Autophagy decreases alveolar epithelial cell injury by regulating the release of inflammatory mediators

To research the impact of autophagy on alveolar epithelial cell inflammation and its possible mechanism in the early stages of hypoxia, we established a cell hypoxia–reoxygenation model and orthotopic left lung ischemia–reperfusion model. Rat alveolar epithelial cells stably expressing GFP-LC3 were treated with an autophagy inhibitor (3-MA) or an autophagy promoter (rapamycin), followed by hypoxia–reoxygenation treatment for 2, 4, and 6 hr in vitro. In vivo, 20 male Sprague Dawley rats were randomly divided into four groups (model group: No blocking of the hilum in the left lung; control group: Blocking of the hilum in the left lung for 1 hr with dimethyl sulfoxide lavage; 3-MA group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of 3-MA (5 μmol/L) solution lavage; and rapamycin group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of rapamycin (250 nmol/L) solution lavage) to establish an orthotopic left lung ischemia model. This study demonstrated that rapamycin significantly suppressed the nuclear factor kappa B signaling pathway and limited the expression of proinflammatory factors. A contrary result was found after the 3-MA pretreatment. These findings indicate that autophagy reduces ischemia–reperfusion injury by repressing inflammatory signaling pathways in the early stages of hypoxia in vitro and in vivo. Autophagy could be a new protective method for application in lung ischemia–reperfusion 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.     

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage.     

细胞自噬在大鼠缺血/再灌注肺损伤中的调控作用

目的: 探讨细胞自噬在大鼠缺血/再灌注肺损伤中的作用。方法: 随机将40只SD大鼠分为5组(n=8),分别为 ① 假手术组(Sham组):只开胸3.5 h;② 缺血/再灌注组(I/R组):开胸夹闭肺门缺血0.5 h后再灌注3 h;③ 溶剂组(DMSO组):术前1 h腹腔注射DMSO溶液;④自噬激动剂组(Rap组):术前腹腔注射雷帕霉素溶液;⑤自噬抑制剂组(3-MA组):术前1 h腹腔注射3-MA溶液;后三组的其余操作同I/R组。实验结束后处死大鼠,取肺组织,记录并计算肺组织湿/干重比(W/D)、总肺含水量变化(TLW) ,光镜和电镜观察肺组织及细胞形态,计算肺泡损伤率(IAR),Western blot检测自噬相关蛋白的表达情况。结果: 相对于sham组,其余四组肺W/D、TLW、IAR均明显升高,自噬相关蛋白表达明显上升,p-AMPK、Beclin 1、LC3 II 蛋白明显增多,p-mTOR、p62蛋白明显减少(P＜0.05或P＜0.01),光镜下其余各组肺组织有不同程度的水肿渗出,肺泡结构紊乱,电镜下细胞超微结构损伤加重,部分可见自噬小体;与DMSO组相比,3-MA组肺W/D、TLW、IAR明显下降(P＜0.05或P＜0.01),自噬相关蛋白表达明显下降,肺间质水肿较轻,细胞渗出较少,细胞超微结构损伤减轻,未见自噬小体。而I/R、DMSO、Rap组的各项指标变化无统计学差异(P＞0.05)。结论: 肺缺血/再灌注可诱发细胞自噬增强,从而引起大鼠肺损伤。     

Autophagy Activation Contributes to the Neuroprotection of Remote Ischemic Perconditioning Against Focal Cerebral Ischemia in Rats

Remote ischemic perconditioning (RIPer) has been proved to provide potent cardioprotection. However, there are few studies on neuroprotection of RIPer. This study aims to clarify the neuroprotective effect of RIPer and the role of autophagy induced by RIPer against cerebral ischemia reperfusion injury in rats. Using a transient middle cerebral artery occlusion (MCAO) model in rats to imitate focal cerebral ischemia. RIPer was carried out 4 cycles of 10 min ischemia and 10 min reperfusion, with a thin elastic band tourniquet encircled on the bilateral femoral arteries at the start of 10 min after MCAO. Autophagy inhibitor 3-methyladenine (3-MA) and autophagy inducer rapamycin were administered respectively to determine the contribution of autophagy in RIPer. Neurologic deficit scores, infarct volume, brain edema, Nissl staining, TUNEL assay, immunohistochemistry and western blot was performed to analyze the neuroprotection of RIPer and the contribution of autophagy in RIPer. RIPer significantly exerted neuroprotective effects against cerebral ischemia reperfusion injury in rats, and the autophagy-lysosome pathway was activated by RIPer treatment. 3-MA reversed the neuroprotective effects induced by RIPer, whereas rapamycin ameliorated the brain ischemic injury. Autophagy activation contributes to the neuroprotection by RIPer against focal cerebral ischemia in rats.     

The effect of transforming growth factor β1 on the crosstalk between autophagy and apoptosis in the annulus fibrosus cells under serum deprivation

Autophagy and apoptosis are important in maintaining the metabolic homeostasis of intervertebral disc cells, and transforming growth factor-β1 (TGF-β1) is able to delay intervertebral disc degeneration. This study determined the effect of TGF-β1 on the crosstalk between autophagy and apoptosis in the disc cells, with the aim to provide molecular mechanism support for the prevention and treatment of disc degeneration. Annulus fibrosus (AF) cells were isolated and cultured under serum starvation. 10 ng/mL TGF-β1 reduced the apoptosis incidence in the cells under serum starvation for 48 h, down-regulated the autophagy incidence in the cells pretreated with 3-methyladenine (3-MA) or Bafilomycin A (Baf A), partly rescued the increased apoptosis incidence in the cells pretreated with 3-MA, while further reduced the decreased apoptosis incidence in the cells pretreated with Baf A. Meanwhile, TGF-β1 down-regulated the expressions of autophagic and apoptotic markers in the cells under starvation, partly down-regulated the expressions of Beclin-1, LC3 II/I and cleaved caspase-3 in the cells pretreated with 3-MA or Baf A, while significantly decreased the expression of Bax/Bcl-2 in the cells pretreated with Baf A. 3-MA blocked the phosphorylation of both AKT and mTOR and partly reduced the inhibitory effect of TGF-β1 on the expression of LC3 II/I and cleaved caspase-3. TGF-β1 enhanced the expression of p-ERK1/2 and down-regulated the expressions of LC3 II/I and cleaved caspase-3. U0126 partly reversed this inhibitory effect of TGF-β1. In conclusion, TGF-β1 protected against apoptosis of AF cells under starvation through down-regulating excessive autophagy. PI3K–AKT–mTOR and MAPK–ERK1/2 were the possible signaling pathways involved in this process.     

Dual role of chloroquine in liver ischemia reperfusion injury: reduction of liver damage in early phase,but aggravation in late phase

The anti-malaria drug chloroquine is well known as autophagy inhibitor. Chloroquine has also been used as anti-inflammatory drugs to treat inflammatory diseases. We hypothesized that chloroquine could have a dual effect in liver ischemia/reperfusion (I/R) injury: chloroquine on the one hand could protect the liver against I/R injury via inhibition of inflammatory response, but on the other hand could aggravate liver I/R injury through inhibition of autophagy. Rats (n=6 per group) were pre-treated with chloroquine (60 mg/kg, i.p.) 1 h before warm ischemia, and they were continuously subjected to a daily chloroquine injection for up to 2 days. Rats were killed 0.5, 6, 24 and 48 h after reperfusion. At the early phase (i.e., 0–6 h after reperfusion), chloroquine treatment ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines and fewer histopathologic changes. In contrast, chloroquine worsened liver injury at the late phase of reperfusion (i.e., 24–48 h after reperfusion). The mechanism of protective action of chloroquine appeared to involve its ability to modulate mitogen-activated protein kinase activation, reduce high-mobility group box 1 release and inflammatory cytokines production, whereas chloroquine worsened liver injury via inhibition of autophagy and induction of hepatic apoptosis at the late phase. In conclusion, chloroquine prevents ischemic liver damage at the early phase, but aggravates liver damage at the late phase in liver I/R injury. This dual role of chloroquine should be considered when using chloroquine as an inhibitor of inflammation or autophagy in I/R injury.     

mTOR and Beclin1: Two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury

Autophagy is the general term of lysosomal degradation of substances in cells, which is considered the key to maintaining the normal structure and function of the heart. It also has a correlation with several heart diseases, in particular, myocardial ischemia/reperfusion (I/R) injury. At the stage of myocardial ischemia, autophagy degrades nonfunctional cytoplasmic proteins providing the critical nutrients for the critical life activities, thereby suppressing cell apoptosis and necrosis. However, autophagy is likely to affect the heart negatively in the reperfusion stage. Mammalian target of rapamycin (mTOR) and Beclin1 are two vital autophagy-related molecules in myocardial I/R injury playing significant roles in different stages. In the ischemia stage, mTOR plays its roles through AMPK/mTOR and phosphoinositide 3-kinase/Akt/mTOR pathway, whereas Beclin1 plays its roles through its upregulation in the reperfusion stage. A possible interaction between mTOR and Beclin1 has been reported recently, and further studies need to be done to find the underlying interaction between the two molecules in myocardial I/R injury     

Gadd45b prevents autophagy and apoptosis against rat cerebral neuron oxygen-glucose deprivation/reperfusion injury

Autophagic (type II) cell death has been suggested to play pathogenetic roles in cerebral ischemia. Growth arrest and DNA damage response 45b (Gadd45b) has been shown to protect against rat brain ischemia injury through inhibiting apoptosis. However, the relationship between Gadd45b and autophagy in cerebral ischemia/reperfusion (I/R) injury remains uncertain. The aim of this study is to investigate the effect of Gadd45b on autophagy. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) model of rat primary cortex neurons, and lentivirus interference used to silence Gadd45b expression. Cell viability and injury assay were performed using CCK-8 and LDH kit. Autophagy activation was monitored by expression of ATG5, LC3, Beclin-1, ATG7 and ATG3. Neuron apoptosis was monitored by expression of Bcl-2, Bax, cleaved caspase3, p53 and TUNEL assay. Neuron neurites were assayed by double immunofluorescent labeling with Tuj1 and LC3B. Here, we demonstrated that the expression of Gadd45b was strongly up-regulated at 24 h after 3 h OGD treatment. ShRNA-Gadd45b increased the expression of autophagy related proteins, aggravated OGD/R-induced neuron cell apoptosis and neurites injury. ShRNA-Gadd45b co-treatment with autophagy inhibitor 3-methyladenine (3-MA) or Wortmannin partly inhibited the ratio of LC3II/LC3I, and slightly ameliorated neuron cell apoptosis under OGD/R. Furthermore, shRNA-Gadd45b inhibited the p-p38 level involved in autophagy, but increased the p-JNK level involved in apoptosis. ShRNA-Gadd45b co-treatment with p38 inhibitor obviously induced autophagy. ShRNA-Gadd45b co-treatment with JNK inhibitor alleviated neuron cell apoptosis. In conclusion, our data suggested that Gadd45b inhibited autophagy and apoptosis under OGD/R. Gadd45b may be a common regulatory protein to control autophagy and apoptosis.     

Autophagy Reduces Neuronal Damage and Promotes Locomotor Recovery via Inhibition of Apoptosis After Spinal Cord Injury in Rats

Autophagy is an intracellular catabolic mechanism that maintains the balance of proteins, lipids and aging organelles. 3-Methyladenine (3-MA) is a selective inhibitor of autophagy, whereas rapamycin, an antifungal agent, is a specific inducer of autophagy, inhibiting the protein mammalian target of rapamycin. In the present study, we examined the role of autophagy, inhibited by 3-MA and enhanced by rapamycin, in a model of acute spinal cord injury in rats. We found that rapamycin could significantly increase the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 at the injury site. At the same time, the number of neurons and astrocytes with LC3 positive in the spinal cord was upregulated with time. In addition, administration of rapamycin produced an increase in the Basso, Beattie and Bresnahan scores of injured rats, indicating high recovery of locomotor function. Furthermore, expression of the proteins Bcl-2 and Bax was upregulated and downregulated, respectively. By contrast, the results for rats treated with 3-MA, which inhibits autophagy, were the opposite of those seen with the rapamycin-treated rats. These results show that induction of autophagy can produce neuroprotective effects in acute spinal cord injury in rats via inhibition of apoptosis.     

Quercetin attenuates renal ischemia/reperfusion injury via an activation of AMP-activated protein kinase-regulated autophagy pathway

Renal ischemia/reperfusion (I/R) is a major cause of acute renal failure. Quercetin, a flavonoid antioxidant, presents in many kinds of food. The molecular mechanism of quercetin on renal protection during I/R is still unclear. Here, we investigated the role of AMP-activated protein kinase (AMPK)-regulated autophagy in renal protection by quercetin. To investigate whether quercetin protects renal cells from I/R-induced cell injury, an in vitro model of I/R and an in vivo I/R model were used. Cell apoptosis was determined by propidium iodide/annexin V staining. Western blotting and immunofluorescence were used to determine the autophagy. AMPK expression was inhibited with appropriate short hairpin RNA (shRNA). In cultured renal tubular cell I/R model, quercetin decreased the cell injury, up-regulated the AMPK phosphorylation, down-regulated the mammalian target of rapamycin (mTOR) phosphorylation and activated autophagy during I/R. Knockdown of AMPK by shRNA transfection decreased the quercetin-induced autophagy but did not affect the mTOR phosphorylation. In I/R mouse model, quercetin decreased the increased serum creatinine level and altered renal histological score. Quercetin also increased AMPK phosphorylation, inhibited the mTOR phosphorylation and activated autophagy in the kidneys of I/R mice. These results suggest that quercetin activates an AMPK-regulated autophagy signaling pathway, which offers a protective effect in renal I/R injury.     

Hydrogen sulfide protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord ischemia-reperfusion injury

Background

Hydrogen sulfide (H2S), a novel gaseous mediator, has been recognized as an important neuromodulator and neuroprotective agent in the nervous system. The present study was undertaken to study the effects of exogenous H2S on ischemia/reperfusion (I/R) injury of spinal cord and the underlying mechanisms.

Methods

The effects of exogenous H2S on I/R injury were examined by using assessment of hind motor function, spinal cord infarct zone by Triphenyltetrazolium chloride (TTC) staining. Autophagy was evaluated by expressions of Microtubule associated protein 1 light chain 3 (LC3) and Beclin-1 which were determined by using Quantitative Real-Time PCR and Western blotting, respectively.

Results

Compared to I/R injury groups, H₂S pretreatment had reduced spinal cord infarct zone, improved hind motor function in rats. Quantitative Real-Time PCR or Western blotting results showed that H2S pretreatment also downregulated miR-30c expression and upregulated Beclin-1 and LC3II expression in spinal cord. In vitro, miR-30c was showed to exert negative effect on Beclin-1 expression by targeting its 3’UTR in SY-SH-5Y cells treated with Oxygen, Glucose Deprivation (OGD). In rat model of I/R injury, pretreatment of pre-miR-30c or 3-MA (an inhibitor for autophagy) can abrogated spinal cord protective effect of H2S.

Conclusion

H2S protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord hemia-reperfusion injury.     

Fenofibrate reduces cisplatin-induced apoptosis by inhibiting the p53/Puma/Caspase-9 pathway and the MAPK/Caspase-8 pathway rather than by promoting autophagy in murine renal proximal tubular cells

The main lesion of cisplatin nephrotoxicity is damage to proximal tubular cells due to increased apoptosis via the mitochondrial and death receptor pathways, which may be alleviated by appropriate promotion of autophagy. Fenofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-α) activator, is recently reported to promote autophagy as well as protect against cisplatin nephrotoxicity, although the mechanisms were only partially analyzed. Here, the detailed mechanisms of these putative protective effects were investigated in a murine renal proximal tubular (mProx) cell line. Fenofibrate attenuated cisplatin-induced apoptosis of mProx cells based on flow cytometry. As for the mitochondrial apoptotic pathway, the reagent reduced cisplatin-stimulated caspase-3 activation by decreasing the phosphorylation of p53, JNK, and 14-3-3, cytosolic and mitochondrial Puma accumulation, cytochrome C release to the cytosol, and resulting cytosolic caspase-9 activation. Fenofibrate also decreased cisplatin-stimulated activation of caspases-8 by suppressing MAPK and NFkB pathways and reducing the gene expression of TNF-α, TL1A, and Fas, main mediators of the death receptor apoptotic pathway. Autophagy defined by p62 reduction and an increase in LC3 II/I was promoted by fenofibrate in mProx cells under starvation. Autophagy inhibition using 3-MA further increased basal and cisplatin-induced caspase-3 and -8 activation, but had no influence on the inhibitory effects of fenofibrate on caspase activation. In conclusion, our study suggests fenofibrate to be a candidate agent to mitigate cisplatin nephrotoxicity by inhibiting the mitochondrial and death apoptotic pathways rather than by promoting autophagy.     

Peroxisome Proliferator-Activated Receptor-γ Agonist 15d-Prostaglandin J2 Mediates Neuronal Autophagy after Cerebral Ischemia-Reperfusion Injury

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has recently emerged as potential therapeutic agents for cerebral ischemia-reperfusion (I/R) injury because of anti-neuronal apoptotic actions. However, whether PPAR-γ activation mediates neuronal autophagy in such conditions remains unclear. Therefore, in this study, we investigated the role of PPAR-γ agonist 15-PGJ₂ on neuronal autophagy induced by I/R. The expression of autophagic-related protein in ischemic cortex such as LC3-II, Beclin 1, cathepsin-B and LAMP1 increased significantly after cerebral I/R injury. Furthermore, increased punctate LC3 labeling and cathepsin-B staining occurred in neurons. Treatment with PPAR-γ agonist 15d-PGJ₂ decreased not only autophagic-related protein expression in ischemic cortex, but also immunoreactivity of LC3 and cathepsin-B in neurons. Autophagic inhibitor 3-methyladenine (3-MA) decreased LC3-II levels, reduced the infarct volume, and mimicked some protective effect of 15d-PGJ₂ against cerebral I/R injury. These results indicate that PPAR-γ agonist 15d-PGJ₂ exerts neuroprotection by inhibiting neuronal autophagy after cerebral I/R injury. Although the molecular mechanisms underlying PPAR-γ agonist in mediating neuronal autophagy remain to be determined, neuronal autophagy may be a new target for PPAR-γ agonist treatment in cerebral I/R injury.     

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。     

Increased leptin by hypoxic-preconditioning promotes autophagy of mesenchymal stem cells and protects them from apoptosis

Autophagy is the basic catabolic progress involved in cell degradation of unnecessary or dysfunctional cellular components.It has been proven that autophagy could be utilized for cell survival under stresses.Hypoxic-preconditioning(HPC)could reduce apoptosis induced by ischemia and hypoxia/serum deprivation(H/SD)in bone marrow-derived mesenchymal stem cells(BMSCs).Previous studies have shown that both leptin signaling and autophagy activation were involved in the protection against apoptosis induced by various stress,including ischemia-reperfusion.However,it has never been fully understood how leptin was involved in the protective effects conferred by autophagy.In the present study,we demonstrated that HPC can induce autophagy in BMSCs by increased LC3-II/LC3-I ratio and autophagosome formation.Interestingly,similar effects were also observed when BMSCs were pretreated with rapamycin.The beneficial effects offered by HPC were absent when BMSCs were incubated with autophagy inhibitor,3-methyladenine(3-MA).In addition,down-regulated leptin expression by leptin-shRNA also attenuated HPC-induced autophagy in BMSCs,which in turn was associated with increased apoptosis after exposed to sustained H/SD.Furthermore,increased AMP-activated protein kinase phosphorylation and decreased mammalian target of rapamycin phosphorylation that were observed in HPC-treated BMSCs can also be attenuated by down-regulation of leptin expression.Our data suggests that leptin has impact on HPC-induced autophagy in BMSCs which confers protection against apoptosis under H/SD,possibly through modulating both AMPK and mTOR pathway.     

热休克蛋白A5介导的自噬在小鼠脑缺血/再灌注损伤中的作用

目的：探讨热休克蛋白A5（HSPA5）诱导的自噬在小鼠脑缺血/再灌注损伤中的作用。方法：将36只BALB/c小鼠随机分为sham、缺血再灌注（I/R）、vehicle + I/R、3-甲基腺嘌呤（3-MA） + I/R、scramble siRNA + I/R和HSPA5 siRNA + I/R组（n=6）。Sham组只进行手术操作,不插入线栓。I/R采用大脑中动脉阻塞（MCAO）60 min后再灌注24 h。Vehicle + I/R组和3-MA + I/R将5μl 0.9% NaCl或3-MA （30 mg/ml）在MCAO前30 min侧脑室注射。scramble siRNA + I/R组和HSPA5 siRNA + I/R组将5μl scramble siRNA或HSPA5 siRNA （2μg/μl）在MCAO前24 h侧脑室注射。检测神经细胞内自噬体、缺血大脑皮层（LC3）-Ⅱ/LC3-I表达、神经元损伤程度及神经功能缺损。结果：显微镜下sham组小鼠大脑皮层神经细胞形态正常;I/R组小鼠缺血大脑皮层神经元胞质中细胞器减少,自噬体形成。与sham组比较,I/R组缺血大脑皮层LC3-Ⅱ/LC3-I蛋白表达水平显著增高（P < 0.05）;与I/R组相比,3-MA + I/R组或HSPA5 siRNA + I/R组缺血大脑皮层LC3-Ⅱ/LC3-I蛋白表达明显减少（P < 0.05）;3-MA + I/R组及HSPA5 siR-NA + I/R组I/R后脑缺血性损伤及神经系统症状加重（P < 0.05）。结论：HSPA5诱导自噬可能在小鼠局灶性I/R损伤中发挥保护作用。     

Renoprotective effects of angiotensin receptor blocker and stem cells in acute kidney injury: Involvement of inflammatory and apoptotic markers

Cisplatin, Cis-diamminedichloroplatinum (CDDP), is a platinum-based chemotherapy drug, and its chemotherapeutic use is restricted by nephrotoxicity. Inflammatory and apoptotic mechanisms play a central role in the pathogenesis of CDDP-induced acute kidney injury (AKI). The aim of this study was to compare the therapeutic potential of candesartan, angiotensin II receptor blocker, versus bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model of CDDP-induced nephrotoxicity. Adult male Wistar rats (n = 40) were divided into four groups; Normal control: received saline injection, CDPP group: received CDDP injection (6 mg/kg single dose), Candesartan group: received candesartan (10 mg/kg/day) for 10 days + CDDP at day 3, and Stem cells group: received CDDP + BM-MSCs intravenously one day after CDDP injection. The rats were sacrificed seven days after CDDP injection. Significant elevation in serum creatinine and urea, renal levels of tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1, renal expressions of nuclear factor kappa B (NF-κB), p38-mitogen-activated protein kinase (MAPK), caspase-3 and Bcl-2-associated x protein (Bax) were found in CDDP-injected rats when compared to normal rats. Both candesartan and BM-MSCs ameliorated renal function and reduced significantly the inflammatory markers (TNF-α , NF-κB, p38-MAPK and MCP-1) and apoptotic markers (caspase-3 and Bax) in renal tissue after CDDP injection. Candesartan as well as BM-MSCs have anti-inflammatory and anti-apoptotic actions and they can be used as nephroprotective agents against CDDP-induced nephrotoxicity. BM-MSCs is more effective than candesartan in amelioration of AKI induced by CDDP.