Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

The Role of Mitochondrial Impairment in Alzheimer´s Disease Neurodegeneration: The Tau Connection

Accumulative evidence has shown that mitochondrial dysfunction plays a pivotal role in the pathogenesis of Alzheimer''s disease (AD). Mitochondrial impairment actively contributes to the synaptic and cognitive failure that characterizes AD. The presence of soluble pathological forms of tau like hyperphosphorylated at Ser396 and Ser404 and cleaved at Asp421 by caspase 3, negatively impacts mitochondrial bioenergetics, transport, and morphology in neurons. These adverse effects against mitochondria health will contribute to the synaptic impairment and cognitive decline in AD. Current studies suggest that mitochondrial failure induced by pathological tau forms is likely the result of the opening of the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial mega-channel that is activated by increases in calcium and is associated with mitochondrial stress and apoptosis. This structure is composed of different proteins, where Ciclophilin D (CypD) is considered to be the primary mediator of mPTP activation. Also, new studies suggest that mPTP contributes to Aβ pathology and oxidative stress in AD.Further, inhibition of mPTP through the reduction of CypD expression prevents cognitive and synaptic impairment in AD mouse models. More importantly, tau protein contributes to the physiological regulation of mitochondria through the opening/interaction with mPTP in hippocampal neurons. Therefore, in this paper, we will discuss evidence that suggests an important role of pathological forms of tau against mitochondrial health. Also, we will discuss the possible role of mPTP in the mitochondrial impairment produced by the presence of tau pathology and its impact on synaptic function present in AD.     

Adaptive changes in autophagy after UPS impairment in Parkinson's disease

Aim:

Ubiquitin-proteasome system (UPS) and autophagosome-lysosome pathway (ALP) are the most important machineries responsible for protein degradation in Parkinson''s disease (PD). The aim of this study is to investigate the adaptive alterations in autophagy upon proteasome inhibition in dopaminergic neurons in vitro and in vivo.

Methods:

Human dopaminergic neuroblastoma SH-SY5Y cells were treated with the proteasome inhibitor lactacystin (5 μmol/L) for 5, 12, or 24 h. The expression of autophagy-related proteins in the cells was detected with immunoblotting. UPS-impaired mouse model of PD was established by microinjection of lactacystin (2 μg) into the left hemisphere of C57BL/6 mice that were sacrificed 2 or 4 weeks later. The midbrain tissues were dissected to assess alterations in autophagy using immunofluorescence, immunoblotting and electron microscopy assays.

Results:

Both in SH-SY5Y cells and in the midbrain of UPS-impaired mouse model of PD, treatment with lactacystin significantly increased the expression levels of LC3-I/II and Beclin 1, and reduced the levels of p-mTOR, mTOR and p62/SQSTM1. Furthermore, lactacystin treatment in UPS-impaired mouse model of PD caused significant loss of TH-positive neurons in the substantia nigra, and dramatically increased the number of autophagosomes in the left TH-positive neurons.

Conclusion:

Inhibition of UPS by lactacystin in dopaminergic neurons activates another protein degradation system, the ALP, which includes both the mTOR signaling pathway and Beclin 1-associated pathway.     

Lactacystin requires reactive oxygen species and Bax redistribution to induce mitochondria-mediated cell death

Background and purpose:

The proteasome inhibitor model of Parkinson''s disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin-induced, mitochondria-mediated apoptotic pathway remain poorly defined.

Experimental approach:

We have used lactacystin as a specific inhibitor of the 20S proteasome in the dopaminergic neuroblastoma cell line SH-SY5Y. We over-expressed a green fluorescent protein (GFP)–Bax fusion protein in these cells to study localization of Bax. Free radical scavengers were used to assess the role of reactive oxygen species (ROS) in these pathways.

Key results:

Lactacystin triggered a concentration-dependent increase in cell death mediated by the mitochondrial apoptotic pathway, and induced a change in mitochondrial membrane permeability accompanied by cytochrome c release. The participation of Bax protein was more critical than the formation of the permeability transition pore in mitochondria. GFP–Bax over-expression demonstrated Bax redistribution from the cytosol to mitochondria after the addition of lactacystin. ROS, but not p38 mitogen-activated protein kinase, participated in lactacystin-induced mitochondrial Bax translocation. Lactacystin disrupted the intracellular redox state by increasing ROS production and depleting endogenous antioxidant systems such as glutathione (GSH). Pharmacological depletion of GSH, using l-buthionine sulphoxide, potentiated lactacystin-induced cell death. Lactacystin sensitized neuroblastoma cells to oxidative damage, induced by subtoxic concentrations of 6-hydroxydopamine.

Conclusions and implications:

The lactacystin-induced, mitochondrial-mediated apoptotic pathway involved interactions between ROS, GSH and Bax. Lactacystin could constitute a potential factor in the development of sporadic PD.     

Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes

Aim:

To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552).

Methods:

Methods: Primary astrocyte cultures were prepared from the cortex of postnatal rat pups. An astrocytes model of Huntington''s disease was established using the astrocytes infected with adenovirus carrying coden gene of N-terminal 552 residues of Huntingtin. The protein levels of glutamate transporters GLT-1 and GLAST, the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the autophagy substrate p62 in the astrocytes were examined using Western blotting. The mRNA expression levels of GLT-1 and GLAST in the astrocytes were determined using Real-time PCR. ³H]glutamate uptake by the astrocytes was measured with liquid scintillation counting.

Results:

The expression of mutant Htt-552 in the astrocytes significantly decreased both the mRNA and protein levels of GLT-1 but not those of GLAST. Furthermore, Htt-552 significantly reduced ³H]glutamate uptake by the astrocytes. Treatment with the autophagy inhibitor 3-MA (10 mmol/L) significantly increased the accumulation of mutant Htt-552, and reduced the expression of GLT-1 and ³H]glutamate uptake in the astrocytes. Treatment with the autophagy stimulator rapamycin (0.2 mg/mL) significantly reduced the accumulation of mutant Htt-552, and reversed the changes in GLT-1 expression and ³H]glutamate uptake in the astrocytes.

Conclusion:

Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.     

Drug Development Targeting the Glycogen Synthase Kinase-3β (GSK-3β)-Mediated Signal Transduction Pathway: Role of GSK-3β in Myocardial Protection Against Ischemia/Reperfusion Injury

Although reperfusion is required to salvage ischemic myocardium from necrosis, reperfusion per se induces myocardial necrosis. In this “lethal reperfusion injury”, opening of the mitochondrial permeability transition pore (mPTP) upon reperfusion is crucially involved. The mPTP primarily consists of adenine nucleotide translocator (ANT) and voltage-dependent anion channel, and its opening is triggered by binding of cyclophilin-D (CyP-D) to ANT, which increases Ca²⁺ sensitivity of the mPTP. Recent studies have shown that inactivation of glycogen synthase kinase-3β (GSK-3β) suppresses mPTP opening and protects cardiomyocytes. Multiple intracellular signals relevant to cardiomyocyte protection converge to GSK-3β and inactivate this kinase by phosphorylation. Although the effect of GSK-3β phosphorylation on mPTP structure and function remains unclear, suppression of ANT–CyP-D interaction by binding of phospho-GSK-3β to ANT and reduction in GSK-3β–mediated phosphorylation of p53 may contribute to elevation of the threshold for mPTP opening. Furthermore, a significant inverse correlation was observed between level of phospho-GSK-3β at the time of reperfusion and the extent of myocardium infarction in heart. Together with the infarct size–limiting effect of GSK-3β inhibitors, this finding indicates that phospho-GSK-3β is a determinant of myocardial tolerance against reperfusion-induced necrosis. Thus, GSK-3β appears to be a target of novel therapy for cardioprotection upon reperfusion.     

Synergistic effects of autophagy/mitophagy inhibitors and magnolol promote apoptosis and antitumor efficacy

Mitochondria as a signaling platform play crucial roles in deciding cell fate. Many classic anticancer agents are known to trigger cell death through induction of mitochondrial damage. Mitophagy, one selective autophagy, is the key mitochondrial quality control that effectively removes damaged mitochondria. However, the precise roles of mitophagy in tumorigenesis and anticancer agent treatment remain largely unclear. Here, we examined the functional implication of mitophagy in the anticancer properties of magnolol, a natural product isolated from herbal Magnolia officinalis. First, we found that magnolol induces mitochondrial depolarization, causes excessive mitochondrial fragmentation, and increases mitochondrial reactive oxygen species (mtROS). Second, magnolol induces PTEN-induced putative kinase protein 1 (PINK1)‒Parkin-mediated mitophagy through regulating two positive feedforward amplification loops. Third, magnolol triggers cancer cell death and inhibits neuroblastoma tumor growth via the intrinsic apoptosis pathway. Moreover, magnolol prolongs the survival time of tumor-bearing mice. Finally, inhibition of mitophagy by PINK1/Parkin knockdown or using inhibitors targeting different autophagy/mitophagy stages significantly promotes magnolol-induced cell death and enhances magnolol's anticancer efficacy, both in vitro and in vivo. Altogether, our study demonstrates that magnolol can induce autophagy/mitophagy and apoptosis, whereas blockage of autophagy/mitophagy remarkably enhances the anticancer efficacy of magnolol, suggesting that targeting mitophagy may be a promising strategy to overcome chemoresistance and improve anticancer therapy.     

海藻糖治疗帕金森病的作用机制研究进展

帕金森病是一种常见的神经变性疾病,特征性病理改变主要是黑质多巴胺能神经元丢失和路易小体的形成。路易小体中主要成分是纤维化的α-突触核蛋白,研究表明多巴胺能神经元中异常的蛋白质沉积可能与溶酶体自噬途径的失调有关。自噬调节剂的治疗潜力已在帕金森病动物模型中得到证实。海藻糖是一种天然双糖,被认为是治疗神经退行性疾病的新候选药物。它具有类似伴侣活性,防止蛋白质错误折叠或聚集,并有助于通过促进自噬去除积聚的蛋白质。总结异常自噬在帕金森病疾病发展过程中的潜在机制,讨论使用海藻糖对抗帕金森病的促进自噬、蛋白质稳定和抗神经炎症作用。     

Ginsenoside Rbl protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening

Aim:

To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.

Methods:

Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 μmol/L CoCl₂), Gs-Rb1 200 μmol/L group (CoCl₂ intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 μmol/L group, wortmannin+Gs-Rb1 group, adenine 9-β-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 μmol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl₂ in high conductance mode. Expression of GSK-3β, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. ΔGSK-3β was defined as the ratio of p-Ser9-GSK-3β to total GSK-3β.

Results:

CoCl₂ significantly stimulated mPTP opening and up-regulated the level of ΔGSK-3β. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and ΔGSK-3β, and between mPTP opening and ΔGSK-3β. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%±2.0%, P<0.001) . Gs-Rb1 caused a 77.3%±3.2% reduction in the expression of GSK-3β protein (P<0.001) and a significant increase of 1.182±0.007–fold (P=0.0001) in p-Ser9-GSK-3β compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and ΔGSK-3β. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%±1.7%, P=0.001), caspase-3 (56.5%±2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%±1.4%, P=0.001).

Conclusion:

Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3β-mediated mPTP opening.     

Lipid emulsion reverses bupivacaine-induced apoptosis of h9c2 cardiomyocytes: PI3K/Akt/GSK-3β signaling pathway

Some findings have suggested that the rescue of bupivacaine (BPV)-induced cardiotoxicity by lipid emulsion (LE) is associated with inhibition of mitochondrial permeability transition pore (mPTP). However, the mechanism of this rescue action is not clearly known. In this study, the roles of phosphoinositide 3-kinase (PI3K)/Akt and glycogen synthase kinase-3β (GSK-3β) in the molecular mechanism of LE-induced protection and its relationship with mPTP were explored. h9c2 cardiomyocytes were randomly divided into several groups: control, BPV, LE, BPV + LE. To study the effect of LE on mPTP, atractyloside (Atr, 20 μM, mPTP opener) and cyclosporine A (CsA, 10 μM, mPTP blocker) were used. To unravel whether LE protects heart through the PI3K/Akt/GSK-3β signaling pathway, cells were treated with LY294002 (LY, 30 μM, PI3K blocker) or TWS119 (TWS 10 μM, GSK-3β blocker). Later mitochondrial respiratory chain complexes, apoptosis, opening of mPTP and phosphorylation levels of Akt/GSK-3β were measured. LE significantly improved the mitochondrial functions in h9c2 cardiomyocytes. LE reversed the BPV-induced apoptosis and the opening of mPTP. The effect of LE was not only enhanced by CsA and TWS, but also abolished by Atr and LY. LE also increased the phosphorylation levels of Akt and GSK-3β. These results suggested that LE can reverse the apoptosis in cardiomyocytes by BPV and a mechanism of its action is inhibition of mPTP opening through the PI3K/Akt/GSK-3β signaling pathway.     

Recent Progress of Imaging Agents for Parkinson's Disease

Parkinson''s disease (PD) is a common progressive, neurodegenerative brain disease that is promoted by mitochondrial dysfunction, oxidative stress, protein aggregation and proteasome dysfunction in the brain. Compared with computer tomography (CT) or magnetic resonance imaging (MRI), non-invasive nuclear radiopharmaceuticals have great significance for the early diagnosis of PD due to their high sensitivity and specificity in atypical and preclinical cases. Based on the development of coordination chemistry and chelator design, radionuclides may be delivered to lesions by attaching to PD-related transporters and receptors, such as dopamine, serotonin, and others. In this review, we comprehensively detailed the current achievements in radionuclide imaging in Parkinson’s disease.     

Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy

Background and purpose:

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP.

Experimental approach:

The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx^5Cv mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg⁻¹), CsA (10 mg kg⁻¹) or placebo. The effects on muscle necrosis and function were measured.

Key results:

In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg⁻¹, respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg⁻¹ relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress.

Conclusions and implications:

Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients.     

RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy

Tumor necrosis factor alpha (TNFα) induces necroptosis and autophagy; however, the detailed molecular mechanism is not fully understood. In this study, we found that TNFα administration caused mitochondrial dysfunction and reactive oxygen species (ROS) production, which led to necroptosis and autophagy in murine fibrosarcoma L929 cells. Notably, the RIP1 (serine–threonine kinase receptor-interacting protein 1, a main adaptor protein of necroptosis) specific inhibitor necrostatin-1 (Nec-1) recovered mitochondrial dysfunction and ROS production due to TNFα administration. Moreover, pan-caspase inhibitor z-VAD-fmk (zVAD) increased RIP1 expression and exacerbated TNFα-induced mitochondrial dysfunction and ROS production, indicating that RIP1 led to mitochondrial dysfunction and ROS production. In addition, cytochrome c release from mitochondria was accompanied with TNFα administration, and Nec-1 blocked the release of cytochrome c upon TNFα administration, while zVAD enhanced the release. These further suggested that RIP1 induced mitochondrial dysfunction accompanied with cytochrome c release. Furthermore, autophagy inhibitor 3-methyladenine (3MA) did not affect RIP1 expression as well as mitochondrial dysfunction and ROS production. Together with our previous publication that autophagy was a downstream consequence of necroptosis, we concluded that TNFα induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death.     

Unravelling mitochondrial pathways to Parkinson's disease

Mitochondria are essential for cellular function due to their role in ATP production, calcium homeostasis and apoptotic signalling. Neurons are heavily reliant on mitochondrial integrity for their complex signalling, plasticity and excitability properties, and to ensure cell survival over decades. The maintenance of a pool of healthy mitochondria that can meet the bioenergetic demands of a neuron, is therefore of critical importance; this is achieved by maintaining a careful balance between mitochondrial biogenesis, mitochondrial trafficking, mitochondrial dynamics and mitophagy. The molecular mechanisms that underlie these processes are gradually being elucidated. It is widely recognized that mitochondrial dysfunction occurs in many neurodegenerative diseases, including Parkinson''s disease. Mitochondrial dysfunction in the form of reduced bioenergetic capacity, increased oxidative stress and reduced resistance to stress, is observed in several Parkinson''s disease models. However, identification of the recessive genes implicated in Parkinson''s disease has revealed a common pathway involving mitochondrial dynamics, transport, turnover and mitophagy. This body of work has led to the hypothesis that the homeostatic mechanisms that ensure a healthy mitochondrial pool are key to neuronal function and integrity. In this paradigm, impaired mitochondrial dynamics and clearance result in the accumulation of damaged and dysfunctional mitochondria, which may directly induce neuronal dysfunction and death. In this review, we consider the mechanisms by which mitochondrial dysfunction may lead to neurodegeneration. In particular, we focus on the mechanisms that underlie mitochondrial homeostasis, and discuss their importance in neuronal integrity and neurodegeneration in Parkinson''s disease.

LINKED ARTICLES

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Quality control gone wrong: mitochondria,lysosomal storage disorders and neurodegeneration

The eukaryotic cell possesses specialized pathways to turn over and degrade redundant proteins and organelles. Each pathway is unique and responsible for degradation of distinctive cytosolic material. The ubiquitin-proteasome system and autophagy (chaperone-mediated, macro, micro and organelle specific) act synergistically to maintain proteostasis. Defects in this equilibrium can be deleterious at cellular and organism level, giving rise to various disease states. Dysfunction of quality control pathways are implicated in neurodegenerative diseases and appear particularly important in Parkinson''s disease and the lysosomal storage disorders. Neurodegeneration resulting from impaired degradation of ubiquitinated proteins and α-synuclein is often accompanied by mitochondrial dysfunction. Mitochondria have evolved to control a diverse number of processes, including cellular energy production, calcium signalling and apoptosis, and like every other organelle within the cell, they must be ‘recycled.’ Failure to do so is potentially lethal as these once indispensible organelles become destructive, leaking reactive oxygen species and activating the intrinsic cell death pathway. This process is paramount in neurons which have an absolute dependence on mitochondrial oxidative phosphorylation as they cannot up-regulate glycolysis. As such, mitochondrial bioenergetic failure can underpin neural death and neurodegenerative disease. In this review, we discuss the links between cellular quality control and neurodegenerative diseases associated with mitochondrial dysfunction, with particular attention to the emerging links between Parkinson''s and Gaucher diseases in which defective quality control is a defining factor.

LINKED ARTICLES

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

基于NLRP3-IL-1β-NF-κB信号轴研究野菊花总黄酮对 脂多糖诱导HK-2细胞炎性反应及自噬的影响

摘要：目的 探究野菊花总黄酮（TFC）对脂多糖（LPS）诱导HK-2细胞炎性反应及自噬的影响,并初步探究其可 能的作用机制。方法 体外培养人肾小管上皮HK-2细胞,将细胞分为阴性对照（Control）组、LPS处理（LPS）组、TFC 预处理（TFC）组、NLRP3激活预处理（4-MET）组、TFC联合4-MET预处理（TFC+4-MET）组。CCK-8法检测细胞增殖 情况;Hoechst 33342染色观察各组细胞形态变化;免疫印迹法检测各组细胞核苷酸结合域样受体蛋白3（NLRP3）、凋 亡相关斑点样蛋白（Asc）、半胱氨酸天冬氨酸蛋白酶1（caspase-1）、p-IκB、IκB、自噬相关蛋白（Beclin1）、微管相关蛋 白轻链3Ⅱ（LC3Ⅱ）、LC3Ⅰ、蛋白表达情况;ELISA法检测细胞中白细胞介素（IL）-1β、IL-18水平;透射电镜观察细胞 自噬情况。结果 与Control组相比,LPS组细胞增殖率降低,细胞核破碎情况加重,细胞凋亡率升高,NLRP3、Asc、 caspase-1蛋白表达升高、IκB蛋白磷酸化水平升高,IL-1β、IL-18水平升高,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低 （P＜0.05）。与LPS组相比,TFC组细胞增殖率升高,细胞核破碎减轻,细胞凋亡率降低,NLRP3、Asc、caspase-1蛋白 表达降低、IκB蛋白磷酸化水平降低,IL-1β、IL-18水平降低,细胞自噬程度增加,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达升 高;4-MET组细胞增殖率降低,细胞凋亡率升高,细胞核破碎情况加重,NLRP3、Asc、caspase-1蛋白表达升高、IκB蛋 白磷酸化水平升高,IL-1β、IL-18水平升高,细胞自噬程度降低,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低（P＜0.05）。 TFC+4-MET组细胞增殖率、细胞自噬程度、LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达高于4-MET组,细胞核破碎情况减轻, 细胞凋亡率、NLRP3、Asc、caspase-1蛋白表达、IκB蛋白磷酸化、IL-1β、IL-18水平低于4-MET组（P＜0.05）。结论 野菊花总黄酮可能通过抑制NLRP3-IL-1β-NF-κB信号通路活化,进而抑制脂多糖诱导HK-2细胞炎性反应,诱导 细胞自噬     

Impairment of autophagy: From hereditary disorder to drug intoxication

At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinson's, Alzheimer's, and Huntington's diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication.     

Naringin Protects against Rotenone-induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

Rotenone, a mitochondrial complex I inhibitor, can induce the pathological features of Parkinson''s disease (PD). In the present study, naringin, a grapefruit flavonoid, inhibited rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We assessed cell death and apoptosis by measuring mitogen-activated protein kinase (MAPKs) and caspase (CASPs) activities and by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Naringin also blocked rotenone-induced phosphorylation of Jun NH2-terminal protein kinase (JNK) and P38, and prevented changes in B-cell CLL/lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) expression levels. In addition, naringin reduced the enzyme activity of caspase 3 and cleavages of caspase 9, poly (ADP-ribose) polymerase (PARP), and caspase 3. These results suggest that naringin has a neuroprotective effect on rotenone-induced cell death in human neuroblastoma SH-SY5Y cells.