Acupuncture ameliorated skeletal muscle atrophy induced by hindlimb suspension in mice

Preventing skeletal muscle atrophy is critical for maintaining quality of life, but it is often a challenging goal for the elderly and patients with severe conditions. We hypothesized that acupuncture in place of exercise training is an alternative non-pharmacological intervention that can help to prevent muscle atrophy. To elucidate the effects of acupuncture on skeletal muscle atrophy caused by hindlimb suspension (HS), we performed acupuncture on mice according to two different methods: acupuncture with electrical stimulation (EA: electroacupuncture) and without electrical stimulation (MA: manual acupuncture). A needle was retained in the gastrocnemius muscle for 30 min every day for 2 weeks in the EA and MA groups. In the EA group, 30 min of repetitive electrical stimulation (1 Hz, 1 ms pulse width, 6.5 mA intensity) was also applied. HS significantly reduced muscle mass and the cross-sectional area of the soleus muscles. This HS-induced reduction was significantly improved in the EA group, although the level of improvement remained insufficient when compared with the control group. We found that the mRNA expression levels of atrogin-1 and MuRF1, which play a principal role in muscle-specific degradation as E3 ubiquitin ligases, were significantly increased in the HS group compared to the control group. EA and MA reduced the HS-induced upregulation of atrogin-1 (p < 0.01 in EA and MA) and MuRF1 (p < 0.01 in EA) mRNAs. We also found that the expression levels of PI3K, Akt1, TRPV4, adenosine A1 receptor, myostatin, and SIRT1 mRNAs tended to be increased by HS. EA and MA further increased the HS-induced upregulation of Akt1 (p < 0.05 in MA) and TRPV4 (p < 0.05 in MA) mRNAs. We concluded that acupuncture partially prevented skeletal muscle atrophy. This effect might be due to an increase in protein synthesis and a decrease in protein degradation.     

Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities

H₂O₂ can freely crosses membranes and in the presence of Fe²⁺ (or Cu⁺) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H₂O₂-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H₂O₂ treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 µM H₂O₂/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.     

Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.     

Hindlimb unloading decreases thioredoxin-related antioxidant proteins and increases thioredoxin-binding protein-2 in rat skeletal muscle

To investigate role(s) of thioredoxin-related antioxidant proteins in disuse muscle atrophy, we examined the levels of thioredoxin-1 (Trx-1), peroxiredoxin-3/SP-22 (Prx-3) and thioredoxin-binding protein-2 (TBP-2) in rat soleus muscle subjected to hindlimb unloading (HU) for 2, 4, 7 or 14 days. The muscle weight loss was initially observed on day 4. The increases in aclorein- and malondialdehyde-modified proteins, and the decreases in the levels of Trx-1, Prx-3 and Mn-SOD were observed in the late phase of muscle atrophy, whereas, the increase in mRNA expression of TBP-2, a negative regulator of thioredoxin, preceded muscle atrophy. These findings suggest that the decrease of those antioxidant proteins, particularly a marked decrease of Trx-1, may be responsible for the enhanced oxidative damage during the late phase of disuse muscle atrophy. Furthermore, the increase in TBP-2 preceding the muscle atrophy may suppress the thioredoxin-mediated redox signaling, which can be an initial trigger leading to disuse muscle atrophy.     

Pterostilbene protects against H2O2-induced oxidative stress by regulating GAS6/Axl signaling in HL-1 cells

Pterostilbene (PTE, trans-3,5-dimethoxy-4′-hydroxystilbene), a natural plant polyphenol, possesses numerous pharmacological effects, including antioxidant, antidiabetic, antiatherosclerotic, and neuroprotective aspects. This study aims to investigate whether PTE plays a protective role against oxidative stress injury by GAS6/Axl signaling pathway in cardiomyocytes. Hydrogen peroxide (H₂O₂)-induced oxidative stress HL-1 cells were used as models. The mechanism by which PTE protected oxidative stress is investigated by combining cell viability, cell ROS levels, apoptosis assay, molecular docking, quantitative real-time PCR, and western blot analysis. GAS6 shRNA was performed to investigate the involvement of GAS6/Axl pathways in PTE's protective role. The results showed that PTE treatment improved the cell morphology and viability, and inhibited the apoptosis rate and ROS levels in H₂O₂-injured HL-1 cells. Particularly, PTE treatment upregulated the levels of GAS6, Axl, and markers related to oxidative stress, apoptosis, and mitochondrial function related. Molecular docking showed that PTE and GAS6 have good binding ability. Taken together, PTE plays a protective role against oxidative stress injury through inhibiting oxidative stress and apoptosis and improving mitochondrial function. Particularly, GAS6/Axl axis is the surprisingly prominent in the PTE-mediated pleiotropic effects.     

Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway

Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H₂O₂) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H₂O₂ and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H₂O₂ by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H₂O₂, which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H₂O₂ via activating the PI3K/Akt signal pathway.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

何首乌苷通过激活BDNF/TrkB信号通路拮抗H2O2诱导的大鼠海马神经元氧化损伤

探讨脑源性神经营养因子/酪氨酸激酶受体B(BDNF/TrkB)信号通路激活参与何首乌苷(PMG)对过氧化氢(H2O2)诱导神经元氧化应激损伤的保护作用。实验采用神经元原代培养,建立大鼠乳鼠海马神经元氧化应激损伤模型。实验结果显示高浓度的H2O2与MTT测定的细胞存活率降低相关,选择细胞存活率在40%~50%之间的200μmol/LH2O2浓度作为氧化应激损伤的实验浓度。与模型组相比,PMG预处理组(200μmol/L)可抑制H2O2诱导的神经元损伤(P<0.001)。TUNEL和β-微管蛋白III荧光染色显示PMG保护H2O2诱导的神经细胞损伤,明显降低细胞凋亡率(P<0.001),细胞骨架形态恢复正常。与PMG+H2O2预处理组相比较,当加入BDNF/TrkB信号转导通路阻断剂K252a后,PMG+H2O2+K252a组神经元细胞存活率大幅度下降(P<0.01),细胞骨架形态呈损伤状态。同时,我们发现PMG预处理恢复H2O2诱导的BDNF和P-TrkB的低表达水平,并且用K252a阻断BDNF/TrkB信号传导抑制了PMG对BDNF和P-TrkB表达水平的影响(P<0.01)。综上所述,何首乌苷可能通过激活BDNF/TrkB信号转导通路及维护神经元骨架的完整,实现对大鼠海马神经元氧化应激损伤的拮抗作用。     

Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells

In the present study, we characterized hydrogen peroxide (H₂O₂)-induced cell apoptosis and related cell signaling pathways in cultured embryonic neural stem/progenitor cells (NS/PCs). Our data indicated that H₂O₂ induced acute cell apoptosis in NS/PC in concentration- and time-dependent manners and selectively, it transiently increased PI3K-Akt and Mek-Erk1/2 in a dose-dependent manner. Inhibition of PI3K-Akt with wortmannin, a PI3-K inhibitor, was found to significantly increase H₂O₂-induced acute apoptosis and dramatically decrease basal pGSK3β levels. The level of pGSK3β remained unchanged with H₂O₂ exposure. We conclude that the transient activation of PI3K-Akt signaling delays the H₂O₂-induced acute apoptosis in cultured NS/PCs in part through maintaining the basal pGSK3β level and activating other downstream effectors.     

Vitamin D3 treatment regulates apoptosis,antioxidant defense system,and DNA integrity in the epididymal sperm of an aged rat model

The present study aimed to investigate the effects of vitamin D3 in the epididymal sperm cells of D ‐gal‐induced aged rats. It is well known that during aging sperm quality and quantity declines and leads to age‐related infertility problems in males. The results of the present study showed that there were elevated levels of oxidative stress and poor DNA integrity of sperm of aged rats. The expression of BCL2 also showed a significant decline in the sperm of aged rats, however, the expression of BAX and active caspase‐3 did not show significant change compared with the control group. The treatment of vitamin D3 at lower doses to aged rats showed increased expression of BAX and active caspase‐3 in the sperm, this finding suggests that increased apoptosis may be responsible for removal of poor quality sperm during aging. Vitamin D3 treatment at both doses showed improvement in the oxidative stress and DNA integrity in the sperm of aged rats. We also investigated the expression of AGER, visfatin, and HSPA1A in the epididymal sperm. It has been found that expression of AGER, visfatin, and HSPA1A increased in the sperm aged rats and vitamin D3 treatments at both doses decreased its expression. Thus, it might be suggested that during aging vitamin D3 treatment would be important for managing the sperm quality by regulating the apoptosis, antioxidant system and DNA integrity via modulation of visfatin and HSPA1A.     

低温和氧化应激产生活酵母细胞衍生物的研究

对低温和H2O2应激条件下产生活性酵母细胞衍生物（Live Yeast Cell Derivative,简称LYCD）进行了研究。结果表明：低温预处理能够增加细胞内谷胱甘肽（GSH）含量,提高超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性,降低MDA含量。低温预处理可以诱导对致死浓度H2O2的抗性。通过0—15℃低温和0．2mmol／L H2O2处理酵母细胞后,提取LYCD并添加到酵母细胞培养液中,发现细胞在致死浓度H2O2作用下的存活率明显提高,说明0—15℃低温和H2O2刺激酵母细胞形成的LYCD对细胞具有抗氧化作用。     

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

Objectives: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity.

Methods: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H₂O₂-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H₂O₂-treated vector control and LCMT1-overexpressing cells.

Results: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H₂O₂-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H₂O₂ treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton.

Discussion: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.     

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts

Background

Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca²⁺ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H₂O₂-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts.

Results

Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H₂O₂ exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H₂O₂-induced injury in H9c2 cells. H₂O₂ exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca²⁺ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H₂O₂-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H₂O₂-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells.

Conclusions

Collectively, these findings suggest that EGCG blunts the H₂O₂-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.     

Moderate O3/O2 therapy enhances enzymatic and non-enzymatic antioxidant in brain and cochlear that protects noise-induced hearing loss

Mitochondrial damage and oxidative stress are known to contribute to the pathogenesis of noise-induced hearing loss (NIHL). In this study, we examined the protective effect of O₂/O₃ mixture (ozone/oxygen) therapy against mitochondrial induced damage and oxidative stress by noise exposure in rat brain and cochlear. For this purpose, rats were divided into four groups: 1 – control group; 2 – noise-exposed group (100?dB); 3 – noise?+?O₂/O₃, and 4 – O₂/O₃ (30 µg/ml). After 14 d, animals were anesthetised. Rat brain and cochlear tissue were removed for evaluation of the histopathological damages, oxidative stress, and mitochondrial dysfunction in both tissues. Our findings indicated that noise caused pathological damage, oxidative stress, and mitochondrial dysfunction in rat brain and cochlear. Also, daily administration of an O₂/O₃ therapy (30 µg/ml intravenous) efficiently increased enzymatic and non-enzymatic antioxidant in brain and cochlear that this action led to inhibition of pathological damages, oxidative stress, reactive oxygen species formation, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release resulting from noise. These findings suggest that the moderate O₂/O₃ therapy enhances the capacity of enzymatic and non-enzymatic antioxidant in brain and cochlear that protects against NIHL.     

The ubiquitin/proteasome pathway: friend or foe in zinc-, cadmium-, and H2O2-induced neuronal oxidative stress

One of the hallmarks of neurodegeneration is the accumulation of ubiquitinated proteins in intraneuronal inclusions in the cytosol, endosomes/lysosomes and nuclei of affected cells. The relationship between inclusion production and cell viability is not well understood. On the one hand inclusions may be beneficial and result from an attempt of the cell to isolate a subclass of ubiquitinated proteins that are not effectively degraded. On the other hand, the inclusions may impede normal cell function contributing to cell death. To address this issue we treated mouse neuronal HT4 cells with three toxic agents cadmium, zinc and H2O2, and investigated their effects on glutathione homeostasis, on accumulation of ubiquitinated proteins and on cell viability. The three treatments induce oxidative stress manifested by decreases in glutathione (GSH) and/or increases in protein mixed disulfides (PrSSG). After an overnight recovery period in the absence of treatment, GSH and PrSSG were restored to almost normal levels. However, the levels of ubiquitinated proteins were significantly increased, and cell viability was sharply reduced. These results suggest that the ubiquitin-proteasome pathway is recruited for removal of proteins that are oxidatively modified. However, if the ubiquitinated proteins are not efficiently degraded, they accumulate in the cell and contribute to a decrease in cell viability.