| Although many research have been report that estrogen can protect myocardial during hypoxia / reoxygenation injury, the study of estrogen on myocardial cells protection mechanism is rarely reported. Therefore, it is necessary to conduct in-depth study. In addition to the promotion of reproductive system development and maintenance of reproductive function, 17β-estradiol can protect cardiovascular and other organs. Its main functions are anti-inflammatory, antioxidant, anti-apoptotic and so on. Myocardial cells have estrogen receptors (ER), cell activity is regulated by 17β-estradiol.In our study, we use the cultured neonatal rat myocardial cells during hypoxia / reoxygenation (H / R) injury model to study the effect of estrogen on myocardial cells in inflammatory and antioxidant mechanism. This study is divided into three parts.The first part is about the protective effect of 17β-estradiol on myocardial cells during hypoxia / reoxygenation.We establish the hypoxia / reoxygenation model to simulate myocardial ischemia / reperfusion injury model. To a certain dose of exogenous estrogen (5μM), we identified myocardial cells by a-Actin immunohistochemical staining, observed the morphology of myocardial cells by inverted microscope and detected CK, LDH, MDA in the culture medium. We use laser scanning confocal microscopy to detect calcium transient and calcium concentration, use MTT and flow cytometry with AnnexinⅤand PI double staining experiments to detect cell apoptosis rate and survival rate. The results showed that: cultured neonatal rat myocardial cells during hypoxia / reoxygenation injury can lead to cell shrinkage, refractive index decreased, cell processes decreased; the contents of LDH, CK and MDA in the culture supernatant compared with the control group increased 5.1 fold, 10.2 fold, 8.2 fold respectively; spontaneous calcium transient increased in amplitude and duration, the calcium was overload obviously; the number of apoptotic cells increased by 10.83% compared with the control group, the cell survival rate decreased by 40.9%.Pretreatment of myocardial cells with E2 can reduce the degree of myocardial hypoxia / reoxygenation injury, significantly reduced morphological damage and the contents of LDH, CK and MDA in the culture supernatant compared with the injury group reduced 2.7 fold, 2.7fold and 2.4fold respectively; calcium transient amplitude value and duration were decreased compared with the injury, but with no significant difference between the control group; the number of apoptosis cells decreased by 9.32% compared with the injured group; cell survival rate increased by 26.45%. E2 has a significant protective effect. Its role is mainly to protect the cell morphology and membrane integrity, reduced enzyme leakage, reduced the calcium transient amplitude and duration increased caused by hypoxia / reoxygenation, inhibit apoptosis, increase myocardial cells activity and myocardial cell viability.The second part, study on the mechanism of the 17β-estradiol effect on the expression of NF-κB, ICAM-1 and VCAM-1 during hypoxia / reoxygenation.We observed the effect of E2 on the expression of ICAM-1 and VCAM-1 by RT-PCR and ELISA.; observed the effect of E2 on NF-κB nuclear translocation and subcellular localization by flow cytometry, Western blot technique and laser scanning confocal microscopy; observed the effect of E2 on the expression of NF-κB inhibitory protein I-κB by RT-PCR and ELISA .The results showed that:E2 inhibited the expression of mRNA and protein of ICAM-1 and VCAM-1(P <0.05), prevented NF-κB nuclear transfer (P <0.05). Intervention by PDTC, we analyze the relationship of the levels of mRNA and protein of ICAM-1 and VCAM-1 and NF-κB nuclear translocation. Summary that the effect of E2 on the expression of mRNA and protein of ICAM-1 and VCAM-1 expression levels of inhibition is achieved by blocking NF-κB nuclear translocation.E2 can not effect the expression of mRNA and protein of NF-κB cytoplasmic inhibitor protein I-κB significantly during hypoxia / reoxygenation. E2 can inhibit the expression of ICAM-1 and VCAM-1, reduce excessive inflammatory response during hypoxia / reoxygenation.E2 prevents NF-ΚB nuclear transfer to reduce the volume of NF-ΚB in the nuclear, inhibit the expression of inflammatory factors, reduce the hypoxia / reoxygenation-induced inflammatory response and inflammatory injury. NF-ΚB control the expression of I-ΚB, while I-ΚB also can regulate the volume of NF-ΚB in the nuclear by the mechanism of negative feedback regulation. E2 can not effect the expression of I-ΚB.The third part, study on the mechanism of the 17β-estradiol effect on the expression of Nrf2, GST and GCL during hypoxia / reoxygenation.We observed the effect of E2 on the expression of GST and GCL by RT-PCR and ELISA.; observed the effect of E2 on Nrf2 nuclear translocation and subcellular localization by Western blot technique and laser scanning confocal microscopy; observed the effect of E2 on the expression of Nrf2 binding protein Keap1 by RT-PCR and ELISA .The results showed that:E2 promoted the expression of mRNA and protein of GST and GCL significantly(P <0.05), increased the capacity of cell antioxidant. The antioxidant role of E2 has a close relationship with Nrf2/ARE antioxidant system. E2 can promote transcription factor Nrf2 into nuclear (P <0.05), increase the expression of phaseⅡdetoxification enzymes and antioxidant enzyme which located in the downstream of ARE, enhance the antioxidant protection capacity of cells.E2 can not effect the expression of mRNA and protein of Nrf2 binding protein Keap1 significantly. E2 can increase the the expression of GST and GCL to enhance the antioxidant capacity of myocardial cells and reduce the excessive oxidative damage to protect cells.E2 can increase the amount of Nrf2 nuclear transfer and enhance the regulation ability of ARE to regulate the gene expression of antioxidant enzymes and phaseⅡdetoxification enzymes. E2 has no effect on the expression of Keap1.
|
PDF Full Text Request