Remote Ischemic Postconditioning Inhibited Mitophagy to Achieve Neuroprotective Effects in the Rat Model of Cardiac Arrest.

Remote ischemic postconditioning (RI-postC) is an effective measure to improve nerve function after cardiac arrest. However, the brain protective mechanism of RI-postC has not been fully elucidated, and whether it is related to mitophagy is unclear. In this study, we used the rat model of cardiac arrest to study the effect of RI-postC on mitophagy and explore its possible signaling pathways. Rats were randomly divided into Sham group, CA/CPR group, Mdivi-1 group and RI-postC group. The animal model of cardiac arrest was established by asphyxia. RI-postC was performed by clamping and loosening the left femoral artery. Mdivi-1 was treated with a single intravenous injection. Levels of TOMM20, TIM23, Mfn1, PINK1 and parkin were detected by western blots. Mitochondrial membrane potential was measured by flow cytometry. Real-time PCR was used to detect relative mitochondrial DNA levels. The apoptosis of hippocampal neurons was detected by flow and TUNEL. In addition, Histopathological tests were performed. The results showed that RI-postC was similar to the mitophagy inhibitor Mdivi-1, which could inhibit the decrease of mitophagy-related protein level, improve mitochondrial membrane potential and up-regulate the ratio of mt-Atp6/Rpl13 after cardiopulmonary resuscitation (CPR). Furthermore, RI-postC could also reduce the rate of hippocampal nerve apoptosis and the damage of hippocampal neurons after CPR. Moreover, RI-postC and Mdivi-1 could reduce the protein levels of PINK1 and parkin in mitochondria after CPR, while increasing PINK1 levels in the cytoplasm. These findings suggested that RI-postC could inhibit the overactivation mitophagy through the PINK1/parkin signaling pathway, thus providing neuroprotective effects.

Mitophagy in the Hippocampus Is Excessive Activated After Cardiac Arrest and Cardiopulmonary Resuscitation.

This study examined the activation of mitophagy following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) and the relationship between the change with time and apoptosis. MAIN METHODS: The male Sprague-Dawley rats were randomized into four groups: Sham group, CPR24h group, CPR48h group, CPR72h group. The rat model of cardiac arrest was established by asphyxiation. We employed western blot to analyze the levels of mitophagy related proteins of hippocampus, JC-1 to detect mitochondrial membrane potential (MMP) and flow cytometry to measure the rate of apoptosis of hippocampal neurons. Moreover, we also intuitively observed the occurrence of mitophagy through electron microscopy. KEY FINDINGS: The results showed that the levels of TOMM20 and Tim23 protein were significantly decreased after CPR, which were more remarkable following 72 h of CPR. However, the protein levels of dynamin related protein 1 (Drp1) and cytochrome C (Cyt-c) were strongly up-regulated after CPR. Meanwhile, the hippocampal MMP decreased gradually with time after CPR. Furthermore, we more intuitively verified the activation of mitophagy through electron microscopy. In addition, the rats of apoptosis rate of hippocampus after CPR were significantly increased, which were gradually enhanced over time from 24 h until at least 72 h following CPR. SIGNIFICANCE: with the enhancement of mitophagy, the apoptosis of hippocampal neurons was gradually enhanced, which suggested mitophagy may be excessive activated and aggravating brain damage after CA and CPR.

Cyclosporine A Plus Ischemic Postconditioning Improves Neurological Function in Rats After Cardiac Resuscitation.

BACKGROUND AND OBJECTIVE: Attenuation of neuronal apoptosis helps maintain neurological function in patients after cardiac arrest. After ischemia-reperfusion, both cyclosporin A (CsA) and ischemic postconditioning independently protect mitochondria and thus reduce nerve injury. This study employed a rat model to evaluate the neuroprotective effect of combining ischemic postconditioning with CsA after cardiopulmonary resuscitation (CPR). METHODS: Rats were apportioned equally to model control, postconditioned, CsA-treated, or CsA + postconditioned groups. Asphyxial cardiac arrest was imposed using modified Utstein-style guidelines. In the appropriate groups, postconditioning was implemented by ischemia and reperfusion (clamping and loosening the left femoral artery); CsA treatment was delivered with a single intravenous dose. Neurological deficits were scored at different times after CPR. Histological evaluation and electron microscopy were used to evaluate tissue damage, and TUNEL and flow cytometry were used to measure the apoptotic rate of hippocampal neurons and size of the mitochondrial permeability transition pore (mPTP) opening. RESULTS: The apoptotic rate was significantly lower in the postconditioned and CsA-treated groups compared with the model control and lowest in the CsA + postconditioned group. By histological evaluation and electron microscopy, the least damage was observed in the CsA + postconditioned group. The neurological deficit score of the CsA + postconditioned group was significantly higher than that of the CsA-treated group, but the size of the mPTP openings of these two groups was comparable. CONCLUSION: Ischemic postconditioning combined with CsA exerted a better neuroprotective effect after CPR than did either postconditioning or CsA alone. Inhibiting the opening of the mPTP is not the only neuroprotective mechanism.

The protective effects of distal ischemic treatment on apoptosis and mitochondrial permeability in the hippocampus after cardiopulmonary resuscitation.

Apoptosis and mitochondrial dysfunction are the main cause of neurological injury after cardiopulmonary resuscitation (CPR). However, the effects of distal ischemic treatments on ischemia induced apoptosis are rarely studied, and the mechanism by which mitochondrial dysfunction contributes to CPR still unclear. A rat model of distal ischemia was established by clipping the right femoral artery. Rats were divided into blank, model, pre distal ischemic treatment, per-treatment, and post-treatment groups. Neurological deficit score was scored to evaluate neurologic function after cardiopulmonary resuscitation for 72 hr. We employed TUNEL and flow cytometry to measure the rate of apoptosis of hippocampal neurons, the integrity of mitochondrial membrane and the degree of mitochondrial permeability transition pore (mPTP) opening. The rate of apoptosis rate of hippocampal CA1 neurons in the pre-treatment and post-treatment groups were significantly lower than that of the model group. Moreover, the integrity of the mitochondrial membrane in the pre-treatment and post-treatment groups was higher than that in the model and per- treatment groups. Furthermore, the degree of mPTP opening was lower in the pre-treatment and post-treatment groups than the untreated and per-treatment groups. Taken together, our results show that ischemic preconditioning and post processing can maintain the integrity of mitochondria, perhaps by inhibiting the opening of mPTP, and reducing apoptosis of hippocampal neurons by regulating expression of apoptosis related proteins after CPR, to improve neurological function. This study highlights a novel target pathway for treatment of CPR.

Surgical treatment of an invasive thymoma extending into the superior vena cava and right atrium.

Although invasive thymoma commonly infiltrates neighbouring mediastinal structures, its extension into the superior vena cava (SVC) and consequent SVC occlusion are rare. In such cases, the urgent removal of the thymoma and radical resection of the infiltrated SVC representreasonable options, since induction therapy is time-consuming and useless for symptom resolution. A case of invasive thymoma extending into the SVC and right atrium (RA) with SVC syndrome is reported. The patient underwent a combined resection of the invasive tumor and SVC under cardiopulmonary bypass (CPB), and the SVC and bilateral brachiocephalic vein (BCV) were reconstructed with an autologous pericardial 'Y' conduit. After 40 months of follow-up, the patient showed a patent graft and no tumor recurrence.

Surgical treatment of aortoesophageal fistula induced by a foreign body in the esophagus: 40 years of experience at a single hospital.

BACKGROUND: Aortoesophageal fistula (AEF) is a rare but usually fatal complication of a foreign body in the esophagus. Little effective therapy exists to cure an AEF induced by esophageal foreign body. This report describes the authors' 40 years of experience treating patients with AEF caused by a foreign body and compares different treatments of patients and their clinical outcomes. METHODS: The treatments of five patients with AEF caused by esophageal foreign body impaction were recorded at Wuhan General Hospital of Guangzhou Command from 1970 to 2011. One of these five patients was managed with nonsurgical measures, whereas three were treated by surgery with cardiopulmonary bypass, and one was treated by surgery with endovascular stent-graft repair. RESULTS: All five AEF cases were confirmed by computed tomography, esophagogastroscopy, surgical findings, or two or both. The nonsurgically treated patient died of fatal hemorrhage. Another patient died during the postoperative period because of ventricular fibrillation (he had a history of coronary heart disease before the operation), and still another patient died of fatal hemorrhage during the surgery. The remaining two patients were completely cured by surgery: the one via traditional open thoracotomy with cardiopulmonary bypass and the other by surgery with endovascular stent-graft repair. CONCLUSIONS: The authors' experience indicates that early diagnosis and an aggressive surgical treatment without delay is the only form of effective therapy for AEF. Endovascular stent-graft repair may be a safe and feasible method for treating patients with AEF that has potential as an improved treatment option for AEF.

Anomalous origin of the left coronary artery from the pulmonary artery confirmed by 320-slice computed tomography.

Anomalous origin the left coronary artery from the pulmonary artery (ALCAPA) is an extremely rare congenital coronary abnormality that may be difficult to diagnose by echocardiography. Most patients present with a potentially fatal illness leading to sudden cardiac death during infancy. This report describes a 15-year-old girl who had 15-year history of cardiac murmur but with no clinical symptoms. Echocardiographic examination was normal, but a 320-slice computed tomographic (CT) scan showed the anomalous origin of the left coronary artery form the pulmonary artery. This case demonstrates that the 320-slice CT scan is a sensitive and reliable technique for establishing the diagnosis of ALCAPA in both symptomatic and asymptomatic patients when it cannot be visualized by echocardiography.

Descending aortic aneurysm associated with coarctation.

A large aneurysm of the descending aorta associated with coarctation is an extremely rare congenital coronary abnormality. This report presents a case of descending aortic large aneurysm associated with coarctation, which was confirmed by 16-slice computed tomography.

Toll-like receptor 4/nuclear factor-kappa B pathway is involved in myocardial injury in a rat chronic stress model.

Chronic stress is considered to predispose to various cardiovascular events such as coronary artery disease, hypertension, and even heart failure. In this study, rats were exposed to stress for 1 day, 1, 2, 3, and 4 weeks to establish a chronic stress model. A specific toll-like receptor 4 (TLR4) antagonist eritoran was used to block the activity of TLR4. On the second day after the last stress exposure, the animals were killed. The expression of TLR4 mRNA and nuclear factor-kappa B (NF-κB) DNA-binding activity in the myocardium were measured using reverse transcriptase polymerase chain reaction and electrophoretic mobility shift assay. The proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL-6) in myocardium were assayed by enzyme-linked immunosorbent assay. Myocardial injury was evident after chronic stress for 2 weeks. The TLR4 mRNA expression reached a peak after stress for 1 week. It was sustained at a stable level after stress exposure for 3 weeks and was restored to a nearly normal level in the fourth week. NF-κB DNA-binding activity was significantly enhanced after the stress for 1 day and markedly enhanced again after a 2-week stress exposure. It was weakened and reached a normal level after stress exposure for 4 weeks. The levels of TNF-α and IL-6 gradually increased and reached peaks after stress for 4 weeks. Meanwhile, eritoran significantly decreased the TLR4 mRNA expression and NF-κB activity in rats from the 2-week stress group. However, it did not downregulate the levels of TNF-α and IL-6. Importantly, it significantly improved the myocardial injury induced by the chronic stress. In conclusion, TLR4/NF-κB participates in myocardial injury during chronic stress.

Remote Ischemic Postconditioning Inhibits Hippocampal Neuronal Apoptosis and Mitophagy After Cardiopulmonary Resuscitation in Rats.

BACKGROUND: Studies have shown that remote ischemic post-conditioning can improve brain damage caused by ischemia and hypoxia. However, the specific mechanism underlying this phenomenon is still unclear. The purpose of this study was to investigate the effects of remote ischemic post-conditioning on neuronal apoptosis and mitophagy after cardiopulmonary resuscitation (CPR) in rats. METHODS: Male Sprague-Dawley rats were used to establish an asphyxia cardiac arrest model by clamping the tracheal duct. First, the expression levels of P53, Cytochrome c (Cytc), and Parkin in the cytoplasm and mitochondria were observed at 3, 6, 24, and 72 h after the restoration of spontaneous circulation (ROSC). Then neurological deficit scores, hippocampal neuron apoptosis, mitochondrial P53 and Parkin, cytoplasmic Cytc, and neuron ultrastructure were evaluated 24 h after ROSC. RESULTS: P53 and Parkin can translocate from the cytoplasm to the mitochondria, promoting the translocation of cytoplasmic Cytc to mitochondria after CPR, reaching a peak at 24 h after the ROSC. The P53 inhibitor Pifithrin-µ reduced apoptosis induced by P53 mitochondrial translocation. Apoptosis was induced after cardiac arrest and attenuated by remote ischemic postconditioning via inhibiting P53 mitochondrial translocation and the release of Cytc to the cytoplasm. In addition, remote ischemic postconditioning could inhibit Parkin-mediated mitophagy. CONCLUSION: Taken together, our results show that remote ischemic post-conditioning improves neural function after CPR by inhibiting P53 mitochondrial translocation-induced apoptosis and Parkin-mediated mitophagy.

Clinical Characteristics and Prognosis of 218 Patients With COVID-19: A Retrospective Study Based on Clinical Classification.

Background: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that has spread worldwide. Methods: This was a retrospective case series involving 218 patients admitted to three tertiary hospitals in the Loudi, Shaoyang, and Xiangtan areas of China from January 21 to June 27, 2020, who were confirmed by RT-PCR to have SARS-CoV-2. The patients' clinical characteristics, laboratory results, treatments, and prognoses based on clinical classification were recorded. Poor outcome was defined as admission to an ICU, the use of mechanical ventilation, or death. Results: The patients were classified into four clinical groups based on disease severity, namely mild (10/218, 5%), moderate (146/218, 67%), severe (24/218, 11%), or critical (14/218, 6%); 24 (11%) asymptomatic cases were also included in the study. The most common symptoms were self-reported cough (162/218, 74%), fever (145/218, 67%), sputum production (99/218, 45%), and fatigue (77/218, 35%). Among the 218 patients, 192 (88%) received lopinavir/ritonavir and interferon-alpha inhalation, and 196 (90%) patients received traditional Chinese medicine. Among the severe and critical patients, 25 (11%) were admitted to an ICU with or without mechanical ventilation, and one patient died. The presence of diabetes [relative risk (RR), 3.0; 95% CI, 1.3-6.8; p = 0.007) or other comorbidities (RR, 5.9; 95% CI, 1.9-17.8; p = 0.002) was independently associated with poor outcome. To date, 20 (9%) patients have retested positive for SARS-CoV-2 RNA after recovering and being discharged. Conclusion: The majority of patients in this case series were clinically classified as having moderate COVID-19. Older patients tended to present with greater levels of clinical severity. The prognosis for patients who were elderly or had diabetes or other chronic comorbidities was relatively poor.

Hypertonic saline infusion suppresses apoptosis of hippocampal cells in a rat model of cardiopulmonary resuscitation.

Hypertonic saline (HS) attenuates cerebral edema, improves microcirculation perfusion and alleviates inflammation. However, whether the beneficial effect of HS on neurological function after cardiopulmonary resuscitation (CPR) in rat model of asphyxial cardiac arrest (CA) is mediated via attenuating apoptosis of neurons is not known. We studied the neuroprotective effect of HS in rats after CA and CPR, and explored the likely underlying mechanisms. Animals were randomly assigned to 4 equal groups (n = 15 each) according to the different infusions administered during resuscitation: control (C), normal saline (NS), hypertonic saline (HS), and hydroxyethyl starch (HES) groups. NDS at 12, 24, 48 and 72 h post-ROSC in the HS group were significantly higher than those in the NS and HES groups. Western blot analysis demonstrated a significant increase in Bcl-2 expression in HS, as compared to that in the NS and HES groups. However, Bax and Caspase-3 expressions in HS were significantly lower than that in the NS and HES groups. The apoptosis rate in HS was significantly lower than that in the NS and HES groups, suggesting HS treatment during resuscitation could effectively suppress neuronal cell apoptosis in hippocampal CA1 post-ROSC and improve neuronal function.

Management of the vertebral artery during thoracic endovascular aortic repair with coverage of the left subclavian artery.

BACKGROUND: The application of thoracic endovascular aortic repair (TEVAR), a minimally invasive operation, in the aortic arch has been a challenge of cardiovascular surgery in recent years. This study aimed to investigate management of the vertebral artery with coverage of the left subclavian artery (LSA) during TEVAR. METHODS: From January 2007 to September 2014 in the Department of Cardiothoracic Surgery at Wuhan General Hospital of Guangzhou Military Region, 160 patients underwent LSA closure or partial coverage during TEVAR of an aortic lesion near the LSA. The vertebral artery treatment, the reason for the surgical approach selection, and the prognosis were analyzed. RESULTS: In 94 patients with partial LSA coverage during TEVAR, no treatment was provided for the vertebral arteries, revealing blood flow of the left vertebral artery forward into the skull after surgery. For 66 patients with full LSA coverage (closure) during TEVAR, right carotid artery-left common carotid artery bypass surgery was performed before TEVAR in ten patients, without any treatment for the vertebral artery, showing reverse blood flow of the left vertebral artery after surgery. Left common carotid artery-LSA bypass surgery was performed before TEVAR in four patients; right common carotid artery-left common carotid artery-LSA bypass surgery was performed before TEVAR in three cases, and 6 out of these 7 patients underwent proximal LSA ligation, showing no obvious blood flow in the left vertebral artery. The closure of the LSA aortic arch opening using an occluder was performed in one patient, preserving the forward blood flow in the left vertebral artery. Among the 160 patients in this study, postoperative recurrent laryngeal nerve injury occurred in one patient after right common carotid artery-left common carotid artery-LSA bypass surgery, and the remaining 159 patients had no significant severe complications or death within 1 postoperative month. CONCLUSIONS: Appropriate management of the aortic arch branch vessels may expand the application of TEVAR to the aortic arch and reduce complications, especially for high-risk patients who have a difficult time tolerating thoracotomy.

A giant symptomatic cardiac lipoma recurring at the fifth year.

Cardiac lipomas are extremely rare in the heart diseases and only few present with a wide spectrum of clinical signs, including life-threatening arrhythmias and sudden death. We report a 48-year-old woman who with a 2-year history of recurrent dyspnea with mild anemia was admitted to our hospital as a huge mass was found in her mediastinum. After complete surgical tumor resection, she was recurred at the fifth year. This case underlines the giant cardiac lipomas had a slightly higher risk of recurrence over the next five years.

TGF-ß1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling.

The vascular smooth muscle cell (VSMC) phenotypic switch is considered to be the key pathophysiological change in various cardiovascular diseases, such as aortic dissection, atherosclerosis, and hypertension. The results in this study showed that TGF-ß1 promotes the proliferation, migration and morphological changes of VSMC.TGF-ß1 promoted the expressions of PI3K, P-PI3K, AKT, P-AKT, ID2, and OPN protein and suppressed the expressions of α-SMA and SM22α protein; the opposite results were observed for TGF-ß1 inhibitor group, AKT inhibitor group and Combined inhibitors group. After the stimulation of TGF-ß1 signaling, the mRNA levels of PI3K, AKT, ID2, and OPN were the highest, while the mRNA levels of α-SMA and SM22α were the lowest; the opposite results were found in the same groups above. These results suggested the PI3K/AKT/ID2 signaling pathway is involved in TGF-ß1-mediated human aortic VSMC phenotypic switching, that is from a contractile to synthetic phenotype, and Combined inhibitors was more effective in inhibiting the phenotypic switch than a single inhibitor. The Combined inhibitors experiments may provide new avenues for the prevention and treatment of thoracic aortic dissection (TAD) that are based on the pathological effects of phenotypic switching.

Feasibility study on the application of fenestrated stent grafts in canine aortic arches.

OBJECTIVE: To validate the feasibility and effectiveness of applying fenestrated stent grafts in canine aortic arches. METHODS: According to the anatomic characteristics of the aortic arches from four adult beagle dogs, a straight-type aortic coated vascular stent system from Lifetech Scientific (Shenzhen) Co., Ltd. was released in vitro, after which a square window was burnt out at the back tendon of the coated vascular stent with an electrocautery pen, and the fenestrated stent grafts were then returned in the catheter and delivery sheath, following the original release path. Endovascular aortic repair (EVAR) was then performed in the canine aorta. Immediately after surgery, digital subtraction angiography (DSA) and computed tomography (CT) angiography were conducted. On day 3, the dressing was changed, and on day 7, the stitches were removed and CT angiography was reviewed. Animal autopsies were performed 2 weeks after surgery. RESULTS: DSA and CT angiography were conducted in 4 beagles immediately after the experiments. The CT angiography reviewed on day 7 after surgery and the animal autopsy performed two weeks after surgery both revealed that the fenestrated stent grafts were anchored in the canine aortic arch, the openings were aligned against the branch vessels above the aortic arch, and in each branch vessel, the blood flow was smooth, without any obvious internal leakage phenomena. CONCLUSION: An ordinary straight-type coated vascular stent, fenestrated in vitro, followed by the performance of EVAR in the canine aortic arch for in vivo stent implantation, was technically feasible. When a branch coated vascular stent cannot meet the individual needs of the wound, this technology may provide a valuable strategy for clinical thoracic aortic trauma emergencies.

Secondary aortoesophageal fistula after thoracic aortic aneurysm endovascular repair: literature review and new insights regarding the hypothesized mechanisms.

BACKGROUND: Endovascular aortic repair was first performed nearly two decades ago and has become a well-established alternative therapy for many thoracoabdominal aortic diseases. Early survival results with the endovascular aortic repair were impressive, but it also brought many complications. Aortoesophageal fistula is little-known and may be underestimated because it is an unusual complication of thoracic endovascular aortic repair. OBJECTIVE: To provide a review of the general features of aortoesophageal fistula as a little-known complication after thoracic endovascular aortic repair and to present a new insight regarding the hypothesized mechanisms of this complication based on clinical experience. METHODS: The new insights regarding the hypothesized mechanisms built on the literature review and clinical experience. Literature Review from PubMed and Web of Knowledge for relevant studies with English paper. Searches were performed without year, and used the combinations of the following key words: "thoracic aortic aneurysm", "endovascular", "aortoesophageal fistula", "complication". RESULTS: The authors' hypothesized mechanisms of aortoesophageal fistula after thoracic aortic aneurysm endovascular repair include the relatively thin vessel wall on thoracic aortic aneurysm hard to prevent the relatively rigid stent graft projecting the aortic and direct erosion into the esophagus. CONCLUSION: Selecting flexibility and appropriate size stent graft, avoiding the thin aortic wall, and identifying the risk factors may reduce the morbidity of complications with aortoesophageal fistula after thoracic aortic aneurysm endovascular repair.

Remote preconditioning, perconditioning, and postconditioning: a comparative study of their cardio-protective properties in rat models.

OBJECTIVE: Ischemia reperfusion injury is partly responsible for the high mortality associated with induced myocardial injury and the reduction in the full benefit of myocardial reperfusion. Remote ischemic preconditioning, perconditioning, and postconditioning have all been shown to be cardioprotective. However, it is still unknown which one is the most beneficial. To examine this issue, we used adult male Wistar rat ischemia reperfusion models to compare the cardioprotective effect of these three approaches applied on double-sided hind limbs. METHODS: The rats were randomly distributed to the following five groups: sham, ischemia reperfusion, remote preconditioning, remote perconditioning, and remote post-conditioning. The ischemia/reperfusion model was established by sternotomy followed by a 30-min ligation of the left coronary artery and a subsequent 3-h reperfusion. Remote conditioning was induced with three 5-min ischemia/5-min reperfusion cycles of the double-sided hind limbs using a tourniquet. RESULTS: A lower early reperfusion arrhythmia score (1.50 + 0.97) was found in the rats treated with remote perconditioning compared to those in the ischemia reperfusion group (2.33 + 0.71). Meanwhile, reduced infarct size was also observed (15.27 + 5.19% in remote perconditioning, 14.53 + 3.45% in remote preconditioning, and 19.84+5.85% in remote post-conditioning vs. 34.47 + 7.13% in ischemia reperfusion, p<0.05), as well as higher expression levels of the apoptosis-relevant protein Bcl-2/Bax following global (ischemia/reperfusion) injury in in vivo rat heart models (1.255 + 0.053 in remote perconditioning, 1.463 + 0.290 in remote preconditioning, and 1.461 +0.541 in remote post-conditioning vs. 1.003 + 0.159 in ischemia reperfusion, p<0.05). CONCLUSION: Three remote conditioning strategies implemented with episodes of double-sided hind limb ischemia/reperfusion have similar therapeutic potential for cardiac ischemia/reperfusion injury, and remote perconditioning has a greater ability to prevent reperfusion arrhythmia.

Effect of 5-aza-2'-deoxycytidine on cell proliferation of non- small cell lung cancer cell line A549 cells and expression of the TFPI-2 gene.

OBJECTIVE: The present study employed 5-aza-2'-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer (NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene. METHODS: Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 µmol/L 5-Aza-CdR, a specific demethylating agent, for 24 ,48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM) to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), real time polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 gene methylation status, mRNA expression and protein expression. RESULTS: MTT assay showed that the growth of A549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group (0 µmol/L 5-Aza-CdR). After treatment with 0, 1, 5, 10 µmol/L 5-Aza-CdR for 72h, FCM showed their proportion in G0/G1 was 69.7±0.99%, 76.1±0.83%, 83.8±0.35%, 95.5±0.55% respectively (P<0.05), and the proportion in S was 29.8±0.43%, 23.7±0.96%, 15.7±0.75%, 1.73±0.45%, respectively (P<0.05), suggesting 5-Aza-CdR treatment induced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 gene was detected in control group (0 µmol/L 5-Aza-CdR), and demethylation appeared after treatment with 1, 5, 10 µmol/L 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were 1±0, 1.49±0.14, 1.86±0.09 and 5.80±0.15 (P<0.05) respectively. Western blotting analysis showed the relative expression levels of TFPI-2 protein were 0.12±0.01, 0.23±0.02, 0.31±0.02, 0.62±0.03 (P<0.05). TFPI-2 protein expression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration. CONCLUSIONS: TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expression in the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation of TFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be one molecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.