Achyranthes bidentata Polypeptide Protects Schwann Cells From Apoptosis in Hydrogen Peroxide-Induced Oxidative Stress

Abstract

ABPPk, the active ingredient separated from Achyranthes bidentata polypeptides, is a traditional Chinese medicine with multiple pharmaceutical properties. In this study, we investigated the molecular mechanisms of ABPPk in protecting Schwann cells (SCs) from H₂O₂-induced cell apoptosis. The viability of SCs pretreated with ABPPk was elevated significantly by MTT assay estimation. Meanwhile, the apoptosis of SCs was reduced which was showed in flow cytometry and transferase-mediated dUTP nick end labeling analysis. Furthermore, the addition of ABPPk also increased the activities of SOD and GSH accompanied with a decrease in MDA and LDH activities. According to Western blot analysis, the upregulation of Bcl-2, also downregulation of Bax and cleaved caspase-3 were demonstrated in SCs which was ABPPk pretreated. Further research showed that PI3K/AKT and ERK1/2 pathways in SCs have been activated after pretreatment of ABPPk. Collectively, results in our study suggested that ABPPk protected SCs from H₂O₂-induced oxidative damage by reducing the expression of apoptotic molecules and enhancing the activities of antioxidant enzymes, which inhibited the apoptosis of SCs modulated by PI3K/AKT and ERK1/2 signaling pathways. In our perspectives, ABPPk as an active factor with its antioxidative activities has potential and promising therapeutic effects in the prevention of neurologic disorders.

Introduction

Schwann cells (SCs), an important component of the peripheral nervous system (PNS) that wrapping both myelinated and unmyelinated nerve fibers, could excrete a large number of growth factors to promote axonal growth and myelinization (Jessen and Mirsky, 1997; Woszczycka-Korczynska et al., 2013). Damage to SCs is likely to induce cell apoptosis and restrict functional recovery of peripheral nerves (Zhao et al., 2017). It is reported in previous studies that apoptosis of SCs caused by oxidative stress is the common and vital mechanism of peripheral neuropathy (Purves et al., 2001). Oxidative stress-induced cell apoptosis is implicated as an important pathogenic factor in many neurodegenerative diseases (Olanow, 1993; Behl, 1999; Finkel and Holbrook, 2000; Choi et al., 2006). H₂O₂ known as a precursor of reactive oxygen species (ROS) (Sherer et al., 2002), can cause dysfunction of DNA synthesis, protein expression, and mitochondria structure, and also complete disruption of cellular integrity and cell function (Pizarro et al., 2009; Fernandez-Checa et al., 2010; Wang C.H. et al., 2013). Accordingly, inhibition of the oxidative damage to SCs will improve the potential ability of protection from damage and regenerative efficacy of peripheral nerve injuries.

Achyranthes bidentata, listed in the Chinese Pharmacopoeia, is a significant medicinal plant with multiple therapeutic effects, such as anti-inflammatory, antipyretic, and diuretic activities (He et al., 2014). In previous research, we found that A. bidentata polypeptides (ABPP) that isolated from the aqueous extract of A. bidentata Blume possesses a protective effect on N-methyl-D-aspartate (NMDA)-induced apoptosis of hippocampal neurons (Shen et al., 2008) and could promote peripheral nerve regeneration in rats and rabbits (Yuan et al., 2010; Wang Y. et al., 2013; Cheng et al., 2014). Furthermore, ABPPk purified by HPLC, exhibits excellent neuron-protective efficiency (Yu et al., 2014; Peng et al., 2018). However, little is known about the mechanism by which ABPPk exerts its protective role on SCs, especially in the aspect of oxidative stress. Consequently, the aim of this research is to validate the hypothesis whether ABPPk has a cytoprotective effect on SCs against oxidative stress damage in vitro and to reveal its underlying molecular mechanism.

In our study, we used H₂O₂-induced cell apoptosis models to investigate the potential cytoprotective activity of ABPPk on SCs. Results revealed that ABPPk treatment significantly protects SCs from apoptosis in oxidative stress induced by H₂O₂. Nevertheless, the beneficial effects of ABPPk against oxidative stress damage likely coupled with the PI3K/AKT and ERK1/2 signaling pathways. It is suggested that ABPPk treatment is potentially a useful intervention of preventing SCs against oxidative stress-induced cell apoptosis.

Materials and Methods

Preparation of ABPPk

ABPP was extracted from A. bidentata Blume, which was obtained from a Chinese medicine grocery, identified by Professor Haoru Zhao from China Pharmaceutical University (Peng et al., 2018). HPLC was further used to purify ABPP and the preparation protocol of ABPPk was previously described (Cheng et al., 2014; Yu et al., 2014).

Cell Treatment

SCs were cultured from the sciatic nerves of 1-day-old SD rats as previously described (Mantuano et al., 2008; Yu et al., 2012). In brief, to remove fibroblasts, anti-Thy1.1 antibody (Sigma, St. Louis, MO, United States) and rabbit complement (Invitrogen, Carlsbad, CA, United States) were further added to SCs isolated. Then SCs was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ and 95% air at 37°C (Li et al., 2015). Primary cultured SCs were identified by immunostaining and flow cytometric analysis (Dubovy et al., 2001; Shen et al., 2012). H₂O₂ was used to establish the apoptosis model as previously reported (Luo et al., 2012). H₂O₂ was freshly diluted from 30% H₂O₂ stock solution with DMEM medium to a 400 μM final concentration prior to each experiment. To determine the effect of ABPPk on H₂O₂-exposed SCs, SCs were pretreated with ABPPk for 12 h followed by co-treatment of ABPPk with H₂O₂ for 24 h. In a single experiment, each treatment was performed in triplicate.

Cell Viability Assay

Cell viability was determined by using MTT assay. SCs onto 96-well plates were treated differently with a density of 2 × 10⁵cells/ml. Then 20% sodium dodecyl sulfide (SDS) was added to dissolve the resulting formazan, following the incubation of SCs with MTT (0.5mg/ml) for 4 h at 37°C, which was described by measuring the absorbance (OD) values at 570 nm using Microplate reader (BioTek, United States) (Shen et al., 2011).

Tunel Staining of Apoptosis Cells

The SCs were seeded at a density of 2 × 10⁵cells/ml to experience different treatments. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay by the DeadEndTM Fluorometric Tunel system (Promega, Madison, WI, United States) was showed according to manufacturer’s instructions to detect the apoptotic cells. Differential interference contrast microscopy images were then obtained randomly (Peng et al., 2018). The ratio of Hoechst positive cells and Tunel positive cells were respectively calculated and at least five images of each sample in different visions were observed (Geoffroy et al., 2017).

Flow Cytometry

The purity of primary cultured SCs was further evaluated by flow cytometric analysis with S100 antibody (He et al., 2012; Shen et al., 2012). And the extent of apoptosis was measured through AnnexinV-FITC apoptosis detection kit (BD Bioscience, San Jose, CA, United States) on the basis of the manufacturer’s instruction. In brief, the SCs were resuspended in 1× Binding Buffer and stained with FITC-Annexin V and propidium iodide (PI) for 15 min at room temperature followed by washing twice with cold PBS. The apoptotic cells were analyzed by flow cytometry (BD Biosciences) (Zhou et al., 2015). The relative ratio of early apoptotic cells was calculated which was repeated more than three times.

LDH Release Assay

As an indicator of cell injury, LDH was released into the cell culture supernatant when cells undergo apoptosis (Chien et al., 2013). The cell viability was estimated by the amount of LDH release using LDH Detection Kit according to the manufacturer’s protocol (Li et al., 2014). The absorbance of samples was surveyed at 450 nm under a microplate reader (BioTek, United States).

Measurements of Intracellular SOD, MDA, and GSH Contents

SCs cells were seeded in 6-well plates and grown overnight. Next, being exposed to H₂O₂ and ABPPk, the cellular SOD, MDA, and GSH were assessed by following the manufacturer’s instructions of assay kits as described previously (Li et al., 2014). The activity of SOD was detected at 550 nm with an ELx-800 microplate reader. The MDA contents in each cultured SCs were measured at a wavelength of 532 nm. The absorbance of GSH was measured at 405 nm by using a microplate reader (BioTek, United States).

Immunofluorescence Staining

The SCs were seeded in 24-well dishes. After treatment and fixation, the cells were incubated with anti-cleaved caspase-3 (1:1000; Abcam) or anti-S100 (1:1000; Abcam) primary antibody solution at 4°C overnight, and then, secondary antibodies (Donkey anti-Mouse IgG-Alex-488, 1:200; Goat anti-Rabbit IgG-Cy3, 1:200; Invitrogen) were added for 1 h at room temperature. Finally, the nuclei were stained with DAPI or Hoechst 33342. The reaction was observed and photographed under a fluorescence microscopy (AxioImager M2, Zeiss).

Western Blotting Analysis

The protein concentration was determined using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, United States) (Yi et al., 2016). Equal amounts of protein samples were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States) (Gu et al., 2015). The membranes were blocked in 5% nonfat dry milk for 2 h and incubated with primary antibody anti-Bcl-2 (1:1000; Abcam), anti-Bax (1:1000; Abcam), anti-caspase-3 (1:1000; Abcam), anti-cleaved caspase-3 (1:1000; Abcam), anti-phospho ERK1/2 (1:1000; Abcam), anti-ERK1/2 (1:1000; Abcam), anti-PI3K (1:1000; Abcam), anti-phospho PI3K (1:1000; Abcam), anti-phospho AKT (1:1000; Abcam), anti-AKT (1:1000;Abcam), and anti-GAPDH (1:5000; Abcam) overnight at 4°C. After three washes with Tris-buffered saline with 0.1% Tween-20 (TBST), HRP-conjugated secondary antibodies (Pierce) were used to incubate the membranes for 1 h at room temperature. The membranes were developed using a chemiluminescence reagent (Roche) after three times washed in TBST and exposed to Kodak exposure films. GAPDH served as an internal control in order to make normalization and relative quantitative analysis of target protein expression (Feng et al., 2016).

Statistical Analysis

All data are presented as mean ± SEM. All experiments were undertaken in triplicates. Statistical significance was conducted by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. A value of P < 0.05 was considered to be statistically significant.

Results

ABPPk Protected SCs Against H₂O₂-Induced Cell Cytotoxicity

The purity of primary cultured SCs was confirmed by immunocytochemistry with anti-S100 antibody (Figure 1A) and flow cytometry data (Figure 1B), which indicated that 97.32% of the cell population was S100-positive. The MTT results showed that H₂O₂ stimulation (100, 200, and 400 μM) could gradually reduce cell activity in a time- and dose-dependent manner. As shown in Figure 1C, an obvious decrease of viability was observed at 24 h with 400 μM H₂O₂ exposure (53.1 ± 4.9%, compared to the control). To evaluate the potential cytoprotective effect of ABPPk against oxidative stress-induced SCs death, SCs were pretreated with ABPPk at various concentrations (0.1, 0.25, and 0.5 μg/ml) for 12 h followed by exposure to H₂O₂ at a final concentration of 400 μM for 24 h. After pretreatment with ABPPk, the cell viability was significantly increased in a dose-dependent manner compared with H₂O₂ treatment alone. Specifically, treatment with ABPPk prior to H₂O₂ exposure at different concentrations (0.1, 0.25, and 0.5 μg/ml) increased the survival to 60.3 ± 2.2, 66.8 ± 3.2, and 87.3 ± 1.1%, respectively (P < 0.01) (Figure 1D). These data suggest that ABPPk protected SCs against oxidative stress in SCs. In addition, ABPPk at these concentrations was not significantly cytotoxic.

FIGURE 1

ABPPk Protected SCs Against H₂O₂-Induced Cell Apoptosis

TUNEL analysis indicated that ABPPk prevented cultured SCs from H₂O₂-induced apoptosis. The TUNEL-positive cells were significantly increased from 3.04 ± 1.02 (control) to 21.86 ± 1.86% by exposure to H₂O₂ alone (P < 0.01). After pretreatment with ABPPk (0.5 μg/ml) for 12 h, H₂O₂ stimulation was added and the number of TUNEL-positive cells significantly reduced to 10.58 ± 4.49% as compared to H₂O₂ stimulation alone (P < 0.01) (Figures 2A,B). The apoptotic rate was also quantitatively analyzed by flow cytometry with AnnexinV-FITC/PI staining and provided further evidence that ABPPk pretreatment prevented cultured SCs from H₂O₂-induced apoptosis. The results of the apoptotic cell rate showed that ABPPk inhibited the early SCs apoptotic rate induced by H₂O₂ (Figures 2C,D). The ability of ABPPk to inhibit the SCs apoptosis under H₂O₂ conditions was exhibited according to the above results.

FIGURE 2

ABPPk Inhibits Apoptosis as Measured by Bax, Bcl-2, and Cleaved Caspase-3 in SCs

To confirm ABPPk protection against H₂O₂-induced apoptosis, apoptosis-associated protein (Bcl-2, Bax, and cleaved caspase-3) levels were measured. Western blot analyses indicated that treatment with ABPPk significantly increased the expression of Bcl-2 and significantly decreased the expression of Bax and cleaved caspase-3, compared with that of H₂O₂ treatment alone (Figures 3A–C). Immunofluorescence staining was performed to detect the activation of caspase-3, and cleaved caspase-3 induced by H₂O₂, while fewer ABPPk-treated cells displayed activated caspase-3 (Figure 3D).

FIGURE 3

ABPPk Attenuates H₂O₂-Induced Oxidative Injury

More and more evidence has shown that H₂O₂-induced oxidative stress and apoptosis plays the crucial role in SCs dysfunction and death. SOD and GSH levels were decreased due to the treatment of the cells by adding H₂O₂ along for 24 h (P < 0.01, compared to the control group). However, co-incubation with ABPPk remarkably attenuated the changes in the content of SOD and GSH (P < 0.01) (Figures 4A,B) when compared to H₂O₂-treated cells. Additionally, treating with H₂O₂ for 24 h, the intracellular MDA and LDH release were increased (P < 0.01, compared to the control group), however, incubation with ABPPk produced a significant decrease in the intracellular level of MDA and LDH compared with the H₂O₂ group (P < 0.01) (Figures 4C,D).

FIGURE 4

PI3K/AKT and ERK1/2 Pathways Are Involved in ABPPk Treatment Suppressing H₂O₂ Induced Apoptosis in SCs

Here, the expression levels of p-PI3K, PI3K, p-AKT, AKT, p-ERK1/2, and ERK1/2 were analyzed by Western blot. Compared with the control group, the p-PI3K/PI3K, p-AKT/AKT, and p-ERK1/2/ERK2 ratios were significantly reduced in the H₂O₂ group at 24 h. ABPPk treatment significantly reversed the decreased expression levels of p-PI3K/PI3K, p-AKT, and p-ERK1/2 in SCs exposed to H₂O₂ for 24 h (Figures 5A,B). These data suggest that ABPPk might suppress H₂O₂-induced apoptosis through the PI3K/AKT and ERK1/2 signaling pathways.

FIGURE 5

To further confirm our presumption that the role of ABPPk in the inhibition of H₂O₂-induced oxidative stress was related to the activation of PI3K/AKT and ERK1/2 pathways, two signal inhibitors, LY294002 and PD98059 (10 μM), respectively, were added to SCs. We found that the increased levels of p-PI3K, p-AKT, and p-ERK1/2 by ABPPk treatment were decreased after inhibitor treatment (Figures 6A,B). The agonists of these two signaling pathways had been performed (Supplementary Figure S1).

FIGURE 6

In terms of the caspase-dependent pattern in our cell model and ABPPk was proved to reduce the caspase-3 activation (Figure 3), we further evaluate if LY294002 and PD98059 could affect the protection of ABPPk according to the change in the protein level of cleaved caspase-3. As described in Western blot, ABPPk treatment significantly prevented the H₂O₂ induced upregulation of cleaved caspase-3 in SCs, which was eliminated after the addition of the inhibitor LY294002 or PD98059 (Figures 7A,B). It was suggested that ABPPk plays a protective role on the cell apoptosis induced by H₂O₂via the PI3K/AKT and ERK1/2 signaling pathways on the basis of the results.

FIGURE 7

Discussion

In this study, H₂O₂ was used to establish an oxidative damage model of SCs. The experimental results demonstrated that ABPPk markedly protects SCs against H₂O₂-induced apoptosis, which may be due to the activation of PI3K/AKT and ERK1/2 signaling pathways. In addition, ABPPk could increase SOD and GSH activity and decrease MDA and LDH level in oxidation damaged SCs. Furthermore, ABPPk inhibited the apoptosis of SCs by increasing the level of Bcl-2 and decreasing the levels of Bax and cleaved caspase-3. In accordance with the results, ABPPk has a potent protective effect on peripheral nerve injuries by protecting SCs from oxidative stress-induced apoptosis.

Achyranthes bidentata was extensively investigated based on its multiple physiological functions, including anti-inflammatory, antirheumatic, antipyretic, and diuretic effects. A large amount of previous studies have discussed the effectiveness of ABPP in nervous system (Shen et al., 2008, 2013). Recently, we separated the ABPP by HPLC and then one fraction was obtained, which was named ABPPk. It exhibits excellent neuron-protective efficiency (Cheng et al., 2014; Yu et al., 2014). In this research, we have found that ABPPk could protect SCs from oxidative stress-induced damage. It is the first time that the protective roles of ABPPk on SCs in oxidative stress were investigated.

Such neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and the like attribute to oxidative stress that induces cell apoptosis are characterized by overloading ROS (Roy et al., 2007; Numakawa et al., 2011; Kurian et al., 2017). Oxidative injuries to SCs bring about demyelination and slow formation of axon, which might play the important role in PNS injury and regeneration. H₂O₂ is widely used as an oxidant for studies in vitro. Previous research showed that H₂O₂ injury was able to induce cell apoptosis with concentration and time dependence (Wang and Huang, 2005; Kaji et al., 2010; Bai et al., 2012). In the present experiment, it is confirmed that SCs treated with 400 μM H₂O₂ exhibits loss of cell viability. Pretreatment with 0.1–0.5 μg/ml ABPPk, however, significantly attenuate the loss of cell viability induced by H₂O₂ in a dose-dependent manner.

The apoptosis process could be regulated by proteins of Bcl-2 family through balancing of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) products (Bone, 1991; Jezek and Plecita-Hlavata, 2009). In view of the results, ABPPk decreased H₂O₂-induced apoptosis of SCs by upregulation of Bcl-2 as well as downregulation of Bax. In addition, caspase 3 acts as an executor in the protease cascade reactions of cell apoptosis (Mao et al., 2007; Hua et al., 2015). The recent work suggested that caspase-3 is important for cell morphology and biochemical events related to the process of apoptosis (Makin, 2016; Zhang et al., 2016). In our study, we confirmed that the cultured SCs showed a remarkable increase of cleaved caspase-3 after exposed to H₂O₂ and pretreatment with ABPPk significantly attenuated activation of caspase-3, which suggest that the potential anti-apoptotic effects of ABPPk against H₂O₂-induced cell apoptosis of SCs which might be regulated by the apoptosis-related caspase-3 protein expression and activation.

During the process of cell apoptosis, the MDA, as a byproduct induced by excessive ROS, is considered to be an indicator for oxidative stress (Wang et al., 2012). SOD and GSH, as antioxidants for the prevention of free-radical damage caused by ROS, provide a repair mechanism for oxidative stress (Wang et al., 2012). In our study, SOD and GSH were decreased after exposure to H₂O₂, indicating the impairment of SCs in oxidative stress. Moreover, the MDA level has an obvious elevation associated with an increase of LDH release. Nonetheless, these H₂O₂-induced cellular events were considerably blocked when SCs were co-cultured with ABPPk. The results reveal that the increase of endogenous antioxidant preservation may represent a potential mechanism of ABPPk for attenuating intracellular oxidative stress.

PI3K/AKT and ERK1/2 signaling, as we know, is vital to cell survival and cell apoptosis under stress exposure (Gerrits et al., 2006; Chen et al., 2015). In this research, the expression levels of p-PI3K, p-AKT, and p-ERK1/2 were reduced under the exposure to H₂O₂ and this situation was partially reversed by the ABPPk treatment. It shows that ABPPk activate the PI3K/AKT and ERK1/2 pathways in SCs resulting in the suppression of effect of H₂O₂ stress induced-apoptosis of SCs. In accordance with the results, the effects of pharmacological inhibitors for PI3K/AKT (LY294002) and ERK1/2 (PD98059) in SCs, and the reduction of cell death by ABPPk depressed by inhibitor treatment were demonstrated.

In summary, our study indicates that ABPPk could protect SCs from H₂O₂-induced oxidative stress and cell apoptosis, as well as activation of PI3K/AKT and ERK1/2 signaling pathways. The results lay a ground work for deeper understanding of the molecular mechanism of ABPPk-regulated anti-apoptotic activity in SCs and also provide valuable information of new strategies for better clinical treatments of peripheral nerve injuries.

References

• 1

  BaiY. H.PengY. M.YinW. Q.LiuH.LiuF. Y.DuanS. B.et al (2012). p-Aminophenol and p-paraphenylenediamine induce injury and apoptosis of human HK-2 proximal tubular epithelial cells.J. Nephrol.25481–489. 10.5301/JN.2011.8495

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 2

  BehlC. (1999). Alzheimer’s disease and oxidative stress: implications for novel therapeutic approaches.Prog. Neurobiol.57301–323. 10.1016/S0301-0082(98)00055-0

  • CrossRef
  • Google Scholar

• 3

  BoneR. C. (1991). The pathogenesis of sepsis.Ann. Intern. Med.115457–469. 10.7326/0003-4819-115-6-457

  • CrossRef
  • Google Scholar

• 4

  ChenX. L.FuJ. P.ShiJ.WanP.CaoH.TangZ. M. (2015). CXC195 induces apoptosis and endoplastic reticulum stress in human hepatocellular carcinoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.Mol. Med. Rep.128229–8236. 10.3892/mmr.2015.4479

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 5

  ChengQ.JiangC.WangC.YuS.ZhangQ.GuX.et al (2014). The Achyranthes bidentata polypeptide k fraction enhances neuronal growth in vitro and promotes peripheral nerve regeneration after crush injury in vivo.Neural Regen. Res.92142–2150. 10.4103/1673-5374.147948

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 6

  ChienW. L.LeeT. R.HungS. Y.KangK. H.WuR. M.LeeM. J.et al (2013). Increase of oxidative stress by a novel PINK1 mutation, P209A.Free Radic. Biol. Med.58160–169. 10.1016/j.freeradbiomed.2012.12.008

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 7

  ChoiJ.SullardsM. C.OlzmannJ. A.ReesH. D.WeintraubS. T.BostwickD. E.et al (2006). Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases.J. Biol. Chem.28110816–10824. 10.1074/jbc.M509079200

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 8

  DubovyP.SvizenskaI.KlusakovaI.ZitkovaA.Houst’AvaL.HaninecP. (2001). Laminin molecules in freeze-treated nerve segments are associated with migrating Schwann cells that display the corresponding alpha6beta1 integrin receptor.Glia3336–44. 10.1002/1098-1136(20010101)33:1<36::AID-GLIA1004>3.0.CO;2-B

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 9

  FengJ.HuZ.ChenH.HuaJ.WuR.DongZ.et al (2016). Depletion of kinesin-12, a myosin-IIB-interacting protein, promotes migration of cortical astrocytes.J. Cell Sci.1292438–2447. 10.1242/jcs.181867

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 10

  Fernandez-ChecaJ. C.FernandezA.MoralesA.MariM.Garcia-RuizC.ColellA. (2010). Oxidative stress and altered mitochondrial function in neurodegenerative diseases: lessons from mouse models.CNS Neurol. Disord. Drug Targets9439–454. 10.2174/187152710791556113

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 11

  FinkelT.HolbrookN. J. (2000). Oxidants, oxidative stress and the biology of ageing.Nature408239–247. 10.1038/35041687

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 12

  GeoffroyM.KleinclaussA.GrandemangeS.HupontS.BoisbrunM.FlamentS.et al (2017). Pro-apoptotic effect of Delta2-TGZ in “claudin-1-low” triple-negative breast cancer cells: involvement of claudin-1.Breast Cancer Res. Treat.165517–527. 10.1007/s10549-017-4378-2

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 13

  GerritsM.WestenbroekC.KochT.GrootkarzijnA.ter HorstG. J. (2006). Increased limbic phosphorylated extracellular-regulated kinase 1 and 2 expression after chronic stress is reduced by cyclic 17beta-estradiol administration.Neuroscience1421293–1302. 10.1016/j.neuroscience.2006.07.014

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 14

  GuY.ChenC.YiS.WangS.GongL.LiuJ.et al (2015). miR-sc8 inhibits schwann cell proliferation and migration by targeting Egfr.PLoS One10:e0145185. 10.1371/journal.pone.0145185

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 15

  HeG.GuoW.LouZ.ZhangH. (2014). Achyranthes bidentata saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK MAPK signaling pathway.Cell Biochem. Biophys.70467–473. 10.1007/s12013-014-9942-3

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 16

  HeQ.ManL.JiY.DingF. (2012). Comparison in the biological characteristics between primary cultured sensory and motor Schwann cells.Neurosci. Lett.52157–61. 10.1016/j.neulet.2012.05.059

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 17

  HuaP.LiuJ.TaoJ.LiuJ.YangS. (2015). Influence of caspase-3 silencing on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells under hypoxia.Int. J. Clin. Exp. Med.81624–1633.

  • Pubmed Abstract
  • Google Scholar

• 18

  JessenK. R.MirskyR. (1997). Embryonic schwann cell development: the biology of Schwann cell precursors and early Schwann cells.J. Anat. 191(Pt 4), 501–505. 10.1046/j.1469-7580.1997.19140501.x

  • CrossRef
  • Google Scholar

• 19

  JezekP.Plecita-HlavataL. (2009). Mitochondrial reticulum network dynamics in relation to oxidative stress, redox regulation, and hypoxia.Int. J. Biochem. Cell Biol.411790–1804. 10.1016/j.biocel.2009.02.014

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 20

  KajiY.KiuchiT.OshikaT. (2010). Carteolol hydrochloride suppresses the generation of reactive oxygen species and rescues cell death after ultraviolet irradiation of cultured lens epithelial cells.Open Ophthalmol. J.460–65. 10.2174/1874364101004010060

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 21

  KurianP.ObisesanT. O.CraddockT. J. A. (2017). Oxidative species-induced excitonic transport in tubulin aromatic networks: potential implications for neurodegenerative disease.J. Photochem. Photobiol. B175109–124. 10.1016/j.jphotobiol.2017.08.033

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 22

  LiJ.ShenF.GuanC.WangW.SunX.FuX.et al (2014). Activation of Nrf2 protects against triptolide-induced hepatotoxicity.PLoS One9:e100685. 10.1371/journal.pone.0100685

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 23

  LiM.ZhangP.LiH.ZhuY.CuiS.YaoD. (2015). TGF-beta1 is critical for Wallerian degeneration after rat sciatic nerve injury.Neuroscience284759–767. 10.1016/j.neuroscience.2014.10.051

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 24

  LuoY.LiuX.ZhengQ.WanX.OuyangS.YinY.et al (2012). Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H2O2-activated calcium signaling pathway in mouse hippocampal neurons.Biochem. Biophys. Res. Commun.425473–477. 10.1016/j.bbrc.2012.07.131

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 25

  MakinS. (2016). Pathology: the prion principle.Nature538S13–S16. 10.1038/538S13a

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 26

  MantuanoE.InoueG.LiX.TakahashiK.GaultierA.GoniasS. L.et al (2008). The hemopexin domain of matrix metalloproteinase-9 activates cell signaling and promotes migration of schwann cells by binding to low-density lipoprotein receptor-related protein.J. Neurosci.2811571–11582. 10.1523/JNEUROSCI.3053-08.2008

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 27

  MaoZ.ZhengY. L.ZhangY. Q.HanB. P.ZhuX. W.ChangQ.et al (2007). The anti-apoptosis effects of daidzein in the brain of D-galactose treated mice.Molecules121455–1470. 10.3390/12071455

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 28

  NumakawaT.MatsumotoT.NumakawaY.RichardsM.YamawakiS.KunugiH. (2011). Protective action of neurotrophic factors and estrogen against oxidative stress-mediated neurodegeneration.J. Toxicol.2011:405194. 10.1155/2011/405194

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 29

  OlanowC. W. (1993). A radical hypothesis for neurodegeneration.Trends Neurosci.16439–444. 10.1016/0166-2236(93)90070-3

  • CrossRef
  • Google Scholar

• 30

  PengS.WangC.MaJ.JiangK.JiangY.GuX.et al (2018). Achyranthes bidentata polypeptide protects dopaminergic neurons from apoptosis in Parkinson’s disease models both in vitro and in vivo.Br. J. Pharmacol.175631–643. 10.1111/bph.14110

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 31

  PizarroJ. G.FolchJ.Vazquez De la TorreA.VerdaguerE.JunyentF.JordanJ.et al (2009). Oxidative stress-induced DNA damage and cell cycle regulation in B65 dopaminergic cell line.Free Radic. Res.43985–994. 10.1080/10715760903159188

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 32

  PurvesT.MiddlemasA.AgthongS.JudeE. B.BoultonA. J.FernyhoughP.et al (2001). A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy.FASEB J.152508–2514. 10.1096/fj.01-0253hyp

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 33

  RoyD.FeltyQ.NarayanS.JayakarP. (2007). Signature of mitochondria of steroidal hormones-dependent normal and cancer cells: potential molecular targets for cancer therapy.Front. Biosci. 12:154–173. 10.2741/2056

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 34

  ShenH.WuX.ZhuY.SunH. (2013). Intravenous administration of Achyranthes bidentata polypeptides supports recovery from experimental ischemic stroke in vivo.PLoS One8:e57055. 10.1371/journal.pone.0057055

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 35

  ShenH.YuanY.DingF.LiuJ.GuX. (2008). The protective effects of Achyranthes bidentata polypeptides against NMDA-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A- and NR2B-containing NMDA receptors.Brain Res. Bull.77274–281. 10.1016/j.brainresbull.2008.08.002

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 36

  ShenM.JiY.ZhangS.ShiH.ChenG.GuX.et al (2012). A proteome map of primary cultured rat Schwann cells.Proteome Sci.10:20. 10.1186/1477-5956-10-20

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 37

  ShenY.ZhangQ.GaoX.DingF. (2011). An active fraction of Achyranthes bidentata polypeptides prevents apoptosis induced by serum deprivation in SH-SY5Y cells through activation of PI3K/Akt/Gsk3beta pathways.Neurochem. Res.362186–2194. 10.1007/s11064-011-0543-x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 38

  ShererT. B.BetarbetR.StoutA. K.LundS.BaptistaM.PanovA. V.et al (2002). An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage.J. Neurosci.227006–7015. 10.1523/JNEUROSCI.22-16-07006.2002

  • CrossRef
  • Google Scholar

• 39

  WangC. H.WuS. B.WuY. T.WeiY. H. (2013). Oxidative stress response elicited by mitochondrial dysfunction: implication in the pathophysiology of aging.Exp. Biol. Med.238450–460. 10.1177/1535370213493069

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 40

  WangY.ShenW.YangL.ZhaoH.GuW.YuanY. (2013). The protective effects of Achyranthes bidentata polypeptides on rat sciatic nerve crush injury causes modulation of neurotrophic factors.Neurochem. Res.38538–546. 10.1007/s11064-012-0946-3

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 41

  WangD.YuanX.LiuT.LiuL.HuY.WangZ.et al (2012). Neuroprotective activity of lavender oil on transient focal cerebral ischemia in mice.Molecules179803–9817. 10.3390/molecules17089803

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 42

  WangY. K.HuangZ. Q. (2005). Protective effects of icariin on human umbilical vein endothelial cell injury induced by H2O2 in vitro.Pharmacol. Res.52174–182. 10.1016/j.phrs.2005.02.023

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 43

  Woszczycka-KorczynskaI.OlakowskaE.MarcolW.Lewin-KowalikJ.Jedrzejowska-SzypulkaH. (2013). Schwann cells in therapy of spinal cord injuries.Postepy Hig. Med. Dosw.67680–689. 10.5604/17322693.1059602

  • CrossRef
  • Google Scholar

• 44

  YiS.YuanY.ChenQ.WangX.GongL.LiuJ.et al (2016). Regulation of Schwann cell proliferation and migration by miR-1 targeting brain-derived neurotrophic factor after peripheral nerve injury.Sci. Rep.6:29121. 10.1038/srep29121

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 45

  YuB.QianT.WangY.ZhouS.DingG.DingF.et al (2012). miR-182 inhibits Schwann cell proliferation and migration by targeting FGF9 and NTM, respectively at an early stage following sciatic nerve injury.Nucleic Acids Res.4010356–10365. 10.1093/nar/gks750

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 46

  YuS.WangC.ChengQ.XuH.ZhangS.LiL.et al (2014). An active component of Achyranthes bidentata polypeptides provides neuroprotection through inhibition of mitochondrial-dependent apoptotic pathway in cultured neurons and in animal models of cerebral ischemia.PLoS One9:e109923. 10.1371/journal.pone.0109923

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 47

  YuanY.ShenH.YaoJ.HuN.DingF.GuX. (2010). The protective effects of Achyranthes bidentata polypeptides in an experimental model of mouse sciatic nerve crush injury.Brain Res. Bull.8125–32. 10.1016/j.brainresbull.2009.07.013

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 48

  ZhangY.DaiC. L.ChenY.IqbalK.LiuF.GongC. X. (2016). Intranasal insulin prevents anesthesia-induced spatial learning and memory deficit in mice.Sci. Rep.6:21186. 10.1038/srep21186

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 49

  ZhaoZ.LiX.LiQ. (2017). Curcumin accelerates the repair of sciatic nerve injury in rats through reducing Schwann cells apoptosis and promoting myelinization.Biomed. Pharmacother.921103–1110. 10.1016/j.biopha.2017.05.099

  • Pubmed Abstract
  • CrossRef
  • Google Scholar

• 50

  ZhouX.JiangZ.ZouY.YinY.ZuoQ.SunL. (2015). Role of SOCS3 in the Jak/stat3 pathway in the human placenta: different mechanisms for preterm and term labor.Acta Obstet. Gynecol. Scand.941112–1117. 10.1111/aogs.12708

  • Pubmed Abstract
  • CrossRef
  • Google Scholar