Autophagy activation attenuates renal ischemia-reperfusion injury in rats

Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future.     

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.     

Sevoflurane preconditioning activates HGF/Met-mediated autophagy to attenuate hepatic ischemia-reperfusion injury in mice

Sevoflurane (SEV) preconditioning plays a protective effect against liver ischemia reperfusion (IR) injury, while the role of autophagy in SEV-mediated hepatoprotection and the precise mechanism is unclear. In the current study, mice were pretreated with SEV or autophagy inhibitor before liver IR injury. In vitro, primary rat hepatocytes were pretreated with SEV and then exposed to hypoxia/reoxygenation (H/R). Liver function was measured by biochemical and histopathological examinations, and markers associated with inflammation, oxidation, apoptosis and autophagy were subsequently measured. We found that SEV preconditioning dramatically reduced hepatic damage, alleviated cell inflammatory response, oxidative stress and apoptosis in mice suffering hepatic IR injury, whereas these protective effects were abolished by the autophagy inhibitor 3-MA. In addition, pretreatment with SEV markedly activated HGF/Met signaling pathway regulation. Besides, pretreatment with an hepatocyte growth factor (HGF) inhibitor or knocking down HGF expression significantly downregulated phosphorylated met (p-met) and autophagy levels, and abolished the protective effects of SEV against hepatic IR or hepatocyte H/R injury. Conversely, HGF overexpression efficiently increased the p-met and autophagy levels and strengthened the protective effects of SEV. These results indicated that sevoflurane preconditioning ameliorates hepatic IR injury by activating HGF/Met-mediated autophagy.     

Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increasedBcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.     

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。     

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.     

Necrostatin-1 Suppresses Autophagy and Apoptosis in Mice Traumatic Brain Injury Model

Traumatic brain injury (TBI) results in neuronal apoptosis, autophagic cell death and necroptosis. Necroptosis is a newly discovered caspases-independent programmed necrosis pathway which can be triggered by activation of death receptor. Previous works identified that necrostatin-1 (NEC-1), a specific necroptosis inhibitor, could reduce tissue damage and functional impairment through inhibiting of necroptosis process following TBI. However, the role of NEC-1 on apoptosis and autophagy after TBI is still not very clear. In this study, the amount of TBI-induced neural cell deaths were counted by PI labeling method as previously described. The expression of autophagic pathway associated proteins (Beclin-1, LC3-II, and P62) and apoptotic pathway associated proteins (Bcl-2 and caspase-3) were also respectively assessed by immunoblotting. The data showed that mice pretreated with NEC-1 reduced the amount of PI-positive cells from 12 to 48?h after TBI. Immunoblotting results showed that NEC-1 suppressed TBI-induced Beclin-1 and LC3-II activation which maintained p62 at high level. NEC-1 pretreatment also reversed TBI-induced Bcl-2 expression and caspase-3 activation, as well as the ratio of Beclin-1/Bcl-2. Both 3-MA and NEC-1 suppressed TBI-induced caspase-3 activation and LC3-II formation, Z-VAD only inhibited caspase-3 activation but increased LC3-II expression at 24?h post-TBI. All these results revealed that multiple cell death pathways participated in the development of TBI, and NEC-1 inhibited apoptosis and autophagy simultaneously. These coactions may further explain how can NEC-1 reduce TBI-induced tissue damage and functional deficits and reflect the interrelationship among necrosis, apoptosis and autophagy.     

细叶远志皂苷在Aβ23-35诱导SH-SY5Y 细胞氧化损伤中的作用及机制研究

为研究细叶远志皂苷(tenuifolin,TEN)在Aβ25-35诱导SH-SY5Y细胞氧化损伤中的作用,并探讨其作用机制。建立Aβ25-35诱导的细胞损伤模型,细叶远志皂苷以及自噬抑制剂3-MA进行干预,显微镜观察细胞形态变化,试剂盒检测细胞氧化应激水平,RT-qPCR和Westernblot检测细叶远志皂苷以及自噬抑制剂干预前后Beclin-1、LC3、mTOR、AMPK和ULK1mRNA及蛋白水平变化。结果发现,TEN改善Aβ25-35诱导的SH-SY5Y细胞形态损伤和细胞活力下降;降低ROS和MDA浓度,并提高SOD、GSH-Px及过氧化氢酶的活性;增加AMPK和ULK1的表达,减少mTOR的表达及增加Beclin-1和LC3-Ⅱ/Ⅰ的表达水平。而加入3-MA会拮抗TEN的作用。总之,TEN可能通过调控AMPK/mTOR/ULK1通路,增加Beclin-1及LC3-Ⅱ/Ⅰ蛋白水平激活自噬,进而改善Aβ25-35诱导的细胞形态损伤和细胞活力下降,提高细胞抗氧化应激能力,发挥神经保护作用。     

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy

BACKGROUNDTo date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol (6-GIN) is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.AIMTo investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.METHODSThe cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN. ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis. Matrix metalloproteinase (MMP) was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay. TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate. Additionally, autophagy-related proteins (Beclin-1, LC-3, and p62), apoptosis-associated proteins (Bcl-2, Bax, and caspase-3), and PI3K/Akt signaling pathway-related proteins (PI3K and Akt) were evaluated by Western blot analysis. Autophagosomes were detected by transmission electron microscopy in NPMSCs. LC-3 was also detected by immunofluorescence. The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction (RT-PCR), and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.RESULTS6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs, decreased hydrogen peroxide-induced intracellular ROS levels, and inhibited cell apoptosis. 6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression. The MMP, Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide. 6-GIN treatment promoted extracellular matrix (ECM) expression by reducing the oxidative stress injury-induced increase in MMP-13 expression. 6-GIN activated autophagy by increasing the expression of autophagy-related markers (Beclin-1 and LC-3) and decreasing the expression of p62. Autophagosomes were visualized by transmission electron microscopy. Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux. The PI3K/Akt pathway was also found to be activated by 6-GIN. 6-GIN inhibited NPMSC apoptosis and ECM degeneration, in which autophagy and the PI3K/Akt pathway were involved.CONCLUSION6-GIN efficiently decreases ROS levels, attenuates hydrogen peroxide-induced NPMSCs apoptosis, and protects the ECM from degeneration. 6-GIN is a promising candidate for treating IDD.     

Autophagy enhanced phagocytosis of apoptotic cells by oridonin-treated human histocytic lymphoma U937 cells

Macrophages rapidly engulf and remove apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune disease or inflammation. Recent developments reveal an important role in autophagy for clearance of apoptotic corpses. However, the relationship between autophagy and phagocytosis remains unclear. In this study we found that low doses of oridonin, an active diterpenoid, enhanced phagocytosis of apoptotic cells by human macrophage-like U937 cells, meanwhile it also induced autophagy in these U937 cells. Moreover, inhibition of extracellular signal-related kinase (ERK), nuclear factor-κB (NF-κB) and caspase-1 significantly suppressed oridonin-induced phagocytosis and autophagy. In addition, oridonin increased the protein levels of p-ERK, NF-κB, caspase-1 and pro IL-1β. Autophagic inhibitor 3-methyladenine (3-MA) decreased phagocytosis and the expression of ERK whereas increased the expression of NF-κB- and caspase-1-mediated IL-1β release. Beclin-1 (known as autophagic regulator) loss also led to the similar results. Pretreatment with autophagic agonist rapamycin caused opposite results. Autophagy-associated proteins, Beclin-1, LC3 and Atg4B, involved in this phagocytosis process. These results demonstrated that autophagy enhanced oridonin-induced phagocytosis through feedback regulation of ERK, NF-κB- and caspase-1-mediated IL-1β release.     

Bel-7402细胞通过自噬逃避FAS/ActD诱导的凋亡

为了探究FAS抗体与放线菌素D（actinomycin D,ActD）诱导肝癌细胞Bel-7402凋亡的作用机制,通过自噬阻断剂3-MA的作用,来探讨自噬与凋亡的关系.利用电子显微镜和流式细胞仪观察细胞自噬及凋亡.结果表明,FAS/ActD在诱导细胞凋亡的同时伴有细胞自噬现象,在3-MA作用下,FAS/ActD所诱导的细胞自噬体减少,而凋亡现象严重.并且通过流式细胞仪分析表明,3-MA明显增高FAS/ActD所诱导的细胞凋亡率. Western印迹分析进一步显示,FAS/ActD能引起caspase-3激活产生断裂,同时刺激LC3和BECN1表达,而3-MA作用后自噬体减少,同时LC3和BECN1表达降低,但是caspase-3断裂带表达明显增加.以上结果提示,FAS/ActD诱导的Bel-7402细胞凋亡的同时伴有细胞自噬,Bel-7402细胞通过自噬逃避FAS/ActD诱导的凋亡.     

Ginsenoside Rg1 inhibits autophagy in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation

Ginsenoside Rg1 promotes antioxidative protection and intracellular calcium homeostasis in cardiomyocytes hypoxia/reoxygenation (H/R) model. However, the pharmacological effects of G-Rg1 on autophagy in cardiomyocytes have not been reported. In this study, we employed H9c2 cardiomyocytes as a model to investigate the effects of G-Rg1 on autophagy in cardiomyocytes under H/R stress. Our results showed that H/R induced increased level of LC3B-2, an autophagy marker, in a time-dependent manner in association with decreased cell viability and cellular ATP content. H/R-induced autophagy and apoptosis were further confirmed by morphological examination. 100 μmol/l Rg1-inhibited H/R induced autophagy and apoptosis, and this was associated with the increase of cellular ATP content and the relief of oxidative stress in the cells. Mechanistically, we found that Rg1 inhibited the activation of AMPKα, promoted the activation of mTOR, and decreased the levels of LC3B-2 and Beclin-1. In conclusion, our data suggest that H/R induces autophagy in H9c2 cells leading to cell injury. Rg1 inhibits autophagosomal formation and apoptosis in the cells, which may be beneficial to the survival of cardiomyocytes under H/R.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.     

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.     

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.     

白花蛇舌草多糖提取物对喉癌Hep-2细胞内质网自噬的影响*

目的:探讨白花蛇舌草多糖提取物(HDPE)对喉癌Hep-2细胞内质网自噬的影响。方法:实验分为对照组、HDPE 100、200、400 mg/L组和3-MA(自噬抑制剂)组,噻唑盐比色法(MTT)检测各组细胞培养24 h、48 h、72 h后增殖抑制率;原位末端转移酶标记法(TUNEL)法检测各组培养48 h细胞凋亡情况;单丹黄酰尸胺(MDC)染色观察各组培养48 h细胞自噬体及自噬溶酶体的变化;透射电镜观察培养48 h细胞内质网周围自噬囊泡的产生情况;蛋白印迹法(Western blot)检测各组培养48 h细胞Beclin-1蛋白(Beclin-1)、微管相关轻链蛋白3Ⅰ(LC3Ⅰ)、微管相关轻链蛋白3Ⅱ(LC3Ⅱ)、葡萄糖调节蛋白 78(GRP78)、活化转录因子6(ATF6)及CCAAT 增强子结合蛋白同源蛋白(CHOP)表达。结果:与对照组比较,HDPE 100、200、400 mg/L组和3-MA组细胞增殖抑制率、凋亡指数AI升高,MDC阳性细胞率量降低,内质网周围自噬囊泡减少,GRP78、ATF6及CHOP表达及LC3Ⅰ/LC3Ⅱ比值升高,Beclin-1表达降低(P＜0.05);与3-MA组比较,HDPE 400 mg/L组细胞增殖抑制率、凋亡指数AI升高,MDC阳性细胞率、GRP78、ATF6及CHOP表达及LC3Ⅰ/LC3Ⅱ比值升高,Beclin-1表达降低(P＜0.05)。结论:HDPE可能通过抑制喉癌Hep-2细胞内质网自噬,促进细胞内质网应激凋亡,进而抑制Hep-2细胞增殖能力。     

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.     

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。     

Autophagy protects against necrotic renal epithelial cell-induced death of renal interstitial fibroblasts

We recently reported that necrotic renal proximal tubular cells (RPTC) can induce the death of renal interstitial fibroblasts. Since autophagy plays either cytoprotective or cytodestructive roles depending on the experimental condition, the present study was carried out to investigate whether necrotic RPTC would induce autophagy of renal interstitial fibroblasts and, if so, whether autophagy would contribute to cell death or exert a protective effect. Exposure of necrotic RPTC supernatant (RPTC-Sup) induced autophagy in renal interstitial fibroblast cells (NRK-49F) in a time- and dose-dependent manner, and its induction was earlier than caspase-3 activation. Inhibition of autophagy with 3-methyladenine (3-MA) or knockdown of Beclin-1, a molecule involved in the initiation of autophagosome formation, with small interference RNA (siRNA) significantly enhanced necrotic RPTC-Sup-induced cell death. Necrotic RPTC-Sup induced phosphorylation of extracellular signal-regulated kinases (ERK1/2), p38, c-Jun NH(2)-terminal kinases (JNKs), and AKT. Treatment with an ERK1/2 pathway inhibitor, but not with specific inhibitors for p38, JNKs, or AKT pathways, blocked NRK-49F autophagy and cell death upon exposure to necrotic RPTC-Sup. Furthermore, knockdown of MEK1 with siRNA also reduced autophagy along with cell death in NRK-49F exposed to necrotic RPTC-Sup. In contrast, overexpression of MEK1/2 increased RPTC-Sup-induced fibroblast cell death without enhancing autophagy. Collectively, this study demonstrates that necrotic RPTC induce both autophagy and cell death and that autophagy plays a cytoprotective or prosurvival role in renal fibroblasts. Furthermore, necrotic RPTC-induced autophagy and cell death in renal fibroblasts is mediated by the activation of the MEK1-ERK1/2 signaling pathway.     

The role of AKT1 and autophagy in the protective effect of hydrogen sulphide against hepatic ischemia/reperfusion injury in mice

Hydrogen sulphide (H 2S) exerts a protective effect in hepatic ischemia-reperfusion (I/R) injury. However, the exact mechanism of H 2S action remains largely unknown. This study was designed to investigate the role of the PtdIns3K-AKT1 pathways and autophagy in the protective effect of H 2S against hepatic I/R injury. Primary cultured mouse hepatocytes and livers with or without NaHS (a donor of H 2S) preconditioning were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. In certain groups, they were also pretreated with LY294002 (AKT1-specific inhibitor), 3-methyladenine (3MA, autophagy inhibitor) or rapamycin (autophagy enhancer), alone or simultaneously. Cell viability, expression of P-AKT1, T-AKT1, LC3 and BECN1 were examined. The severity of liver injury was measured by the levels of serum aminotransferase and inflammatory cytokine, apoptosis and histological examination. GFP-LC3 redistribution and transmission electron microscopy were used to test the activity of autophagy. H 2S preconditioning activated PtdIns3K-AKT1 signaling in hepatocytes. LY294002 could abolish the AKT1 activation and attenuate the protective effect of H 2S on hepatocytes A/R and hepatic I/R injuries. H 2S suppressed hepatic autophagy in vitro and in vivo. Further reducing autophagy by 3MA also diminished the protective effect of H 2S, while rapamycin could reverse the autophagy inhibitory effect and enhance the protective effect of H 2S against hepatocytes A/R and hepatic I/R injuries, consequently. Taken together, H 2S protects against hepatocytic A/R and hepatic I/R injuries, at least in part, through AKT1 activation but not autophagy. An autophagy agonist could be applied to potentiate this hepatoprotective effect by reversing the autophagy inhibition of H 2S.