The role of quercetin on the survival of neuron-like PC12 cells and the expression of α-synuclein

Both genetic and environmental factors are important in the pathogenesis of Parkinson's disease. As α-synuclein is a major constituent of Lewy bodies, a pathologic hallmark of Parkinson's disease, genetic aspects of α-synuclein is widely studied. However, the influence of dietary factors such as quercetin on α-synuclein was rarely studied. Herein we aimed to study the neuroprotective role of quercetin against various toxins affecting apoptosis, autophagy and aggresome, and the role of quercetin on α-synuclein expression. PC12 cells were pre-treated with quercetin(100, 500, 1,000 μM) and then together with various drugs such as 1-methyl-4-phenylpyridinium(MPP+; a free radical generator), 6-hydroxydopamine(6-OHDA; a free radical generator), ammonium chloride(an autophagy inhibitor), and nocodazole(an aggresome inhibitor). Cell viability was determined using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertazolium bromide(MTT) assay. Apoptosis was detected by annexin V-fluorescein isothiocyanate and propidium iodide through the use of fluorescence activated cell sorter. α-Synuclein expression was detected by western blot assay and immunohistochemistry. The role of α-synuclein was further studied by knocking out α-synuclein using RNA interference. Cell viability increased at lower concentrations(100 and 500 μM) of quercetin but decreased at higher concentration(1,000 μM). Quercetin exerted neuroprotective effect against MPP+, ammonium chloride and nocodazole at 100 μM. MPP+ induced apoptosis was decreased by 100 μM quercetin. Quercetin treatment increased α-synuclein expression. However, knocking out α-synuclein exerted no significant effect on cell survival. In conclusion, quercetin is neuroprotective against toxic agents via affecting various mechanisms such as apoptosis, autophagy and aggresome. Because α-synuclein expression is increased by quercetin, the role of quercetin as an environmental factor in Parkinson's disease pathogenesis needs further investigation.     

Schwann cells differentiated from skin-derived precursors provide neuroprotection via autophagy inhibition in a cellular model of Parkinson’s disease

Autophagy has been shown to play an important role in Parkinson’s disease.We hypothesized that skin-derived precursor cells exhibit neuroprotective effects in Parkinson’s disease through affecting autophagy.In this study,6-hydroxydopamine-damaged SH-SY5Y cells were pretreated with a culture medium containing skin-derived precursors differentiated into Schwann cells(SKP-SCs).The results showed that the SKP-SC culture medium remarkably enhanced the activity of SH-SY5Y cells damaged by 6-hydroxydopamine,reduced excessive autophagy,increased tyrosine hydroxylase expression,reducedα-synuclein expression,reduced the autophagosome number,and activated the PI3K/AKT/mTOR pathway.Autophagy activator rapamycin inhibited the effects of SKP-SCs,and autophagy inhibitor 3-methyladenine had the opposite effect.These findings confirm that SKP-SCs modulate the PI3K/AKT/mTOR pathway to inhibit autophagy,thereby exhibiting a neuroprotective effect in a cellular model of Parkinson’s disease.This study was approved by the Animal Ethics Committee of Laboratory Animal Center of Nantong University(approval No.S20181009-205)on October 9,2018.     

Protective effect of alpha-synuclein knockdown on methamphetamine-induced neurotoxicity in dopaminergic neurons

The over-expression of α-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson's disease. In the present study, α-synuclein knockdown rats were created by injecting α-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraper-itoneally with methamphetamine to establish the model of Parkinson's disease. Expression of α-synuclein mRNA and protein in the right striatum of the injected rats was significantly down-regulated. Food intake and body weight were greater in α-synuclein knockdown rats, and water intake and stereotyped behavior score were lower than in model rats. Striatal dopamine and tyrosine hydroxylase levels were significantly elevated in α-synuclein knockdown rats. Moreover, superoxide dismutase activity was greater in α-synuclein knockdown rat striatum, but the levels of reactive oxygen species, malondialdehyde, nitric oxide synthase and nitrogen monoxide were lower compared with model rats. We also found that α-synuclein knockdown inhibited metham-phetamine-induced neuronal apoptosis. These results suggest that α-synuclein has the capacity to reverse methamphetamine-induced apoptosis of dopaminergic neurons in the rat striatum by inhibiting oxidative stress and improving dopaminergic system function.     

Neurotrophin-3 reduces apoptosis induced by 6-OHDA in PC12 cells through Akt signaling pathway

Previous work has demonstrated that 6-hydroxydopamine (6-OHDA) induces apoptosis in PC12 cells. The goal of the present study was to investigate the mechanisms underlying the protection by neurotrophin-3 (NT-3) against 6-OHDA-induced apoptosis in PC12 cells. Treatment of PC12 cells with 6-OHDA resulted in activation of caspase-3 and subsequent apoptosis, as detected by TUNEL staining. In addition, Akt phosphorylation was decreased following 6-OHDA treatment. Pretreatment with NT-3 reduced the percentage of apoptotic cells and caspase-3 activity induced by 6-OHDA and suppressed the cleavage of caspase-3 and Poly(ADP-ribose) polymerase (PARP) with a significant decrease in cell viability. Moreover, Akt phosphorylation was enhanced and 6-OHDA-induced chromatin condensation was suppressed by NT-3. Such NT-3-evoked suppression in chromatin condensation was reversed by anti-TrkA antibody receptor blockade. Further study revealed that LY294002, an inhibitor of PI3-kinase (a molecule upstream of Akt), enhanced 6-OHDA-induced apoptosis. These data indicate that NT-3 prevents 6-OHDA-induced apoptosis in PC12 cells via activation of PI3-kinase/Akt pathway.     

EPO-Dependent Activation of PI3K/Akt/FoxO3a Signalling Mediates Neuroprotection in In Vitro and In Vivo Models of Parkinson’s Disease

Erythropoietin (EPO) may become a potential therapeutic candidate for the treatment of the neurodegenerative disorder — Parkinson’s disease (PD), since EPO has been found to prevent neuron apoptosis through the activation of cell survival signalling. However, the underlying mechanisms of how EPO exerts its neuroprotective effect are not fully elucidated. Here we investigated the mechanism by which EPO suppressed 6-hydroxydopamine (6-OHDA)-induced neuron death in in vitro and in vivo models of PD. EPO knockdown conferred 6-OHDA-induced cytotoxicity. This effect was reversed by EPO administration. Treatment of PC12 cells with EPO greatly diminished the toxicity induced by 6-OHDA in a dose- and time-dependent manner. EPO effectively reduced apoptosis of striatal neurons and induced a significant improvement on the neurological function score in the rat models of PD. Furthermore, EPO increased the expression of phosphorylated Akt and phosphorylated FoxO3a, and abrogated the 6-OHDA-induced dysregulation of Bcl-2, Bax and Caspase-3 in PC12 cells and in striatal neurons. Meanwhile, the EPO-dependent neuroprotection was notably reversed by pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Our data suggest that PI3K/Akt/FoxO3a signalling pathway may be a possible mechanism involved in the neuroprotective effect of EPO in PD.     

Erythropoietin attenuates 6-hydroxydopamine-induced apoptosis via glycogen synthase kinase 3β-mediated mitochondrial translocation of Bax in PC12 cells

The aim of this study was to determine the mechanism by which erythropoietin (EPO) suppressed 6-hydroxydopamine (6-OHDA)-induced apoptosis. Our results showed that 6-OHDA remarkably decreased phosphorylation of glycogen synthase kinase 3β (GSK3β) as well as enhanced the level of Bax in the mitochondria. Besides, 6-OHDA decreased the mitochondrial expression of Bcl-2 without altering the cytoplasmic expression of Bcl-2. In line with these results, 6-OHDA treatment enhanced the apoptosis and caspase 3 activity in PC12 cells. These findings indicated that mitochondrial dysfunction was involved in the neurotoxicity of 6-OHDA and GSK3β might act upstream of Bax/Bcl-2 and the caspase 3 pathways in 6-OHDA-treated PC12 cells. Furthermore, EPO reduced 6-OHDA-induced growth inhibition. Western blot exhibited that GSK3β inhibitor 4-benzyl-2-methyl-1, 2,4-thiadiazolidine-3, 5-dione (TDZD8) and EPO not only increased the phosphorylation of GSK3β but also inhibited the mitochondrial translocation of Bax. In agreement with these results, EPO and TDZD8 obviously increased the mitochondrial expression of Bcl-2. Finally, TDZD-8 and EPO significantly suppressed the enhanced apoptosis and activity of caspase 3 induced by 6-OHDA. Taken together, GSK3β-mediated mitochondrial cell death pathway is involved in the neuroprotective effect of EPO against 6-OHDA-induced apoptosis.     

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1(SIAH-1) in PC12 cells. 1-Methyl-4-phenylpyridinium(MPP+) treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP+ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson's disease.     

Elevated Levels of α-Synuclein Oligomer in the Cerebrospinal Fluid of Drug-Na?ve Patients with Parkinson's Disease

Background and Purpose

The detection of α-synuclein in the body fluids of patients with synucleinopathy has yielded promising but inconclusive results, in part because of conformational changes of α-synuclein in response to environmental conditions. The aim of this study was to determine the feasibility of using α-synuclein as a biological marker for Parkinson''s disease (PD).

Methods

Twenty-three drug-naïve patients with PD (age 62.4±12.7 years, mean±SD; 11 males) and 29 age- and sex-matched neurologic control subjects (age 60.1±16.2 years; 16 males) were recruited. The levels of oligomeric and total α-synuclein in the cerebrospinal fluid (CSF) and plasma were measured using two simultaneous enzyme-linked immunosorbent assays.

Results

The level of α-synuclein oligomer in the CSF of PD patients was significantly higher in PD patients than in neurological controls, but other findings (plasma α-synuclein oligomer and total α-synuclein in CSF and plasma) did not differ significantly between the two groups. When the control subjects were divided into a symptomatic control group (11 patients who complained of parkinsonian symptoms and were diagnosed with hydrocephalus and drug-induced or vascular parkinsonism) and a neurologic control group (10 normal subjects and 8 patients with diabetic ophthalmoplegia), the level of α-synuclein oligomer in the CSF was still significantly higher in PD patients than in both of the control subgroups.

Conclusions

These findings provide further evidence for a pathogenic role of the α-synuclein oligomer and suggest that CSF levels of α-synuclein oligomer can be a reliable marker for PD.     

Ghrelin protects spinal cord motoneurons against chronic glutamate-induced excitotoxicity via ERK1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β pathways

Excitotoxic degeneration of spinal cord motoneurons has been proposed as a pathogenic mechanism in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R) 1a, functions as a neuroprotective factor in various animal models of neurodegenerative diseases. In this study, the potential neuroprotective effects of ghrelin against chronic glutamate-induced cell death were studied by exposing organotypic spinal cord cultures (OSCC) to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration. Ghrelin receptor was expressed on spinal cord motoneurons. Exposure of OSCC to THA for 3 weeks resulted in a significant loss of motoneurons. However, THA-induced loss of motoneurons was significantly reduced by treatment of ghrelin. Exposure of OSCC to the receptor-specific antagonist D-Lys-3-GHRP-6 abolished the protective effect of ghrelin against THA. Treatment of spinal cord cultures with ghrelin caused rapid phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and glycogen synthase kinase-3β (GSK-3β). The effect of ghrelin on motoneuron survival was blocked by the MEK inhibitor PD98059 and the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. Taken together, these findings indicate that ghrelin has neuroprotective effects against chronic glutamate toxicity by activating the MAPK and PI3K/Akt signaling pathways and suggest that administration of ghrelin may have the potential therapeutic value for the prevention of motoneuron degeneration in human ALS. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β in motoneurons contributes to the protective effect of ghrelin.     

CDNF基因修饰的大鼠骨髓间充质干细胞对6-OHDA诱导PC12细胞凋亡的抑制作用

目的探讨CDNF对6-羟多巴胺(6-OHDA)诱导的细胞凋亡效应的抑制作用。方法应用Li-po2000将重组真核表达质粒pDsRed-C1-CDNF转染至BMSCs,经G418筛选后获得稳定表达CDNF的MSCs细胞株(重命名为CDNF-MSCs细胞株)。应用MTT法和AnnexinⅤ-FITC细胞凋亡分析法分别检测CDNF-MSCs细胞上清液对6-OHDA诱导的PC12细胞凋亡作用的影响。结果通过G418筛选成功获得稳定表达CDNF的MSCs转染细胞,CDNF-MSCs细胞上清液预处理的PC12细胞对6-OHDA诱导的凋亡效应具有抑制作用,能有效的降低细胞的凋亡率。结论 CDNF作为一种分泌性蛋白,可显著抑制6-OHDA诱导PC12细胞凋亡。     

Olfactory ensheathing cells conditioned medium prevented apoptosis induced by 6-OHDA in PC12 cells through modulation of intrinsic apoptotic pathways

Olfactory ensheathing cells (OECs) express a high level of growth factors which play a very important role as neuronal support. Recent evidence in literatures showed that transplantation of OECs may improve functional restoration in 6-OHDA-induced rat model of Parkinson's disease (PD). However, the biological function of various factors released from OECs in Parkinson' disease is still unclear. In this study, we examined the effects of newborn rat OECs conditioned medium (CM) on PC12 cells. Cells treated with 6-OHDA underwent cytotoxicity and apoptotic death determined by MTT assay and Hoechst 33342/PI staining. OECs CM was able to reduce the cellular damage in PC12 cells. Further investigation results showed that CM inhibited the disruption of mitochondrial transmembrane potential, up-regulation of Bcl-2 and down-regulation of Bax. Taken together, this study indicates that CM has a neuroprotective effect on 6-OHDA induced apoptosis of PC12 cells, which is through up-regulation of the Bcl-2/Bax ratio and protection for mitochondrion.     

Overexpression of human α-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine neurons in the substantia nigra and the progressive loss of these neurons. Recently, mutations in the α-synuclein gene have been identified in early-onset familial PD, and α-synuclein has been shown to be a major component of LB in all patients. Yet, the pathophysiological function of α-synuclein remains unknown. In this report, we have investigated the toxic effects of adenovirus-mediated α-synuclein overexpression on dopamine neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell line – the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27). Adenovirus-transduced cultures showed high-level expression of α-synuclein within the cells. Overexpression of human mutant α-synuclein (Ala₅₃Thr) selectively induced apoptotic programmed cell death of primary dopamine neurons as well as N27 cells. The mutant protein also potentiated the neurotoxicity of 6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human α-synuclein was not directly neurotoxic but did increase cell death after 6-OHDA. Overexpression of wild-type rat α-synuclein had no effect on dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that overexpression of human mutant α-synuclein directly leads to dopamine neuron death, and overexpression of either human mutant or human wild-type α-synuclein renders dopamine neurons more vulnerable to neurotoxic insults.     

Neuroprotective effects of SMAds in a rat model of cerebral ischemia/reperfusion

Previous studies have shown that up-regulation of transforming growth factor β1 results in neuroprotective effects. However, the role of the transforming growth factor β1 downstream molecule, SMAD2/3, following ischemia/reperfusion remains unclear. Here, we investigated the neuroprotective effects of SMAD2/3 by analyzing the relationships between SMAD2/3 expression and cell apoptosis and inflammation in the brain of a rat model of cerebral ischemia/reperfusion. Levels of SMAD2/3 m RNA were up-regulated in the ischemic penumbra 6 hours after cerebral ischemia/reperfusion, reached a peak after 72 hours and were then decreased at 7 days. Phosphorylated SMAD2/3 protein levels at the aforementioned time points were consistent with the m RNA levels. Over-expression of SMAD3 in the brains of the ischemia/reperfusion model rats via delivery of an adeno-associated virus containing the SMAD3 gene could reduce tumor necrosis factor-α and interleukin-1β m RNA levels, down-regulate expression of the pro-apoptotic gene, capase-3, and up-regulate expression of the anti-apoptotic protein, Bcl-2. The SMAD3 protein level was negatively correlated with cell apoptosis. These findings indicate that SMAD3 exhibits neuroprotective effects on the brain after ischemia/reperfusion through anti-inflammatory and anti-apoptotic pathways.     

Activation of Wnt/β-catenin Pathway by Exogenous Wnt1 Protects SH-SY5Y Cells Against 6-Hydroxydopamine Toxicity

Wnt1, initially described as a modulator of embryonic development, has recently been discovered to exert cytoprotective effects in cellular models of several diseases, including Parkinson's disease (PD). We, therefore, examined the neuroprotective effects of exogenous Wnt1 on dopaminergic SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA). Here, we show that 10–500 μM 6-OHDA treatment decreased cell viability and increased lactate dehydrogenase (LDH) leakage. SH-SY5Y cells treated with 100 μM 6-OHDA for 24 h showed reduced Wnt/β-catenin activity, decreased mitochondrial transmembrane potential, elevated levels of reactive oxidative species (ROS) and phosphatidylserine (PS) extraversion, increased levels of Chop and Bip/GRP78 and reduced level of p-Akt (Ser473). In contrast, exogenous Wnt1 attenuated 6-OHDA-induced changes. These results suggest that activation of the Wnt/β-catenin pathway by exogenous Wnt1 protects against 6-OHDA-induced changes by restoring mitochondria and endoplasmic reticulum (ER) function.     

Neuroprotective effects of novel phosphatidylglycerol-based phospholipids in the 6-hydroxydopamine model of Parkinson's disease

Administration of VP025 (Vasogen Inc.), a novel drug formulation based on phospholipid nanoparticles incorporating phosphatidylglycerol, has previously been shown to have a neuroprotective effect in the brain. We examined the effect of VP025 in a rat model of Parkinson's disease, the 6-hydroxydopamine (6-OHDA) lesion of the medial forebrain bundle. VP025 or phosphate-buffered saline (PBS) was administered to rats 14 days, 13 days and 1 day before the unilateral 6-OHDA lesion. Functional integrity of nigrostriatal dopaminergic neurons was assessed 7 and 21 days later by amphetamine-induced rotational testing and we observed that rotational counts were significantly less in rats that were pretreated with VP025 compared with PBS-pretreated 6-OHDA-lesioned rats. Neurochemical analysis at 10 and 28 days after lesion revealed that VP025 protected against a 6-OHDA-induced decrease in concentrations of striatal dopamine and its metabolites. Immunocytochemical studies of the ipsilateral substantia nigra showed that VP025 significantly inhibited 6-OHDA-induced loss of dopaminergic neurons. We also observed that increases in immunostaining for activated microglia and for activated p38 in dopaminergic neurons of 6-OHDA-lesioned rats were prevented by VP025. This study shows that VP025 has significant protective effects on the 6-OHDA-lesioned nigrostriatal pathway and may therefore have potential for the treatment of Parkinson's disease.     

The neurological effects of ghrelin in brain diseases: Beyond metabolic functions

Ghrelin, a peptide released by the stomach that plays a major role in regulating energy metabolism, has recently been shown to have effects on neurobiological behaviors. Ghrelin enhances neuronal survival by reducing apoptosis, alleviating inflammation and oxidative stress, and accordingly improving mitochondrial function. Ghrelin also stimulates the proliferation, differentiation and migration of neural stem/progenitor cells (NS/PCs). Additionally, the ghrelin is benefit for the recovery of memory, mood and cognitive dysfunction after stroke or traumatic brain injury. Because of its neuroprotective and neurogenic roles, ghrelin may be used as a therapeutic agent in the brain to combat neurodegenerative disease. In this review, we highlight the pre-clinical evidence and the proposed mechanisms underlying the role of ghrelin in physiological and pathological brain function.     

Ceftriaxone Blocks the Polymerization of α-Synuclein and Exerts Neuroprotective Effects in Vitro

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Ghrelin protects MES23.5 cells against rotenone via inhibiting mitochondrial dysfunction and apoptosis

Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor and has several important physiological functions. Recently, particular attention has been paid to its neuroprotective effect. Rotenone is used to investigate the pathogenesis of Parkinson's disease (PD) for its ability to inhibit mitochondrial complex I. The current study was carried out to investigate the neuroprotective effects of ghrelin against rotenone in MES 23.5 dopaminergic cells and explored the possible mechanisms underlying this protection. Our results showed that rotenone induced significant decrease in cell viability which was counteracted by ghrelin treatment. In addition, rotenone challenge reduced mitochondrial membrane potential, inhibited the activity of mitochondrial complex I and depressed cytochrome C release from mitochondria. This mitochondrial dysfunction was reversed by ghrelin treatment. Furthermore, our results demonstrated that ghrelin protected MES23.5 cells against rotenone-induced apoptosis by inhibiting activation of caspase-3. Overall, our findings showed ghrelin provided protective effects on MES23.5 dopaminergic cells against rotenone via restoring mitochondrial dysfunction and inhibiting mitochondrial dependent apoptosis.     

Down-regulation of Bcl-2, activation of caspases,and involvement of reactive oxygen species in 6-hydroxydopamine-induced thymocyte apoptosis

OBJECTIVE: Our previous work showed that 6-hydroxydopamine (6-OHDA) induced mouse thymocytes to undergo apoptosis both in vivo and in vitro. In the present study, we further investigated the mechanisms of 6-OHDA-induced apoptosis in vitro. METHODS: Naive mouse thymocytes were cultured with 6-OHDA. The percentages of apoptotic cells were quantified by propidium iodide staining, and DNA fragmentation was detected by agarose gel electrophoresis. Intracellular Bcl-2 was analyzed by immunofluorescence staining. Cu/Zn superoxide dismutase (Cu/Zn-SOD) activities were measured by the SOD-525 method. RESULTS: The apoptotic effect of 6-OHDA was blocked by desipramine, a catecholamine uptake blocker. Treatment with 6-OHDA caused a reduction in Bcl-2 expression. VAD-FMK, a broad-spectrum caspase inhibitor, and DEVD-CHO, a potent inhibitor of caspase-3, could block 6-OHDA-induced thymocyte apoptosis. However, the specific caspase-1 (ICE) inhibitor YVAD-CMK had no effect. This cell death process was prevented by the protein synthesis inhibitor cycloheximide and by antioxidants. The level of Cu/Zn-SOD activities also decreased after cells were exposed to 6-OHDA. CONCLUSION: These results suggest an apoptotic effect of 6-OHDA via the uptake of this neurotoxin by thymocytes, and that down-regulation of Bcl-2, activation of caspases, such as caspase-3 but not caspase-1, generation of reactive oxygen species, and new synthesis of proteins are involved in this process.