Introduction

Oxidative stress (OS) has been confirmed as a contribution to the pathogenesis and pathophysiology of many neurological disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Simon et al. 2000). However, the degree of oxidative stress depends on the balance between reactive oxygen species (ROS) production and antioxidant defenses (Finkel and Holbrook 2000). Overproduction of ROS can attack membrane lipids, DNAs, and proteins, which can promote mitochondrial injury and cell death by activating several signaling pathways, such as p53, PI(3)K, and MAPKs (Annicchiarico et al. 2007; Finkel and Holbrook 2000; Choi et al. 2014). Antioxidants are proposed as free-radical scavenger against oxidative injury by preventing oxidation of proteins and lipid peroxidation. Numerous studies have demonstrated that antioxidants can scavenge ROS, inhibit the formation of ROS, and bind metal ions needed for catalysis of ROS generation (Gulcin et al. 2002; Pisoschi and Pop 2015). Especially, the brain is believed to be particularly vulnerable to oxidative stress due to the low capacity of antioxidants (Uttara et al. 2009). Therefore, there is a need to identify new natural antioxidants for use as safe and effective neuroprotective agents in the prevention and treatment of oxidative stress-related neurodegenerative disease.

As a popular vegetable, burdock is widely planted in China for hundreds of years and recommended as a healthy and nutritious food. Increasing evidence has shown that burdock roots have hepatoprotective, anti-inflammatory, and free-radical scavenging activities, many of the biological properties can be attributed to its components with anti-oxidative activity such as caffeic acid derivatives (Ferracane et al. 2010; Predes et al. 2011). In our previous studies, we demonstrated that the extract of burdock roots displayed significant neuroprotection against glutamate-induced and H2O2-induced cytotoxicity in SH-SY5Y cells (Tian et al. 2014a, b). Recently, we have demonstrated that caffeoylquinic acid derivatives, abundantly found in burdock roots, displayed distinct antioxidative effects and some of which exhibited significant neuroprotective effects against glutamate or NMDA-induced SH-SY5Y cells injury (Jiang et al. 2016; Jun-peng et al. 2015; Tian et al. 2015). However, the structure–activity relationships (SARs) between the physiological functions of these compounds (Fig. 1) and the potential mechanisms against oxidative injury have not been elucidated. Therefore, the objectives of this study are to determine their structural requirements as new neuroprotective agents and investigate the potential mechanism.

Fig. 1
figure 1

The structures of 1–10

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Experimental Section

General Experimental Procedures

The caffeoylquinic acid derivatives mentioned in this paper were isolated from the roots of burdock as previous described (purity of ≥90 %) (Jiang et al. 2016). MTT, N-acetyl-l-cysteine (NAC), and Hoechst 33342 were obtained from Sigma-Chemical (St. Louis, MO, USA). We purchased fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) from Hyclone (Logan, UT, USA). H2O2 was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Annexin V-FITC kit was purchased from Biotool (Houston, USA). The kits for lactate dehydrogenase (LDH) , mitochondrial membrane potential (MMP), 2′,7′-dichlorofluorescin diacetate (DCF-DA), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), Bicinchoninic Acid (BCA), and chemiluminescence were all acquired from Beyotime (Shanghai, China). Antibodies for Bax and Bcl-2 were purchased from Abcam (Cambridge, UK). Antibodies for cytochrome c, caspase-3, caspase-9, and p53 were purchased from ZSGB-Bio (Beijing, China). Antibodies for Bad, PARP, cleaved PARP, p-ERK1/2, t-ERK, p-JNK, t-JNK, p-p38, t-p38, p-AKT, and t-AKT were purchased from Cell Signaling Technology (Boston, USA). Antibodies for cleaved caspase-3, cleaved caspase-9, and β-actin were purchased from Santa Cruz Biotechnology (CA, USA).

Cell Culture

SH-SY5Y cells were cultured in complete DMEM medium containing 10 % (v/v) FBS at 37 °C in 5 % CO2. At 80 % confluence, cells suspension were added to the culture medium to obtain a density of 1 × 104 cells/well into 96-well plates (for MTT assays and LDH assays) or 2 × 105 cells/well into 6-well plates (for Hoechst 33342 staining, Annexin V-FITC and propidium iodide (PI) double staining, the measurement of GSH-Px, SOD, MDA, and Western blotting assays). After growth in the incubator at 37 °C for 24 h, the cells were pretreated with different concentration of compounds for 12 h, change medium, then incubated with 400 μM H2O2 for 3 h. Then, the supernatant or the cells were collected to perform the various assays.

MTT Assay

Cell viability was evaluated by measuring MTT reduction as previously reported (Tian et al. 2014b). After the indicated treatments, MTT solution (0.5 mg/mL) was added into each well and the commixture was incubated for 4 h at 37 °C. After incubation, the medium was replaced and 150 μL of DMSO was added. The absorbance of each sample was measured at 490 nm with a microplate reader (ELX 800, Bio-tek, USA). The concentration (μM) that caused 50 % protective effect relative to the vehicle control (EC50) was determined from an average of three independent experiments each conducted with six points (calculated in GraphPad Prism 5).

LDH Assay

LDH activity was evaluated in the culture medium. According to the manufacturer’s instructions, the supernatants were collected from each well after the indicated treatments and incubated with the reagent mixture. The absorbance of each sample was determined at 490 nm in a microplate reader. The LDH release activity was presented as the percentage of cytotoxicity.

Hoechst 33342 Staining

Hoechst 33342 staining was used to detect changes in the nucleus of apoptotic cells. As previously reported (Tian et al. 2014a), after the indicated treatments, cells were washed with PBS solution three times and stained with a final concentration of 10 μg/mL Hoechst 33342 in the dark for 30 min at 37 °C. The cells were then detected under a fluorescent microscope (IX71, Olympus, Japan).

Annexin V-FITC and PI Double-Staining Assay

The apoptotic and necrotic cells were evaluated and quantified by an Annexin V-FITC kit according to the manufacturer’s instructions as previously reported. (Tian et al. 2014a) After the indicated treatments, cell mass was collected and resuspended in 195 μL of Annexin V-FITC buffer. Annexin V-FITC (5 μL ) and PI (5 μL ) were added to 1 × 105 cells, mixed with cells suspension to uniformity, and incubated for 15 min, then put in an ice bath in the dark, and measured by flow cytometer (Becton–Dickinson, USA).

Measurement of Mitochondrial Membrane Potential (MMP)

JC-1 was used to measure ΔΨm of SH-SY5Y cells as previously reported (Wang et al. 2013). Total cells were incubated with JC-1 for 20 min at 37 °C. The fluorescence intensity was detected with a fluorescence microscope at an excitation of 490 nm and an emission of 530 nm (green fluorescent monomers) and 590 nm (red fluorescent aggregates), respectively. An analysis software (Image J) was used for the quantitative determination.

Measurement of Intracellular ROS Levels

The intracellular ROS levels were evaluated using the fluorescent probe DCFH-DA as previously reported (Tian et al. 2014a). According to the manufacturer’s instructions, cells were washed twice with PBS and loaded with DCFH-DA (final concentration 10 μM). After being incubated for 20 min at 37 °C, the cells were washed three times and observed with a fluorescence microscope at wavelengths of 488 nm for excitation and 525 nm for emission. NAC is used as a positive drug. An analysis software (Image J) was used for the quantitative determination.

Measurement of GSH-Px, SOD Activities, and MDA Levels

The levels of GSH-Px, SOD, and MDA were determined as previously reported (Tian et al. 2014a). Briefly, SH-SY5Y cells were collected after the indicated treatments. Protein concentration was determined by the BCA protein assay kit. The detection method of GSH-Px, SOD, and MDA was conducted according to the manufacturer’s instructions. The absorbance was measured at wavelengths of 340, 450, and 532 nm in a microplate reader, respectively.

Western Blotting Analysis

Western blotting analysis was performed as described previously (Tian et al. 2014a, 2015). In brief, cell lysates were separated on 12 % SDS polyacrylamide gel transferred onto PVDF (Millipore Corporation) membranes, and stained with primary antibodies against Bax, Bcl-2 (1:5000), cytochrome c, caspase-3, caspase-9, p53, Bad (1:800), cleaved caspase-3, cleaved caspase-9 (1:500), PARP, cleaved PARP, p-ERK1/2, t-ERK, p-JNK, t-JNK, p-p38, t-p38, p-AKT (Ser473), and t-AKT (1:1000) over night at 4 °C. The membrane was washed with TBST three times, followed by reaction with anti-rabbit or anti-mouse IgG secondary antibody for 1 h at room temperature. The results were visualized by chemiluminescence using ECL advance reagent. The β-actin (1:1200) protein served as an internal control in Western blotting analysis. An image analysis software (Image J) was used to analyze the gray degree values of the results of Western blotting.

Statistical Analysis

All data were represented as the mean ± SEM. Data were analyzed using one-way ANOVA followed by a Tukey’s multiple comparison Test (calculated in GraphPad Prism 5). Significant difference was considered to be at p < 0.05.

Results

MDCQA Decreased H2O2-Induced Cytotoxicity of SH-SY5Y Cells

We measured the effects of all compounds (Fig. 1) against H2O2-induced SH-SY5Y cells death. The MTT results indicated that all of the compounds showed no cytotoxicity to the SH-SY5Y cells under the concentration of 200 μM (data not shown). Results shown in Table 1 indicated that most of the compounds showed cytoprotective effects, but the effects of those were not better than 2 (MDCQA) and 4 with EC50 values of 15.36 ± 0.52 and 29.88 ± 0.49 μM, respectively. We also obtained 5 and 6 that also exerted notable cytoprotective effects, with EC50 values of 72.74 ± 1.01 and 52.58 ± 1.27 μM, respectively.

Table 1 Neuroprotective effects of compounds 1–10
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Possible effect of MDCQA against H2O2-induced cytotoxicity was further confirmed morphologically by an inverted microscope. As shown in Fig. 2a, the shrunken cells induced by H2O2 were effectively recovered in MDCQA pretreatment groups. In Fig. 2b, no morphological changes were observed after 24 h pretreatment of MDCQA at different concentrations (25–200 μM). The effect of MDCQA against H2O2-induced SH-SY5Y cells injury is shown in Fig. 2c; cell viability was reduced to 48.78 ± 2.31 % of the control group after exposed to 400 μM of H2O2 for 3 h. MDCQA pretreatment significantly attenuates H2O2-induced cells injury compared to H2O2 treatment alone. The cell viability was significantly increased to 52.16 ± 2.20, 65.32 ± 1.42, and 76.15 ± 3.10 % as compared with the control group, respectively.

Fig. 2
figure 2

Effects of MDCQA on H2O2-induced cytotoxicity of SH-SY5Y cells. a The morphological changes of SH-SY5Y cells were observed under a microscope (magnification ×200). b Cytotoxicity to cell viability after treated alone with MDCQA (25–200 μΜ). c The cell viability was measured by MTT assay. d The release of LDH was assessed using the LDH assay. Data are showed as mean ± SEM (n = 3). &&& p < 0.001 versus the control group; ***p < 0.001 versus the H2O2-treated group

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The effect of MDCQA on LDH release was evaluated in the culture medium and was quantified under the same experimental conditions. As shown in Fig. 2d, the LDH release in H2O2-stimulated group was increased to 63.00 ± 4.78 % of the control group (Triton X-100). However, MDCQA decreased the LDH release to 56.67 ± 2.60, 36.33 ± 2.84, and 14.03 ± 1.41 % of the positive control group, respectively.

MDCQA Attenuated H2O2-Induced Apoptosis in SH-SY5Y Cells

To verify whether MDCQA attenuated H2O2-induced SH-SY5Y cell apoptosis, the Hoechst 33342 staining, and Annexin V-PI double staining were performed. As shown in Fig. 3a, the H2O2-stimulated group revealed massive chromatin condensation, nuclear shrinkage, and fragmentation under fluorescence microscope. In contrast, pretreatment with MDCQA ameliorated these characteristics of apoptosis significantly in SH-SY5Y cells.

Fig. 3
figure 3

Effects of MDCQA on attenuating H2O2-induced apoptosis in SH-SY5Y cells. a Morphological changes of nuclear chromatin by Hoechst 33342 staining were observed using a fluorescence microscope (magnification ×200). b Quadrant analysis the fluorescence characteristics of four panels: viable cells on the lower left, Annexin V(−)/PI(−); necrotic cells on the upper left, Annexin V(−)/PI(+); late apoptotic cells on the upper right, Annexin V(+)/PI(+) and early apoptotic cells on the lower right, Annexin V(+)/PI(−). c Quantitative analysis of the bar graphs showed the percentage of late apoptotic and total cells. Data are showed as mean ± SEM (n = 3), &&& p < 0.001 versus the control group, ***p < 0.001 versus the H2O2-treated group

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The effect of MDCQA was further evaluated by Annexin V-PI double-staining assay. As shown in Fig. 3b and c, the percentage of early apoptotic cells in MDCQA groups (right low quadrant) decreased from 9.94 ± 3.96 to 4.05 ± 5.61 %, and the percentage of late apoptotic and necrotic cells (right upper quadrant) was also decreased from 33.56 ± 2.14 to 12.70 ± 1.12 %. Taken together, these results indicated that MDCQA protects SH-SY5Y cells by attenuating H2O2-induced apoptosis.

ΔΨm were measured by determining the red/green fluorescence ratio of JC-1. As shown in Fig. 4a and b, treating with 400 μM H2O2 resulted in significant ΔΨm loss compared with control group. In contrast, ΔΨm loss induced by H2O2 were significantly attenuated by pretreatment with 7.5–30 μM MDCQA. All the results indicated that MDCQA attenuated apoptosis by increasing the ΔΨm in SH-SY5Y cells.

Fig. 4
figure 4

Effects of MDCQA on ameliorating ΔΨm loss in H2O2-induced in SH-SY5Y cells. a Mitochondrial membrane potential fluorescence images visualized by a fluorescence microscope (magnification ×400). b Quantitative analysis of the bar graphs showed the red/green ratio. Data are showed as mean ± SEM (n = 3), &&& p < 0.001 versus the control group, ***p < 0.001 versus the H2O2-treated group (Color figure online)

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MDCQA Attenuated H2O2-Induced ROS Generation in SH-SY5Y Cells

We investigated the ability of MDCQA to counteract H2O2-induced intracellular ROS production by the DCFH-DA assay. As shown in Fig. 5a–g, the percentage of intracellular ROS levels in H2O2-simulated group (300.17 ± 24.56 %) was much higher than that of control group. By contrast, ROS generation tended to decrease significantly by pretreatment with MDCQA in a dose-dependent manner. The levels of ROS were decreased to 246.90 ± 27.96, 153.53 ± 16.53, and 109.31 ± 10.53 % of control group, respectively. As a positive drug, the levels in NAC group were 96.79 ± 5.46 %. These results indicated that MDCQA exerted notable ability to scavenge ROS during the H2O2-induced oxidative injury in SH-SY5Y cells.

Fig. 5
figure 5

Neuroprotection of MDCQA on attenuating ROS generation, improved the GSH-Px and SOD activities, and decreased the MDA level in SH-SY5Y cells induced by H2O2. ROS production was assessed with DCFH-DA fluorescence dye (magnification ×400). a Control; b 400 μM H2O2; c 400 μM H2O2 + 7.5 μM MDCQA; d 400 μM H2O2 + 15 μM MDCQA; e 400 μM H2O2 + 30 μM MDCQA; f 400 μM H2O2 + 10 μM NAC; g Quantitative analysis of the bar graphs showed the percentage of DCF fluorescence intensity. Effects of MDCQA on modulated levels of GSH-Px (h), SOD (i) and MDA (j) in SH-SY5Y cells induced by H2O2. Data are showed as mean ± S.E.M. (n = 3). && p < 0.01 and &&& p < 0.001 versus the control group; *p < 0.05, **p < 0.01, and ***p < 0.001 versus the H2O2-treated group

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MDCQA Improved the GSH-Px and SOD Activities and Attenuated the MDA Level in SH-SY5Y Cells Induced by H2O2

In order to confirm whether the ROS production is affected by regulating the antioxidant defenses, we investigated the activities of GSH-Px, SOD, and MDA level. As shown in Fig. 5h, i, in the presence of 400 μM H2O2, activities of GSH-Px and SOD were decreased to 39.86 ± 2.54 and 12.89 ± 1.49 U/mg, respectively. Pretreatment with MDCQA significantly improved the activities of GSH-Px and SOD in a dose-dependent manner. These results suggested that MDCQA could prevent the decrease of GSH-Px and SOD activities in H2O2-induced SH-SY5Y cells. Furthermore, as one of the most important product of membrane lipid peroxidation, the MDA level was also tested after the exposure to H2O2. From Fig. 5j, we observed a significant increase of MDA level in H2O2-stimulated group, about 16.52 ± 1.52 nmol/mg. However, pretreatment with 7.5–30 μM MDCQA decreased the content of MDA to 14.60 ± 1.09, 10.85 ± 0.82, and 8.19 ± 0.67 nmol/mg, respectively. The results indicated that pretreatment with MDCQA ameliorates H2O2-induced lipid peroxidation in SH-SY5Y cells.

MDCQA Modulated the Expression of Apoptotic-Associated Proteins in SH-SY5Y Cells Induced by H2O2

Upon observing that MDCQA significantly provides cytoprotection against H2O2-induced apoptotic death in SH-SY5Y cells, we conducted immunoblotting analysis to investigate the effects of MDCQA on the apoptotic-associated proteins. As shown in Fig. 6a, Western blotting assay was performed to assess the levels of Bcl-2, Bax, Bad, cytochrome c, procaspase-3, procaspase-9, PARP, and p53. After exposed to 400 μM H2O2, the ratio of Bcl-2/Bax and the expressions of Bad and cytochrome c were decreased (Fig. 6b–d). However, these trends can be reversed by MDCQA in a dose-dependent manner. Similarly, the up-regulation of procaspase-3 and procaspase-9 were significantly suppressed by MDCQA (Fig. 6e, f). In contrast, the high levels of cleaved caspase-3, cleaved caspase-9, PARP, cleaved PARP, and p53 were decreased in a dose-dependent manner by pretreating with MDCQA (Fig. 6g, h).

Fig. 6
figure 6

Neuroprotection of MDCQA on the levels of apoptosis-related proteins in H2O2-treated SH-SY5Y cells. The expression of Bax, Bcl-2, Bad, cytochrome c, procaspase-3/9, cleaved caspase-3/9, PARP, and p53. Data are showed as mean ± SEM (n = 3). & p < 0.05 versus the control group, &&& p < 0.001 versus the control group; *p < 0.05 versus the H2O2-treated group, **p < 0.01 versus the H2O2-treated group, and ***p < 0.001 versus the H2O2-treated group

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MDCQA Modulated the H2O2-Induced Phosphorylation of p38, JNK, ERK 1/2 MAPKs, and AKT in SH-SY5Y Cells

We conducted immunoblotting analysis to investigate the effects of MDCQA on the main signal transduction pathways involved in the induction of apoptosis in neuronal cells. MDCQA significantly decreased the phosphorylation of p38, JNK, and ERK 1/2 MAPKs in a dose-dependent manner (Fig. 7a). Moreover, the effects of MDCQA on phosphorylation of AKT were investigated. As shown in Fig. 7b, contrast to the H2O2-stimulated group, the amounts of AKT phosphorylation were increased in a dose-dependent manner in MDCQA groups. Based on these results, we suggest that MDCQA could exert a protective effect by inhibiting MAPKs activation and increasing AKT phosphorylation on Ser473.

Fig. 7
figure 7

Neuroprotection of MDCQA on the levels of MAPKs and AKT in H2O2-treated SH-SY5Y cells. Levels of phosphorylated and total a p38, JNK, ERK 1/2 and b AKT proteins were determined by Western blot analysis. β-actin was used as an internal control. All analyses were performed in triplicate

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Discussion

The present study demonstrated that MDCQA, a caffeoylquinic acid (CQA) derivatives from burdock roots, protects cells against H2O2 injury by reducing oxidative stress, increasing intracellular GSH-Px, and SOD levels, reducing the phosphorylation of MAPK signaling pathways (ERK1/2, JNK, and p38) and increasing phosphorylation of AKT.

In our study, we found that the intensity of the anti-oxidative stress ability of CQAs against H2O2 injury in SH-SY5Y cells strongly depends on its chemical structure and malonyl groups could obviously increase the neuroprotection. Di-CQAs (6–9) demonstrated a high cytoprotective activity under these conditions as shown in Table 1. However, substitution of the proton at C-1 in the di-CQA by malonyl groups caused a marked increase of neuroprotection. For example, the EC50 value of MDCQA was much lower than that of 7. In addition, the effects were also affected by the location of caffeoyl and malonyl groups of di-CQA, such as 4 (with an EC50 value of 29.88 ± 0.49 μM) and MDCQA (with an EC50 value of 15.36 ± 0.52 μM). Although the structures of 1 and 3 are very similar to MDCQA, the substitutions (5-hydroxypentanoyl and succinoyl) did not make a contribution to their neuroprotective activity. Therefore, we considered that the malonyl group was essential to di-CQA to display protective effects against H2O2-induced SH-SY5Y cells death. In addition, the effect was also dependent upon the position of caffeoyl and malonyl groups on the quinic acid, which may be due to the stereo-hindrance effect. These results show that the neuroprotective activity of CQA derivatives is markedly influenced by the presence of the malonyl group of the C-1 in the di-CQA molecule which may serve as a basis for the design and synthesis of other antioxidants.

Further studies are intensively studied to clarify how MDCQA protect neurons from H2O2-induced SH-SH5Y cells injury. As known to all, overproduction of ROS may cause serious injury to the cells and lead to the augmented pathogenesis of various diseases (Guizani et al. 2011). As a major ROS product during oxidative stress, H2O2 has been implicated in the pathophysiology of various neurodegenerative conditions (Kumar and Khanum 2013). We proposed an intrinsic mechanism of MDCQA for the protection on H2O2-induced SH-SY5Y cells apoptosis in this study, which was confirmed by the cytoprotective action of MDCQA such as decreasing ROS generation, enhancing antioxidant defense, and modulating the expression of apoptosis proteins, MAPKs, and AKT in cytosol.

As the major endogenous enzymatic antioxidants, SOD and GSH-Px work together to defend against ROS through enzymatic reactions. SOD can catalyze the dismutation of intracellular superoxide anion to H2O2, which is the critical conversion for attenuating oxidative stress. Subsequently, H2O2 can be converted to H2O and O2 by GSH-Px, and the oxidized membrane components can be repaired by the combined action of these two enzymes (Jia et al. 2014). MDA, an end product of lipid peroxidation of ROS, is an important biomarker of membrane injury and can be scavenged by endogenous antioxidant enzymes (Xiao et al. 2008). Therefore, we measured the levels of such enzymes. Our data showed that pretreatment with MDCQA decreased the levels of ROS, increased the activities of GSH, and SOD; in addition, it decreased the level of MDA in SH-SY5Y cells incubated with 400 μM H2O2. This indicated that MDCQA alleviated H2O2-induced oxidative injury by enhancing the activities of the antioxidant enzymes.

Previous study has shown that the loss of MMP appears to be initially a transient event, followed by permanent collapse later during apoptotic cascade (Wlodkowic et al. 2011). In addition, MMP regulated by proteins of Bcl-2 family is considered as irreversible apoptotic pathway (Hsu et al. 2007). The balance between the expression levels of Bcl-2 and Bax is regarded to be crucial for cell survival and death, because the decrease in Bcl-2/Bax ratio leads to the release of cytochrome c from mitochondria to the cytosol and activation of intrinsic apoptotic pathway (Monika Raisova et al. 2001). Once activated, cytochrome c acts as a co-factor and interacts with Apaf-1 and procaspase-9, which in turn activates caspase-9 (Ghate et al. 2014), and caspase-9 activates caspase-3. Activated caspase-3 is responsible for the proteolytic cleavage of PARP, and the expression of p53 is also decreased due to the positive linear correlation between PARP and p53 during the apoptotic process (Wang et al. 1998). Then, the proapoptotic factor p53 could further decrease the Bcl-2/Bax ratio. In our study, MDCQA prevented H2O2-induced intracellular ROS production and ΔΨm loss. Furthermore, MDCQA could not only significantly increase the Bcl-2/Bax ratio, procaspase-9, and procaspase-3, but also reduce the expressions of Bad, cytochrome c, cleaved caspase-9/3, PARP, and p53. Altogether, the present data indicated that MDCQA provides neuroprotective effects through the protection of mitochondrial function in H2O2-stimulated SH-SY5Y cells.

The MAPKs signally pathway is involved in regulating many cellular events such as oxidative stress-induced cell injury (Yuan et al. 2014). Therefore, we investigated the effect of MDCQA on H2O2-induced phosphorylative p38, JNK, and ERK 1/2. Our results demonstrated that MDCQA significantly inhibited H2O2-induced phosphorylation of ERK1/2, JNK, and p38, highlighting the ability of MDCQA to directly modulate the MAPK signaling pathway. The serine/threonine kinase AKT has received widespread attention as an important anti-apoptotic protein through which various survival signals suppress cell apoptosis (Wang et al. 2000). As a downstream target of PI3 K, AKT is a key factor for the regulation of cell survival following cerebral ischemia. Our results show that MDCQA could increase the expression of p-AKT and inactivation of caspases. Based on these results, we suggest that the protective effect of MDCQA may be via the inhibition of the activation of MAPKs and increasing phosphorylation of Akt on Ser473.

Conclusion

In conclusion, our findings pin down the neuroprotective activity of CQAs to the presence of the malonyl group of the C-1 in the quinic acid molecule and may therefore serve as a basis for the design and synthesis of other CQAs antioxidants. On the basis of intensive studies on MDCQA, the protective effects of MDCQA may be mediated by the decrease of ROS production as well as enhancement of antioxidant defense and the regulation of the phosphorylation of MAPKs and AKT. Consequently, MDCQA might be useful for the development of promising neuroprotective agents in preventing/counteracting oxidative stress injury involved in neurodegenerative diseases, which requires further studies.