction in human cardiovascular pathophysiology since it occurs in a wide variety of ischemic cardiovascular disorders such as myocardial ischemia and stroke. Microcirculation dysfunction is an important etiological component of MIRI. Accumulating evidence suggests that I/R injury can induce ECs malfunction which is the most important reason of microcirculation dysfunction. Moreover, in the very early stages of reperfusion, ECs apoptosis is firstly observed, which precede myocyte cell apoptosis. Some soluble inflammation mediators from damaged ECs are shown to induce myocyte apoptosis. However, the mechanism of ECs dysfunction induced by MIRI is still not well investigated.Toll-like receptor (TLR) family consists of highly conserved pattern recognition receptors. Among the 10 TLRs identified in human, TLR-4 is the most extensively investigated since its recognition of lipopolysaccharide (LPS). However, evidences show that the ligands of TLR-4 are not only LPS, but also some specific endogenous molecules, such as fibronectin, fibrin, extracellular matrix fragments, heat-shock proteins 60 (HSP-60), HSP-70 and so on. TLR-4, initially found in monocytes, has been shown to be expressed in many tissues, including cardiomyocytes and ECs. Recently, the myocardial MIRI has been viewed as an innate immune response which is mediated in part through TLR-4 signal pathway. The TLR-4 deficient mice had reduced myocardial MIRI injury. Moreover, inhibition of TLR-4 signal with eritoran also attenuated cardiac dysfunction induced by MIRI injury. Previous clinical studies indicate that TLR-4 activation plays an important role in pathology process of atherosclerosis and heart failure.Although several studies have shown that TLR-4 takes part in MIRI of heart, most current studies focused on the relationship between TLR-4 and global heart dysfunction or cadiomyocyte apoptosis. The effect of TLR-4 on CMEC in MIRI is not clear so far. We hypothesized that TLR-4 signaling modulates CMEC dysfunction post MIRI.MethodsPart I: CMEC were isolated from adult male Sprague-Dawley rat hearts. Cultured CMEC, after being replaced with hypoxic buffer, were exposed to hypoxia in anaerobic system at 37℃for 6h. For reoxygenation, CMEC, after hypoxia treatment, were removed from the anaerobic chamber and then maintained in a regular incubator for 2h, 12h and 24h respectively. The proliferation of CMEC was assessed by MTT assay, the apoptosis of CMEC was detected by Hocheset staining, the migration of CMEC was detected by cell scratch wound healing assay and the level of nitric oxide (NO) was detected by an NO assay kit according to the manufacturer's protocol.Part II: After CMEC were treated with hypoxia for 6h followed by reoxygenation 2h, 12h, 24h respectively, the expressions of TLR-4, Nuclear Factor-kappa B (NF-κB) were analyzed by Western blotting. And the levels of interleukin-6 (IL-6), Tumor necrosis factor-α(TNF-α) were detected by ELISA kits.Part III: CMEC were divided into four groups: (1) control group, (2) H/R group, (3) TLR-4 blocking group, (4) TLR-4 blocking and H/R group. The TLR-4 signal of CMEC was blocked by administration with the neutralizing antibody MST510 (10ug/ml). The H/R treatment was performed as hypoxia 6h followed with reoxygenation 2h. Like the methods used in Part I, the proliferations of CMEC in all groups were assessed by MTT assay, the apoptosis of CMEC was detected by Hocheset staining, the migrations of CMEC were detected by cell scratch wound healing assay, the levels of NO, IL-6, TNF-αwere detected by kits.Results1. CMEC were isolated and cultured in vitro successfully.2. Proliferation and migration of CMEC were impaired after H/R injury (P<0.05 vs. control). Moreover, H/R injury increased the apoptosis of CMEC (P<0.05 vs. control), and decreased the level of NO secretion (P <0.05 vs. control).3. Compared with control, H/R injury increased TLR-4 expression after 2 h or 12 h reoxygenation (P<0.05 vs. control). The level of NF-κB increased after 2 h and 24 h reoxygenation (P<0.05 vs. control). H/R injury also enhanced IL-6 and TNF-αsecretion as compared with control group (P<0.05 vs. control).4. Blocking TLR-4 signal pathway by administration of the neutralizing antibody MTS510 prior to H/R treatment attenuated the proliferation and migration of CMEC after H/R injury (P<0.05 vs. H/R). Moreover, TLR-4 inhibition decreased the apoptosis of CMEC after H/R injury (P<0.05 vs. H/R), and increased the NO level (P<0.05 vs. H/R).Conclusions1. H/R injury can increase the apoptosis of CMEC, and impair the proliferation. Moreover, H/R also induces CMEC dysfunction of migration and secretion of NO.2. In the early state of H/R injury, TLR-4/NF-κB signal pathway in CMEC is activated and the activation of TLR-4/NF-κB pathway enhances the secretions of IL-6 and TNF–α, which may participate in the injury and dysfunction of CMEC caused by H/R.3. Blocking TLR-4 signal decreases the apoptosis of CMEC and attenuates dysfunction caused by H/R, suggesting a possible mechanism by which H/R injury lead to CMEC dysfunction.
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