AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

AMPK Mediates Autophagy during Myocardial Ischemia In Vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.

Addendum to:

Distinct Roles of Autophagy in the Heart During Ischemia and Reperfusion: Roles of AMP-Activated Protein Kinase and Beclin 1 in Mediating Autophagy

Y. Matsui, H. Takagi, X. Qu, M. Abdellatif, H. Sakoda, T. Asano, B. Levine and J. Sadoshima

Circ Res 2007; 100:914-22     

Nitric oxide-induced cell death in the heart: the role of autophagy

There is unequivocal evidence of autophagy in the heart, both in human hearts from patients who experienced heart failure and in experimental models of myocardial ischemia and reperfusion. Whether autophagy is involved in the pathophysiology of these conditions is controversial as studies suggest inhibition of Beclin 1 can increase or decrease cardiomyocyte cell injury. Increased beclin 1 expression, however, has been consistently identified in myocardial ischemia/reperfusion. Because of the role of nitric oxide (NO) in myocardial ischemia/reperfusion as well as in heart failure, we sought to determine whether NO and its byproduct peroxynitrite alter the expression of some genes involved in autophagy in the heart. Neonatal mouse cardiomyocytes were treated with SIN-1 (3-morpholinosydnonimine), which releases NO and accelerates formation of peroxynitrite. Gene expression was evaluated using RNA labeled and hybridized to cDNA microarrays. SIN-1 treatment induced significant changes in five caspases. In contrast, there were no changes in three genes involved in autophagy, namely beclin 1, Atg5l and Atg12l. Several different time periods were examined; a short time period, 2h, to more closely model myocardial ischemia reperfusion and a long time period, 20 h, that more closely represents sustained injury. In summary, evidence to date suggests that NO is not involved in increased beclin 1 expression in ischemia/reperfusion injury in the heart and would be unlikely to account for the signs of autophagy in the hearts of patients with heart failure.     

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia–reperfusion injury in hypertrophic heart

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91^phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.     

Propofol protects the autophagic cell death induced by the ischemia/reperfusion injury in rats

Autophagy has been implicated in cardiac cell death during ischemia/reperfusion (I/R). In this study we investigated how propofol, an antioxidant widely used for anesthesia, affects the autophagic cell death induced by the myocardial I/R injury. The infarction size in the myocardium was dramatically reduced in rats treated with propofol during I/R compared with untreated rats. A large number of autophagic vacuoles were observed in the cardiomyocytes of I/R-injured rats but rarely in I/R-injured rats treated with propofol. While LC3-II formation, an autophagy marker, was up-regulated in the I/R-injured myocardium, it was significantly down-regulated in the myocardial tissues of I/R-injured and propofol-treated rats. Moreover, propofol inhibited the I/R-induced expression of Beclin-1, and it accelerated phosphorylation of mTOR during I/R and Beclin-1/Bcl-2 interaction in cells, which indicates that it facilitates the inhibitory pathway of autophagy. These data suggest that propofol protects the autophagic cell death induced by the myocardial I/R injury.     

Protective Role of AMP-Activated Protein Kinase-Evoked Autophagy on an In Vitro Model of Ischemia/Reperfusion-Induced Renal Tubular Cell Injury

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.     

Targeting autophagy in cardiac ischemia/reperfusion injury: A novel therapeutic strategy

Acute myocardial infarction (AMI) is one of the leading causes of morbidity worldwide. Myocardial reperfusion is known as an effective therapeutic choice against AMI. However, reperfusion of blood flow induces ischemia/reperfusion (I/R) injury through different complex processes including ion accumulation, disruption of mitochondrial membrane potential, the formation of reactive oxygen species, and so forth. One of the processes that gets activated in response to I/R injury is autophagy. Indeed, autophagy acts as a “double-edged sword” in the pathology of myocardial I/R injury and there is a controversy about autophagy being beneficial or detrimental. On the basis of the autophagy effect and regulation on myocardial I/R injury, many studies targeted it as a therapeutic strategy. In this review, we discuss the role of autophagy in I/R injury and its targeting as a therapeutic strategy.     

UCP2 protect the heart from myocardial ischemia/reperfusion injury via induction of mitochondrial autophagy

Uncoupling protein 2 (UCP2), located in the mitochondrial inner membrane, is a predominant isoform of UCP that expressed in the heart and other tissues of human and rodent tissues. Nevertheless, its functional role during myocardial ischemia/reperfusion (I/R) is not entirely understood. Ischemic preconditioning (IPC) remarkably improved postischemic functional recovery followed by reduced lactate dehydrogenase (LDH) release with simultaneous upregulation of UCP2 in perfused myocardium. We then investigated the role of UCP2 in IPC-afforded cardioprotective effects on myocardial I/R injury with adenovirus-mediated in vivo UCP2 overexpression (AdUCP2) and knockdown (AdshUCP2). IPC-induced protective effects were mimicked by UCP2 overexpression, while which were abolished with silencing UCP2. Mechanistically, UCP2 overexpression significantly reinforced I/R-induced mitochondrial autophagy (mitophagy), as measured by biochemical hallmarks of mitochondrial autophagy. Moreover, primary cardiomyocytes infected with AdUCP2 increased simulated ischemia/reperfusion (sI/R)-induced mitophagy and therefore reversed impaired mitochondrial function. Finally, suppression of mitophagy with mdivi-1 in cultured cardiomyocytes abolished UCP2-afforded protective effect on sI/R-induced mitochondrial dysfunction and cell death. Our data identify a critical role for UCP2 against myocardial I/R injury through preventing the mitochondrial dysfunction through reinforcing mitophagy. Our findings reveal novel mechanisms of UCP2 in the cardioprotective effects during myocardial I/R.     

Protective Effect of Sevoflurane Postconditioning against Cardiac Ischemia/Reperfusion Injury via Ameliorating Mitochondrial Impairment,Oxidative Stress and Rescuing Autophagic Clearance

Background and Purpose

Myocardial infarction leads to heart failure. Autophagy is excessively activated in myocardial ischemia/reperfusion (I/R) in rats. The aim of this study is to investigate whether the protection of sevoflurane postconditioning (SPC) in myocardial I/R is through restored impaired autophagic flux.

Methods

Except for the sham control (SHAM) group, each rat underwent 30 min occlusion of the left anterior descending coronary (LAD) followed by 2 h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by hemodynamics and echocardiography. The activation of autophagy was evaluated by autophagosome accumulation, LC3 conversion and p62 degradation. Potential molecular mechanisms were investigated by immunoblotting, real-time PCR and immunofluorescence staining.

Results

SPC improved the hemodynamic parameters, cardiac dysfunction, histopathological and ultrastructural damages, and decreased myocardial infarction size after myocardial I/R injury (P < 0.05 vs. I/R group). Compared with the cases in I/R group, myocardial ATP and NAD⁺ content, mitochondrial function related genes and proteins, and the expressions of SOD2 and HO-1 were increased, while the expressions of ROS and Vimentin were decreased in the SPC group (P < 0.05 vs. I/R group). SPC significantly activated Akt/mTOR signaling, and inhibited the formation of Vps34/Beclin1 complex via increasing expression of Bcl2 protein (P < 0.05 vs. I/R group). SPC suppressed elevated expressions of LC3 II/I ratio, Beclin1, Atg5 and Atg7 in I/R rat, which indicated that SPC inhibited over-activation of autophagy, and promoted autophagosome clearance. Meanwhile, SPC significantly suppressed the decline of Opa1 and increases of Drp1 and Parkin induced by I/R injury (P < 0.05 vs. I/R group). Moreover, SPC maintained the contents of ATP by reducing impaired mitochondria.

Conclusion

SPC protects rat hearts against I/R injury via ameliorating mitochondrial impairment, oxidative stress and rescuing autophagic clearance.     

Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.     

Zinc and myocardial ischemia/reperfusion injury

As an important trace element, zinc is required for the normal cellular structure and function, and impairment of zinc homeostasis is associated with a variety of health problems including cardiovascular disease. Zinc homeostasis is regulated through zinc transporters, zinc binding molecules, and zinc sensors. Zinc also plays a critical role in cellular signaling. Studies have documented that zinc homeostasis is impaired by ischemia/reperfusion in the heart and zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. Both exogenous and endogenously released zinc may play an important role in cardioprotection against ischemia/reperfusion injury. The goal of this review is to summarize the current understanding of the roles of zinc homeostasis and zinc signaling in myocardial ischemia/reperfusion injury.     

Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling

The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre‐treatment of DSS significantly improved cell survival and protected against I/R‐induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy‐ and apoptosis‐related proteins (including p62, LC3‐II, Beclin‐1, Bax, and Caspase‐3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre‐treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation.     

Inhibition of 12/15 lipoxygenase by baicalein reduces myocardial ischemia/reperfusion injury via modulation of multiple signaling pathways

12/15-Lipoxygenase (LOX) is a member of the LOX family that catalyzes the step from arachidonic acid to hydroxy-eicosatetraenoic acids (HETEs). Previous studies demonstrated that 12/15-LOX plays a critical role in the development of atherosclerosis, hypertension, heart failure, and other diseases; however, its role in myocardial ischemic injury was contraversal. Here, we investigated the inhibition of 12/15-LOX by baicalein on acute cardiac injury and dissected its molecular mechanism. In a mouse model of acute ischemia/reperfusion (I/R) injury, 12/15-LOX was significantly upregulated in the peri-infarct area surrounding the primary infarction. In cultured cardiac myocytes, baicalein suppressed apoptosis and caspase 3 activity in response to simulated ischemia/reperfusion (I/R). Moreover, administration of 12/15-LOX inhibitor, baicalein, significantly attenuated myocardial infarct size induced by I/R injury. Moreover, baicalein treatment significantly inhibited cardiomyocyte apoptosis, inflammatory responses and oxidative stress in the heart after I/R injury. The mechanisms underlying these effects were associated with the activation of ERK1/2 and AKT pathways and inhibition of activation of p38 MAPK, JNK1/2, and NF-kB/p65 pathways in the I/R-treated hearts and neonatal cardiomyoctes. Our data indicated that 12/15-LOX inhibitor baicalein can prevent myocardial I/R injury by modulation of multiple mechanisms, and suggest that baicalein could represent a novel therapeutic drug for acute myocardial infarction.     

细胞自噬在大鼠肺缺血/再灌注引发肝脏损伤中的作用*

目的: 探讨肺缺血/再灌注(LI/R)时肝脏损伤的影响,并初步探索细胞自噬(Autophagy)在其中发挥的作用。方法: 构建大鼠缺血/再灌注肺损伤(LI/RI)模型,模型制备方法为大鼠麻醉后切开气管进行机械通气,使用动脉夹将肺门夹闭模拟缺血过程,30 min后松开动脉夹,恢复灌注3 h。24只大鼠随机分为伪手术组(Sham组)、缺血/再灌注组(I/R组)、溶剂组(DMSO组)和自噬抑制剂组(3-MA组),每组均6只,后2组大鼠术前分别腹腔注射DMSO和3-MA,造模结束后使用肺湿/干重比判断造模是否成功;抽取静脉血测定肝脏转氨酶指标ALT与AST;取肝脏组织,光镜下观察肝脏形态改变,以及电镜下观察肝细胞超微结构;使用RT-qPCR和Western blot实验分别检测肝脏组织细胞中自噬相关蛋白的基因mRNA表达水平和蛋白表达水平。结果: 与Sham组相比,其余各组肺湿/干重比均升高;血AST和ALT均有大幅升高且肝脏组织损伤明显,其中以I/R组升高最为明显,光镜下组织形态学及电镜下细胞微细结构均有不同程度的破坏;肝脏中自噬相关蛋白的基因表达水平与蛋白表达水平均有明显不同,表现为自噬上升 (P＜0.01或P＜0.05)。I/R组和DMSO组肝脏组织均有较重损伤,肝细胞结构破坏严重,自噬小体形成,而AST、ALT、自噬相关蛋白转录和表达水平等各项指标均无统计学差异(P＞0.05)。而相较于DMSO组,3-MA组肝脏组织损伤有所减轻,肝细胞微细结构损伤程度低,且无自噬小体形成,血中AST和ALT下降,肝脏组织内自噬水平均下降 (P＜0.05)。结论: 肺缺血/再灌注可引起大鼠肝损伤;细胞自噬可介导大鼠肺缺血/再灌注引起的肝损伤,抑制细胞自噬可以有效减轻大鼠LI/R引起的肝损伤。     

sRAGE抑制缺血/再灌注引起的心肌细胞自噬

可溶性晚期糖基化终末产物受体（soluble receptor for advanced glycation end products,sRAGE）作为内源性保护物质,能够拮抗心肌缺血/再灌注(ischemia/reperfusion,I/R）损伤发生,重要机制是减轻心肌细胞凋亡。而近年来随着细胞死亡研究的深入,细胞自噬被认为是一种新的细胞程序性死亡。sRAGE是否可以抑制缺血/再灌引起的心肌细胞自噬尚未见报道。本文研究证明,sRAGE可抑制缺血/再灌注引起的心肌细胞自噬。以心肌细胞缺氧/复氧模拟心肌细胞缺血/再灌注模型,蛋白质印迹检测自噬门户蛋白beclin-1的表达,激光共聚焦显微镜检测自噬小体及自噬溶酶体的形成。心肌再灌注期间,心肌细胞自噬小体增加,而自噬溶酶体下降。细胞内自噬小体堆积,说明心肌细胞缺血/再灌注使自噬小体与溶酶体结合受损,清除发生障碍。与缺血/再灌注（I/R）组比较,缺血/再灌+sRAGE（I/R+sRAGE）组的自噬流减弱。此外,自噬门户蛋白beclin-1也表达下降。以上结果从细胞形态学和蛋白水平两方面说明,sRAGE抑制了I/R引起的心肌细胞自噬。换言之,sRAGE可以直接作用于心肌细胞拮抗缺血/再灌注损伤,其保护性作用可能与抑制心肌细胞自噬有关。     

Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.     

Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy

Cerebral ischemia/reperfusion (I/R) typically occurs after mechanical thrombectomy to treat ischemic stroke, generation of reactive oxygen species (ROS) after reperfusion may result in neuronal insult, ultimately leading to disability and death. Regulated in development and DNA damage responses 1 (REDD1) is a conserved stress response protein under various pathogenic conditions. Recent research confirms the controversial role of REDD1 in injury processes. Nevertheless, the role of REDD1 in cerebral I/R remains poorly defined. In the current study, increased expression of REDD1 was observed in neurons exposed to simulated I/R via oxygen glucose deprivation/reoxygenation (OGD/R) treatment. Knockdown of REDD1 enhanced OGD/R-inhibited cell viability, but suppressed lactate dehydrogenase (LDH) release in neurons upon OGD/R. Simultaneously, suppression of REDD1 also antagonized OGD/R-evoked cell apoptosis, Bax expression, and caspase-3 activity. Intriguingly, REDD1 depression abrogated neuronal oxidative stress under OGD/R condition by suppressing ROS, MDA generation, and increasing antioxidant SOD levels. Further mechanism analysis corroborated the excessive activation of autophagy in neurons upon OGD/R with increased expression of autophagy-related LC3 and Beclin-1, but decreased autophagy substrate p62 expression. Notably, REDD1 inhibition reversed OGD/R-triggered excessive neuronal autophagy. More importantly, depression of REDD1 also elevated the expression of p-mTOR. Preconditioning with mTOR inhibitor rapamycin engendered not only a reduction in mTOR activation, but also a reactivation of autophagy in REDD1 knockdown-neurons upon OGD/R. In addition, blocking the mTOR pathway muted the protective roles of REDD1 inhibition against OGD/R-induced neuron injury and oxidative stress. Together these data suggested that REDD1 may regulate I/R-induced oxidative stress injury in neurons by mediating mTOR-autophagy signaling, supporting a promising therapeutic strategy against brain ischemic diseases.     

High mobility box 1 mediates neutrophil recruitment in myocardial ischemia-reperfusion injury through toll like receptor 4-related pathway

This study aimed to explore the role of high mobility box 1 (HMGB1) and its receptor toll like receptor 4 (TLR4) on neutrophils in myocardial ischemia reperfusion (I/R) injury. We constructed TLR4-mutant (C3H/HeJ) and control (C3H/HeN) mouse models of myocardial I/R injury and subjected the mice to 30min of ischemia and 6h of reperfusion. Light microscope was used to observe structural changes in the myocardium. HMGB1 levels were measured using quantitative real-time PCR and immunohistochemistry. Neutrophil accumulation, TNF-a expression and IL-8 levels were analyzed via myeloperoxidase (MPO) biochemical studies, quantitative real-time PCR and ELISA, respectively. The results demonstrated that fewer neutrophils infiltrated in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency markedly decreased the ischemic injury caused by ischemia/reperfusion, and inhibited the expression of HMGB1, TNF-a, and IL-8, all of which were up-regulated by ischemia/reperfusion. These findings suggest that HMGB1 plays a central role in recruiting neutrophils during myocardial I/R leading to worsened myocardial I/R injury. This recruitment mechanism is possibly due to its inflammatory and chemokine functions based on the TLR4-dependent pathway.     

Kaempferol Mediated AMPK/mTOR Signal Pathway Has a Protective Effect on Cerebral Ischemic-Reperfusion Injury in Rats by Inducing Autophagy

Ischemia/reperfusion (I/R) caused by ischemic stroke treatments leads to brain injury and its pathological mechanism is related to autophagy. The underlying mechanism of kaempferol on cerebral I/R injury needs to be explored. To establish I/R injury, we used a middle cerebral artery occlusion-reperfusion (MCAO) model in rats. MCAO rats were treated with the same amount of saline (I/R group); Treatment group rats were treated orally with kaempferol (50, 100, 200 mg/kg) for 7 days before surgery. After reperfusion for 24 h, the scores of neurological deficits and infarct volume in each group were evaluated. LC3, Beclin-1 p62, AMPK and mTOR protein expression levels were examined by TTC staining, immunofluorescence staining, qRT-PCR and western blotting assay. H&E and TTC staining showed that compared with model group, the infarction size of rats in kaempferol group was markedly reduced. Meanwhile, the results showed that kaempferol had a dose-dependent nerve function repairability. Nissl and TUNEL staining showed that kaempferol could reduce neuronal apoptosis and ameliorate neuronal impairment after I/R. Western blotting and qRT-PCR results showed that kaempferol could protect the brain from ischemia reperfusion by activating autophagy. In addition, add AMPK inhibitor, western blotting and immumohistochemical staining showed that kaempferol mediated AMPK/mTOR signal pathway in MCAO rats. Kaempferol could mediate the AMPK signal pathway to regulate autophagy and inhibit apoptosis to protect brain against I/R injury.

     

Inhibition of mitochondrial autophagy protects donor lungs for lung transplantation against ischaemia‐reperfusion injury in rats via the mTOR pathway

Impaired mitochondrial function is a key factor attributing to lung ischaemia‐reperfusion (IR) injury, which contributes to major post‐transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3‐MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia‐reoxygenation (H/R) were monitored after 3‐MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3‐MA inhibited cell apoptosis of donor lung to alleviate I/R‐induced lung injury as well as inhibited H/R‐induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R‐ and H/R‐mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R‐induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury.