Effects Of Peripheral Benzodiazepine Receptor On Cardiocytes Injury Induced By Hypoxia And Reoxegenation In Rats And Its Mechanism

IntroductionCardial ischemia reperfusion injury(IRI) is still hardly avoided when the following conditions happened such as thrombolysis,unstable angina,open heart operation,heart lung transplantation and cardiopulmonary resuscitation.The prevention of organ IRI is also an important question to be solved urgently.The phenomenon of ischemic precondition(IPC) was firstly reported in myocardial cells by Murry and et al.The subsequent investigations found drug precondition(DPC) can produce the similar effect of IPC.Now the role of mitochondria on organ protection in IRI is known weU.The injury of mitochondria was a important factor for the irreversible injury in myocardiocytes,and the alteration of mitochondria permeability,which was regulated by permeability transition pore,played an important role in apoptosis.The early phase of apoptosis in which mitochondria participated was the opening of PTP,making the elevation of H⁺ disappeared,decoupling the respiratory chain.The destroy of mitochondria membrane and inducing the release of cytoC and the other activated protein, which further triggered the cascade reaction of caspase made the cell show the character of apoptosis.Peripherial benzodiazepine receptor(PBR) is a type of protein located in the outer membrane of mitochondria,which composed mitochondria PTP with voltage-dependent anion channel(VDAC) and adenine nucleotide translocator (ANT).PBR distributed widely,and expressed abundantly in heart,lung,liver, kidney and blood cells.Some investigations indicated that PBR took part in the regulation of opening the PTP,so determination of the effect of PBR and its ligands on mitochondria,cell and IRI is becoming a hot spot. Our study planned to measured the expression of PBR in the model of myocardial hypoxia and reoxygenation and the effect on apoptosis.We explained the mechanism of PBR involved in ceUular IRI by observing the effect of PBR on the cellular concentration of calcium,the expression of protein kinase C(PKC) and the apoptosis involved factors such as Bcl-2/Bax,Caspase-3 and so on.We further observed the effect of PBR on apoptosis by adapting the commonly used benzodiazepines anesthetic:midazolam,and referred a potential basis for the clinical choice of anesthetics.Materials1.Animals:the neonate Wister rats of 2 to 4 days were provided by the experimental animal center of China Medical University.2.Chemical and reagents:PK11195(Alexis,USA);Ro5-4864(Biomol,USA);Midazolam(Roche, Shanghai);polyclonal antibody(Santa,USA);Caspase-3,PKC polyclonal antibody (Neomarker,USA);SP immunohistochemistry kit,DAB kit(Zhongsan, Beijing);Trypsin(1:250)(Amresco,USA);DMEM(Gibco,USA);Fetal calf serum(Haoyang,Tianjing);FITC-Annexin V(Boehringer Mannheim,German); Fura-2/AM,propidium iodide(Sigma,USA);Trizol(Invitrogen,USA); RT-PCR kit(Promega,USA);PVDF(Bio-Rad,USA).3.Experiment instruments:inverted phase contrast microscope(OLYMPUS,Japan);desk centrifuge (SIGMA 1-13,USA);PTC-100 PCRamplificator(USA);electrophoresis image analysator(Alphairmotech ChemiImager 5500,USA);CO₂ incubator(KENDRO/HERAEUS, German);super clean bench(Suzhou,China);PowerPac200 electrophoretic apparatus(BIO-RAD,USA);MDF-U ultra deep-freeze equipment(SANYO,Japan); micro image analysis system(Olympus AX70/Coolsnapfx/MetaMorph, Japan). MethodsCulture and assessment of myocardial cellsThe myocardial cells of neonatal rats were separated by improved Simpson method,in which 0.08%trypsin and 0.05%collagenase were used,and then the cells were cultured in DMEM medium including 20%fetal calf serum and incubated in a 95%air/5%CO₂ incubator at 37℃.To select enrich myocytes, dissociated cells were preplated for an hour to allow nonmyocytes to attach to the bottom of the culture dish.BrdU(0.1mmol/L) was added during the first two days to prevent proliferation of nonmyocytes.The cells were devided into groups randomly when grown in subsequent confluence state and showed beat in common. Cultured cells were further confirmed by the identification of myocardial inocoma under electron microscope.The establishment of hypoxia and reoxigenation modelTo induce hypoxic stress,culture medium was aspirated off and cells were replaced with hypoxic myocardial growth medium before introduced into an hypoxia chamber.The cells were removed from the hypoxic chamber after 30min, the medium was replaced by warm fresh medium and then the cells were placed in a 95%air/5%CO₂ incubator at 37℃for 2h to perform an reoxygenation phase.Normoxic control cells were treated by replacement of culture medium and incubated in a 95%air/5%CO₂ incubator at 37℃.Experiment designCells were devided into seven groups,C group(control group),HR group (hypoxia for 30min and reoxygenation for 2h),P group(PK11195 group),R group(Ro5-4864 group),M group(Midazolam group),PR group(PK11195 and Ro5-4864 group) and PM group(PK11195 and Midazolam group).The groups were added PK11195,Ro5-4864 and Midazolam singlely or both respectively at a concentration of 10^-4mol/L before hypoxia and then treated with hypoxia and reoxygenation.One part of the cells were fixed by paraform for immunohistochemical staining and the other part were used directly for measured the intracellular calcium.Some cells were also cryopreserved for the assay by RT-PCR and Western-Blot methods.Detection index(1) Apoptotic cells assayApoptotic cells were measured by cytometry using FITC-Annexin V/PI Staining.(2) The expression of Bcl-2/Bax and PKCThe expression of Bcl-2/Bax and PKC were measured by Western blot, and were analyzed by electrophoresis image analysator.(3) The expression of Caspase-3The quantitation of Caspase-3 was measured by immunohistochemistry staining and calculated the mean values by optical density detected by MetaMorph 4.5 image analysis software.(4) The intracellular calcium concentrationMarked by fura-2,the calcium was calculated through fluorescence intensity by Meta Flour software.(5) The expression of PBR mRNAQuantitative reverse transcriptase-polymerase chain reaction(RT-PCR) was used.Statistical analysisAll values in the text and figures were presented as mean SE and subjected to one-factor analysis of variance using SPSS 12.0 software.Homoscedasticity was checked by LSD method and the Dunnett T3 was adopted to check the heterogeneity of variance Differences were considered significant at P＜0.05.Results1.The amount of apoptosis in the HR group increased significantly compared with the C group(P＜0.01),and was attenuated in the R group compared with the HR.group(P＜0.01).PK11195 the antiapoptotic effect was blocked in the P group and the result showed a statistic difference compared with the R group(P＜0.01).midazolam decreased the apoptosis(compared with the HR group P＜0.05) and the effect can not be blocked by PK11195(P＞0.05). 2.The expression of PBR in the HR group was downregulated compared with the C group(P＜0.05),and 10^-4mol/L Ro5-4864 the expression of PBR was upregulated in the R group compared with the HR group(P＜0.01). PK11195 significantly blocked the upregulation compared with the R group(P＜0.01).Midazolam had no effect on the expression of PBR compared with the HR group(P＞0.05).3.The expression of Bcl-2 was significantly downregulated after hypoxia and reoxygenation treatment compared with the C group(P＜0.05),but The expression of Bax protein was significantly upregulated compared with the C group (P＜0.05).10^-4 mol/L Ro5-4864 significantly upregulated Bcl-2 and downregulated Bax compared with the HR group(P＜0.01),but the effect was completely reversed by PK11195(P＜0.01).midazolam upregulated the expression of Bcl-2,but did not affect the expression of Bax,and PK11195 significantly attenuated its effect on Bcl-2 compared with the M group(P＜0. 05).4.The expression of Caspase-3 was enhanced significantly after hypoxia and reoxygenation treatment compared with the C group(P＜0.01),10^-4moL/L Ro5-4864 significantly downregulated Caspase-3 compared with the HR group (P＜0.05),but the effect was reversed by PK11195 compared with the R group (P＜0.05).midazolam downregulated the expression of Caspase-3,but it was not significant compared with the HR group(P＞0.05).The effect was not blocked by PK11195.5.The concentration of intracellular calcium increased significantly induced by hypoxia and reoxygenation compared with the C group(P＜0.01). 10^-4mol/L Ro5-4864 significantly inhibited the increasing compared with the HR group(P＜0.01),but PK11195 blocked its effect.Midazolam had no effect on the concentration of intracellular calcium compared with the HR group(P＞0.05).6.Hypoxia and reoxygenation decreased the expression of PKC compared with the C group(P＜0.01).10^-4mol/L Ro5-4864 significantly inhibited the decreasing of PKC induced by hypoxia and reoxygenation compared with the HR group(P＜0.01) and PK11195 blocked its effect compared with the R group(P ＜0.01).midazolam had no effect on the expression of PKC compared with the HR group(P＞0.05).DiscussionApoptosis is a kind of programmed death directed by gene,and the change of mitochondria membrane permeability played an important role.The increasing of mitochondria membrane permeability induced the breakdown of membrane potential, decoupling of respiration chain,interruption of energy production and increasing ROS production,which at last resulted in the rupture of outmembrane, cyto C release and such apoptosis induced factors.These factors can activated multiple catabolic enzymes including triggering pro-Caspase-3 to Caspase-3,which participated in several links for the cellular necrosis and/or apoptosis such as regulation,signal transduction,repair of DNA and so on.PTP played a key role in regulating the permeability of mitochondria.PBR is a type of membrane protein located in the outmembrane of mitochondria, and is the key part of PTP.The analysis of mitochondria fragment showed VDAC and ANT connected with PBR tightly.The function of PBR related to VDAC and ANT and took part in the opening of PTP.Caspase-3 is also called CPP32(cysteine protease protein),which pertained to the family of CPP,and became the most important final executor of apoptosis. So the inhibition of caspase may be an the effective method for prevention IRI.The sub-family of Bcl-2 can decrease the release of cyto C and showed a potential antiapoptotic character,which prevented apoptosis by an caspase-dependant and caspase-independent manner.The sub-family of Bax can form multimer,and affected the integrity of mitochondria in order to promote apoptosis.PKC is a type of phospholipids dependent serine/threonine protein kinase which activated by calcium ion in cytoplasma.It elicited a wide physiologic function by phosphorylating numerous substrate albumen located on membrane and cytoplasma,and became the important link for prevention IRI. Calcium ion is a unique second message which can go into mitochondria and regulated multiple functions of cell.The increasing concentration of calcium in mi- tochondria and cytoplasma played an important role in cell and membrane injury. It was regulated by many factors and PKC was the most important one.We adopted the cultured neonate rat myocardial cells for inducing the model of hypoxia and reoxygenation injury,which was considered as a real manner for reflecting the course of IR,and depleted the interruption of nerve,humor, and hormone,which difficult to remove in the experiments in vivo and in vitro models.Our study showed hypoxia and reoxygenation injured myocytes,destroyed the structure and increased the apoptosis accompanied by decreased expression of Bcl-2 and increased expression of Bax.Hypoxia and reoxygenation enhanced the expression of Caspase-3.Ro5-4864,a specific agonist of PBR, prevented hypoxia and reoxygenation injury by significantly attenuating the apoptosis. These effects were related to the inhibition of Caspase-3 activation,upregulation of Bcl-2 and downregulation of Bax,activating PKC,maintaining the calcium homeostasis and inhibition of calcium overload.The protective effect was reversed by PK11195,an classic antagonist of PBR,which indicated the effects was dependent on PBR.PBR elicited anti-apoptosis through the effects on mitochondria in the early phase including the permeability decreased,the release of cyto C,activation of Caspase-3 and breakage of DNA and et al.These results were coincide with the protection of PBR observed in the prevention of hematopoietic cell apoptosis treated by H₂O₂,which also showed that transfection of PBR increased the cellular resistance to H₂O₂.The similar investigation indicated the agonist of PBR prevented the oxidative injury induced by H₂O₂ in human lymph cell line U937. The specific agonists SSR180575 and Ro5-4864 can prevent the oxidative injury induced by H₂O₂ and protected mitochondria.Investigation also proved that PBR played an important role in the signaling passway of regulating the necrosis and apoptosis of tubular cells after IR,and SSR180575 had protective effect in rat renal IRI.Midazolam,the clinical commonly used benzodiazepines anesthetic, showed protection in myocardial apoptosis induced by hypoxia and reoxygenation besides its sedation,hypnosis,antianxiety and anticonvulsion characters.The results indicated the effect was independent of PBR and had no effect on the concentration of intraceUular calcium and the expression of PKC.It may be related to the link of signaling passway including inhibition of Caspase-3 activation, upregulating Bcl-2 expresion and downregulating Bax.The agonist of PBR elicited protection by maintaining the integrity of mitochondria, decreasing the injury and apoptosis.This result elucidated the mechanism of apoptosis after reperfusion was related to this receptor and referred a possible intervention for prevention and therapy of IRI.Further investigations were also demanded to explain the mechanism because the regulation signaling passway of PBR in stress was not clearly.Conclusion1.It was documented PBR participated in regulating the myocardial apoptosis induced by hypoxia and reoxygenation by adopting specific agonist and antagonist. The expression of PBR was opposite to the result of apoptosis and showed an anti-apoptotic character.2.The anti-apoptotic character of PBR may be related to the mechanism of inhibiting the activation of Caspase-3,upregulating the expression of Bcl2/downregulating Bax,and activating PKC for inhibiting of the intracellular calcium overload to maintain homeostasis.3.Midazolam prevented myocardial apoptosis induced by hypoxia and reperfusion and the effect was independent of PBR.