Cistanche Deserticola Polysaccharides Have A Neuroprotective Effect

for more information:Ali.ma@wecistanche.com

Yue Liu, et al

Abstract:

Ischemia stroke is a disease with high morbidity and mortality. Cistanche deserticola polysaccharides (CDP) possess a wide range of beneficial effects, including hepatoprotection and immune homeostasis. As far as we know, the protective effect of CDP (Cistanche deserticola polysaccharides) on neurons injured by oxygen-glucose deprivation/reperfusion (OGD/RP) has not been investigated. In this study, OGD/RP injured a PC12 cell model. Briefly, CDP (Cistanche deserticola polysaccharides) (0.05, 0.5, and 5 μg/ml) was administered before reperfusion. The protective effect of CDP (Cistanche deserticola polysaccharides) was then evaluated on the basis of cell viability, lactate dehydrogenase (LDH) leakage, [Ca2+] I, mitochondrial membrane potential (MMP)and cell apoptosis, and redox status after reperfusion was evaluated by assaying reactive oxygen species (ROS), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity. Based on the fact that Parkinson’s disease-associated protein DJ-1 participates in endogenous antioxidation and performs neuroprotective effects after ischemia stroke, we investigated the interaction between CDP (Cistanche deserticola polysaccharides) and DJ-1. DJ-1 expression was detected through ELISA and Western blot analysis, and the translocation of DJ-1 was evaluated through immunofluorescence. Results showed that CDP (0.05, 0.5, and 5 μg/ml) attenuated PC12 cell death, preserved MMP and calcium homeostasis; inhibited oxidative stress, and decreased cell apoptosis. Moreover, CDP (5 μg/ml) markedly stimulated DJ-1 secretion and expression. Overall, the results suggested that CDP (Cistanche deserticola polysaccharides) exerts a neuroprotective effect against OGD/RP-induced injury by inhibiting oxidative stress and regulating the DJ-1 pathway

Keywords: Cistanche deserticola, Polysaccharides, Neuroprotective, Oxygen glucose deprivation/reperfusion, Oxidative stress DJ-1

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1. Introduction

Ischemic stroke is the second leading cause of mortality and the primary cause of long-term disability globally [1]. Death due to stroke is expected to rise to 7.8 million in 2030 [2]. In China, 58 to 142 per 100,000 people die from stroke each year. Thus, effective strategies that reverse ischemia-reperfusion injury must be developed. Extensive evidence support that oxidative stress is a major factor that promotes the development and progression of cerebral infarction during reperfusion [3,4]. Therefore, discovering effective neuroprotective agents capable of reducing ROS and suppressing oxidative stress has attracted considerable interest in research. Parkinson’s disease-associated protein DJ-1 mediates neuroprotection by stimulating anti-apoptotic and antioxidative gene expression [4,5]. DJ-1 can be translocated into the mitochondria by oxidative stress and mitogen stimulation and can be secreted to the extracellular matrix under pathological conditions, such as breast cancer, melanoma, and OGD/RP injury [6]. Moreover, DJ-1 is useful in therapeutic targeting for ischemic neurodegeneration owing to its critical role in anti-oxidation [4,7]. In the past 10 years, several groups have confirmed that DJ-1 has neuroprotective effects on in vivo focal cerebral ischemia models and in vitro OGD/RP models [6–9]. Meanwhile, some drugs, including cyclosporine A and sodium phenylbutyrate, abrogate neuronal cell death via DJ-1 upregulation in ischemia stroke [9,10].

Cistanche deserticola, the integral dried plant of Cistanche deserticolaY.C.Ma and Cistanche tubulosa Wight, is produced mainly in the desert areas of north and northwest China [11]. C. deserticola is edible and has been honored as the’ Ginseng of the deserts’. In acupoint prescription, it is used as a treatment for chronic renal disease, sexual impotence, female infertility, leucorrhea, metrorrhagia, and senile constipation [12]. C. deserticola polysaccharides, a major active component isolated fromCistanche deserticola. CDP, modulate immune function and lipid balance and provide protection against aging, oxidation, and liver damage. Moreover, CDP (Cistanche deserticola polysaccharides) prevents ischemia-reperfusion injury in the heart and liver [13,14]. Recent studies reported that CDP (Cistanche deserticola polysaccharides) are nontoxic [12,15,16], and other phytochemicals extracted from C. Deserticola, including echinacoside, isoacteoside, acteoside, and salidroside, exhibitneuroprotective effects [11,12]. However, the potential use of CDP for the amelioration of ischemic stroke damage has not been reported so far.

In the present study, we hypothesis that CDP (Cistanche deserticola polysaccharides) inhibits oxidative stress for neuroprotection. The potential protective effects of CDP (Cistanche deserticola polysaccharides) in ischemia stroke damage were examined by using OGD/RP injured PC12 cells. Underlying interactions associated with DJ-1 were then explored.

2. Methods

2.1. Cell culture and OGD/RP model

PC12 cells were cultured in RPMI 1640 medium containing 10% FBS at 37 °C in a normoxic incubator containing 5% CO2. The medium was replaced every 48 h. OGD/RP model was prepared. The PC12 cells were washed and exposed to Earle’s balanced salt solution. Then, the cells were transferred to an anaerobic chamber containing 5% CO2 and 95% N2 at 37 °C for 4 h and then allowed to undergo reoxygenation. During this step, the same volume of culture medium was added to the cells. After reoxygenation, the cells were placed on a normoxic incubator for 24 h.

2.2. Drug administration

CDP (Cistanche deserticola polysaccharides) was purchased from Yuanye Biological Technology Co., Shanghai, China (C23J7Y18405, > 98% purity). Nimodipine, a first-line preventive drug for ischemia stroke, was used as a positive control compound [17–19]. Nimodipine injection was from Bayer Company. Cells were randomly divided into six groups, namely, the control, vehicle (OGD/RP), Nimo (5 μg/ml), CDP (Cistanche deserticola polysaccharides) (0.05 μg/ml), CDP (Cistanche deserticola polysaccharides) (0.5 μg/ml) and CDP (5 μg/ml). Nimodipine and CDP (Cistanche deserticola polysaccharides) were added before reperfusion. The control group was cultured in a normal medium and incubated under a normoxic condition.

2.3. Cell Viability: MTT and NRU assays

PC12 cells (6 × 103 per well) were seeded in a 96-well culture plate. The cells were treated in accordance with the requirement of each group. MTT (Solarbio, China) was added to the cells at a concentration of 5 mg/ml and incubated for 4 h at 37 °C. Formazan is the reduction product of succinate dehydrogenase in living cells [20]. DMSO was added for the dissolution of generated blue formazan. Absorbance at a wavelength of 490 nm was detected with Microplate Reader (Thermo, USA) after the cells were shaken for 10 min at 25 °C. Cell viability results were manifested as a percentage of the control group [21].

Neutral red uptake (NRU) assay was performed according to a previously published protocol [22]. When reperfusion periods were completed, neutral red (Solarbio, China) was added to the cells at a concentration of 50 μg /ml. The mixture was incubated for 3 h. Then, the cells were washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. The cells were subsequently added to a mixture containing 1% ethylic acid and 50% anhydrous ethanol. Plates were read at 540 nm absorbance after the cells were shaken for 20 min at 37 °C. The results of cell viability were manifested as a percentage of the control group.

Cistanche deserticola polysaccharides have a Neuroprotective effect

2.4. Assessment of cytotoxicity

LDH is a cytoplasmic enzyme catalyzing the oxidation of lactate to pyruvate. LDH is rapidly released into the extracellular fluid when the cell membrane is damaged. Therefore, LDH detection is usually performed for cytotoxicity evaluation. [23]. The experimental procedure followed commercial kit instructions supplied by the LDH assay kit (Jiancheng, China). The absorbance of each sample was detected at 440 nm with a microplate reader. Percent of cell death was calculated by using the following formula: Viability (%) = (OD Treatment − OD Treatment blank)/(OD Max LDH activity − OD Max LDH activity blank) × 100%.

2.5. Detection of intracellular Ca2+ concentration

The intracellular concentration of Ca2+ was measured with Fluo-3/ AM [15]. The PC12 cells were washed with PBS three times and then incubated with 2 μM Fluo-3/AM (Beyotime, China) for 30 min at 37 °C in the dark. The cells were then washed with PBS three times for the purging of extracellular dye. The fluorescence intensity of the Fluo-3/ AM was determined on a laser scanning confocal microscope (Olympus, Japan). Software package Olympus FV10-ASW 4.1 Viewer and ImageJ were used for the calculation of gray-scale values

2.6. Measurement of MMP

JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Beyotime, China) is a convenient voltage-sensitive probe generally used for staining cells and evaluating MMP [24]. The PC12 cells were washed twice with cold PBS and then incubated with a mixture containing 50% medium and 50% working liquid for 15 min at 37 °C in the dark. Then, the mixture was discarded and replaced by a fresh medium. Red and green fluorescence images were captured through laser scanning confocal microscopy. The relative proportion of red and green fluorescence signals was used for MMP measurement [25]. Software package Olympus FV10-ASW 4.1 Viewer and ImageJ were used for the calculation of gray-scale values.

2.7. Apoptosis detection assay, Hoechest33342, and flow cytometry analysis

Hoechst33342, a DNA binding dye, was used for the examination of cell apoptosis [26]. After reperfusion, the PC12 cells were washed with PBS three times and incubated with Hoechst33342 (Beyotime, China) at a concentration of 5 μg/ml for 15 min at 37 °C in the dark. Then, the cells were observed under a laser scanning confocal microscope. The apoptotic cells exhibited intense blue fluorescence and nucleus condensation. For each staining experiment, three random fields were captured and quantified. Olympus FV10-ASW 4.1 Viewer and ImageJ were used for the calculation of gray-scale values. The percentages of the apoptotic cells were calculated through the following formula: apoptotic amount/total amount × 100% [27].

2.8. Measurement of ROS generation

The fluorescent probe 2′,7′- dichlorofluorescein-diacetate (DCFH-DA; Jiancheng, China) can penetrate cell membranes and can be hydrolyzed to nonfluorescent DCFH. DCFH is oxidized by intracellular ROS to fluorescent DCF [29]. After reperfusion, the cells were washed twice with PBS and then incubated with DCFH-DA at a final concentration of 10 μM for 45 min at 37 °C in the dark. Fluorescence was observed by a fluorescence spectrophotometer with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

Cistanche deserticola polysaccharides have a Neuroprotective effect

2.9. Determination of oxidative stress indicators: CAT, GSH-Px, and T-AOC levels

The cells were scraped and resuspended in PBS. The obtained cell suspensions were sonicated 45 times on ice and centrifuged at 1000 r/min for 10 min at 4 °C. The supernatants were retained and used for detection [24]. The total protein content of the cell supernatant was measured with a BCA protein assay reagent kit (KeyGEN, China). The activities of CAT, GSH-Px, and T-AOC levels were measured according to the manufacturers’ instructions (Jiancheng, China). The absorbance of each sample was detected with a microplate reader.

2.10. Measurement of extracellular DJ-1 concentration

PC12 cells (6 × 103 per well) were seeded in 96-well culture plates. Following reperfusion, the cell supernatant was collected and analyzed by DJ-1/PARK-7 ELISA Kit (Raybio, USA) according to the manufacturer's instruction [9]. The absorbance of each sample was detected with Microplate Reader at a dual-wavelength of 450 nm or 540 nm.

2.11. Western blot analysis

Cellular proteins were extracted with ice-cold lysis buffer according to the manufacturers’ instructions (KeyGEN, China). Protein concentration was determined with a BCA protein assay reagent kit. Equal amounts of protein lysates (20 μg) from each group were resolved in 12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto a nitrocellulose membrane. The membrane was incubated with PBS containing 5% skim milk for 2 h at room temperature and then probed with anti-PARK7/DJ-1 antibodies(ab76008, Abcam; dilution 1:1000) overnight at 4 °C. Then, the membrane was washed three times with PBST and incubated with secondary antibody goat anti-rabbit IgG (SA00001-2, Proteintech Group; dilution1:5000) at room temperature for 2 h. The antibody anti-α-Tubulin(10759-1-AP, Proteintech Group; dilution 1:1000) was considered as the control. Protein bands were visualized by enhanced chemiluminescence(ECL) reaction reagents. Band densities were measured with QuantityOne Analysis software v.4.6.9.

2.12. Immunocytochemistry analysis

The PC12 cells were fixed in 4% paraformaldehyde for 15 min at room temperature after the reperfusion treatment [9]. After excessive paraformaldehyde was removed, the cells were incubated in PBS containing 0.3% Triton X-100 for 10 min at room temperature. Then, the cells were washed with PBS three times before they were incubated with 5% normal goat serum for 1 h. The cells were then incubated with rabbit mouse monoclonal anti-ATP synthase β-chain (MABS1304, EMD Millipore, dilution 1:200) for the staining of mitochondria and subsequently incubated with anti-PARK7/DJ-1 antibody (dilution 1:100) for the probing of protein DJ-1. The cells were washed and incubated with goat anti-rabbit IgG-Alexa 488 (green; A11034, Invitrogen; dilution 1:1000) and goat anti-mouse IgG-Alexa 594 (red; A11032; Invitrogen; dilution 1:1000) for 90 min. Finally, the cells were washed three times with PBS and mounted on glass slides with a mounting medium containing DAPI. Immunofluorescent images were visualized by using a Laser Scanning Confocal Microscope.

Cistanche deserticola polysaccharides have a Neuroprotective effect

2.13. Data/statistical analysis

All values were shown as mean ± SD. Significance was determined by a One-Way analysis of variance (ANOVA), followed by Dunnett's multi-comparison test. p < .05 was considered as statistical significance. Experimental data were analyzed using SPSS 17.0 statistical software.

3. Results

3.1. CDP (Cistanche deserticola polysaccharides) inhibited OGD/RP-induced cell damage

The PC12 cells exposed to OGD/RP exhibited a signifificant decrease in number. Cell shape was changed and the cell membrane was broken. In the MTT assay, the cell viabilities in the CDP (Cistanche deserticola polysaccharides) groups (0.05, 0.5 and 5 μg/ml) were 58.91% ± 5.40%, 61.13% ± 3.81% and 68.57% ± 3.24% respectively, p < .05 (Fig. 1A), and that with vehicle had 51.68% ± 3.89%. These values indicated that CDP (Cistanche deserticola polysaccharides) protected the cells against OGD/RP injury and decreased cell death. Furthermore, cell viability in the Nimo group was 77.02% ± 5.22%, which is nearly equal to that in the 5 μg/ml CDP (Cistanche deserticola polysaccharides) treated group.

In the NRU assay, the cell viabilities in the CDP (Cistanche deserticola polysaccharides) groups (0.05, 0.5, and 5 μg/ml) were 52.03% ± 4.72%, 58.49% ± 2.50% and 69.17% ± 3.91%, respectively, (Fig. 1B) and that with vehicle had 47.14% ± 2.88%. The values indicated the concentration-dependent protective effect of CDP.

In contrast to a vehicle (44.73% ± 3.30%), LDH release was significantly reduced in the CDP (Cistanche deserticola polysaccharides) groups. In CDP (Cistanche deserticola polysaccharides) groups (0.05, 0.5 and 5 μg/ml), LDH leakage rates were 32.41% ± 3.70%, 27.13 ± 2.79% and 23.13% ± 4.59% respectively, p < .01 (Fig. 1C). Moreover, LDH leakage rates in the Nimo group were 21.99% ± 4.02%, which was nearly equal to that in the 5 μg/ml CDP treated group.

3.2. CDP (Cistanche deserticola polysaccharides) attenuated OGD/RP-induced increase of [Ca2+]i

The intracellular Ca2+ levels in the PC12 cells were enhanced to4146.60 ± 195.97 after OGD/RP injury in contrast to the control group (373.62 ± 75.69). The CDP (Cistanche deserticola polysaccharides) groups (0.05, 0.5 and 5μg/ml) could dose-dependently decrease the intracellular Ca2+ level to 2921.25 ± 222.19, 2015.85 ± 230.53 and 1768.43 ± 426.12, respectively (Fig. 2A and C). Moreover, the Ca2+ level in the 5 μg/ml CDP treated group decreased to a level comparable to that in the Nimo group (1591.19 ± 213.21).

3.3. CDP (Cistanche deserticola polysaccharides) attenuated OGD/RP-induced dissipation of MMP

Compared with the control group (1.67 ± 0.89), the vehicle group (0.48 ± 0.10) exhibited more green FL fluorescence than red FL fluorescence (Fig. 2B and D). The ratios between red and green FL fluorescence were decreased to 1.11 ± 0.26 and 1.18 ± 0.16 in the 0.5 and 5 μg/ml CDP (Cistanche deserticola polysaccharides) treated groups respectively, p < .01. Green FL fluorescence was significantly decreased.

3.4. CDP (Cistanche deserticola polysaccharides) prevented OGD/RP-induced cell apoptosis

Cells exhibiting condensed chromatins or fragmented nuclei were scored as apoptotic cells [30]. Hoechst33342 assay results revealed the appearance of condensed nuclei after OGD/RP (Fig. 3A and C). A high FL fluorescence intensity was observed in the nuclei in the vehicle group. The FL fluorescence intensity of nuclei was weakened in the 5 μg/ml CDP (Cistanche deserticola polysaccharides) treated group. We analyzed apoptotic rate in the PC12 cells by fellow cytometry using FITC-Annexin V/PI double staining (Fig. 3B and D). The control group had a 4.16% ± 0.24% cell apoptotic rate, whereas the vehicle group had 22.98% ± 0.66%. In the 5 μg/ml CDP-treated group, the cell apoptosis rate was 7.86% ± 1.16%.

3.5. CDP (Cistanche deserticola polysaccharides) attenuated OGD/RP-induced intracellular ROS accumulation and preserved redox status

The effects of CDP (Cistanche deserticola polysaccharides) on oxidative stress were demonstrated on the basis of ROS generation, GSH-Px, CAT activities, and T-AOC levels. The ROS levels in the OGD/RP-injured PC12 cells increased and became significantly higher than that in the control group (p < .01). Meanwhile, the ROS levels in the CDP (Cistanche deserticola polysaccharides) groups (0.05, 0.5 and 5 μg/ml) decreased in a dose-dependent manner (p < .01; Fig. 4A). Further, CDP also significantly increased the T-AOC (p < .05; Fig. 4B), CAT (p < .05; Fig. 4C) and GSH-Px (p < .05; Fig. 4D) levels compared with vehicle group. The activities of endogenous antioxidants in CDP (Cistanche deserticola polysaccharides) groups increased.

3.6. CDP (Cistanche deserticola polysaccharides) stimulated the secretion of DJ-1 and enhanced the expression of DJ-1

DJ-1 concentration changes in the cell supernatant were detected and revealed the possible relationship between DJ-1 and CDP (Cistanche deserticola polysaccharides). DJ-1 release was observed from the OGD/RP-injured PC12 cells (Fig. 5). DJ-1 was considerably up-regulated in the 5 μg/ml CDP (Cistanche deserticola polysaccharides)-treated group (38.66 ± 8.44 pg/ml) and became significantly increase than that in the vehicle group (18.33 ± 3.80 pg/ml, p < .01) and control group (9.67 ± 3.96 pg/ml, p < .01).

To investigate the effects of CDP (Cistanche deserticola polysaccharides) on the intracellular expression of DJ-1, we measured the DJ-1 protein levels by Western blotting. DJ-1 protein expression levels were significantly increased 24 h after reperfusion. By contrast, the 5 μg/ml CDP post-treatment significantly enhanced the overexpression of DJ-1 compared with the vehicle group (p < .01, Fig. 6).

3.7. CDP (Cistanche deserticola polysaccharides) enhanced DJ-1 translocation into the mitochondria

The PC12 cells were stained with anti-PARK7/DJ-1 and anti-ATP synthase β-chain antibodies (Figs. 7 and 8). The anti-ATP synthase β- chain antibody links with mitochondrial complex I, and the binding antigen were localized in the mitochondrial inner membrane. Results revealed that DJ-1 translocated to the mitochondrial inner membrane following OGD/RP insult (Fig. 8). Meanwhile, the DJ-1 and mitochondria double staining results revealed that the CDP (Cistanche deserticola polysaccharides) facilitated DJ- 1 mitochondrial translocation and co-localization (Fig. 8). These results indicated direct interaction between DJ-1 and neuroprotective effect of CDP (Cistanche deserticola polysaccharides) (Fig. 9).

4. Discussion

In this study, we reported firsthand that CDP (Cistanche deserticola polysaccharides) effectively reducesOGD/RP-induced PC12 cell damage via the upregulation of DJ-1 protein. CDP (Cistanche deserticola polysaccharides) improved cell viability, reduced cell membrane damage, maintained intracellular Ca2+ homeostasis, prevented MMP loss, decreased cell apoptosis, suppressed oxidative stress, promoted expression of DJ-1, and enhanced DJ-1 translocation into the mitochondria. DJ-1 has been recently implicated in the regulation of neuroprotective effect and mitochondrial integrity. These results provided convincing evidence of the neuroprotective effects of CDP (Cistanche deserticola polysaccharides) against ischemia. Furthermore, the interaction between CDP and DJ-1 plays a neuroprotective role in OGD/RP-induced PC12 cell damage. The above results may develop our understanding of the beneficial effect of CDP and may provide useful information for the therapy of stroke in the future.

Ischemia stroke has been attributed to a complex series of biochemical and molecular mechanisms, including excitotoxicity, calcium overload, and oxidative stress [31]. OGD/RP is widely used as an in vitro model of ischemia-reperfusion and is used for the exploration of neurological and biochemical changes. The PC12 cell line is sensitive to OGD/RP injury [2]. It exhibits a number of properties and characteristics of sympathetic neurons [32] and thus widely used neuronal cell lines for research related to the mechanisms of neurological damage due to stroke [33,34]. Numerous studies have indicated that oxidative stress plays a vital role in stroke and brain ischemia damage [35–37]. Parkinson’s disease-associated protein DJ-1 can inhibit ischemic neurodegeneration and behavioral dysfunction by reducing ROS-mediated neuronal injury [7]. Therefore, we used the OGD/RP-injury PC12 cell model to investigate the neuroprotective effect of CDP (Cistanche deserticola polysaccharides).

The degree of injury in PC12 cells induced by OGD/RP was evaluated by MTT, NRU, and LDH release assays. Administration of CDP (Cistanche deserticola polysaccharides) (0.05, 0.5, and 5 μg/ml) improved cell survival and decreased LDH release. Beneficial effects of CDP treatment also included a decrease of intracellular calcium overload and preservation of MMP. Abnormalities of intracellular Ca2+ concentration, especially mitochondrial calcium overload, have been linked to neuronal apoptosis and death induced by ischemia stroke [38]. MMP can reflect the efficiency of the electron transport chain and has been indicated as a pathological disorder index of this system [39]. Both elevations of intracellular Ca2+ concentration and MMP loss can lead to destabilization of the neuron structure and eventually result in cell damage or cell death [21,40]. In this study, results demonstrated that CDP significantly inhibited the intracellular Ca2+ overload and increased MMP levels in OGD/RP-injury PC12 cells. Apoptosis plays a vital role in the complex pathophysiology of cerebral ischemia-reperfusion injury. Increasing data suggested that apoptosis has been implicated in mitochondrial dysfunction; the dissipation of MMP is an early event resulting in cell apoptosis [8,39]. Apoptosis is a process of energy-dependent programmed cell death to dispose of redundant cells. Many neurons in the ischemic stroke will undergo apoptosis [41]. In this study, we performed Hoechst33342 and Annexin V-FITC/PI staining to examine the effect of CDP on apoptosis [23,28]. The results showed that CDP maintains the integrity of the nucleus and decreases cell apoptosis rate. All these results indicated that the anti-apoptotic activity of CDP contributed to the beneficial effect in neuronal injury in PC12 cells.

Cells undergoing ischemia damage caused by excessive ROS accumulation, which can lead to lipid peroxidation, cell membrane damage, calcium overload, and mitochondrial dysfunction, ultimately resulting in cell apoptosis and death [42]. The consumption of endogenous antioxidants, such as SOD, CAT, and GSH-Px, also indicated the occurrence Fig. 9. Possible mechanisms for the protective effects of CDP (Cistanche deserticola polysaccharides) against OGD/RP-induced PC12 cell damage. Treatment of CDP suppresses oxidative stress, stabilizes Ca2+ concentration and MMP, enhances DJ-1 expression and localization in the mitochondria. Y. Liu et al. Biomedicine & Pharmacotherapy 99 (2018) 671–680 678of oxidative stress during ischemia-reperfusion. In this study, the OGD/ RP group showed a signifificant increase in ROS and a decrease in CAT, GSH-Px, and T-AOC compared with the 5 μg/ml CDP-treated group. However, we observed substantial decreases in rates of ROS generation and consumption of endogenous antioxidants (CAT, GSH-Px, and T-AOC) in the CDP groups. The beneficial effects in the 5 μg/ml CDP group were close to those in the Nimo group. The results suggested that CDP inhibits the generation of ROS and restores enzymatic antioxidant defense.

DJ-1 is a key redox-reactive neuroprotective protein implicated in the regulation of oxidative stress in stroke. The beneficial effects of DJ-1 over-expression include cell survival from ischemia-reperfusion and preservation of mitochondrial function and morphology, such as precipitating mitochondria permeability transition pore opening [39,43]. Previous studies have indicated that the protective effect of DJ-1 in cells exposed to OGD/RP is related to its antioxidant properties and mitochondria translocation [43–45]. DJ-1 performs efficient endogenous neuroprotection in mitigating mitochondrial defects via translocation [46]. Several studies have indicated that DJ-1 translocation may have an effect on mitochondrial movement, enhance cell-cell interaction, and promote other cell pro-survival processes.

DJ-1 affects several processes, such as cell migration and adhesion, chemotaxis, proliferation, and apoptosis, in both in vitro and in vivo models [6,9,47,48]. Using the OGD/RP injured PC12 cells stroke model, we explored a potential relationship between CDP (Cistanche deserticola polysaccharides) and DJ-1. DJ- 1 overexpression was identified by ELISA and Western blotting. Along with this function, a major finding in the experiments demonstrated that the signifificant over-expression of DJ-1 occurred when CDP (Cistanche deserticola polysaccharides) (5 μg/ ml) was administrated before reperfusion. This suggested a possible neuroprotective effect in the relationship of CDP and DJ-1. Moreover, we have observed that DJ-1 located in the mitochondria under conditions of OGD/RP and abundant expression of DJ-1 decreased cellular sensitivity to ROS and inhibited oxidative stress [45]. The results implied that CDP regulates and enhances DJ-1, which acts as a molecular link between mitochondria dysfunction and oxidative stress and provides a progression of secondary cell death inherent in stroke [5,47,49–51]. In tandem with stabilization of DJ-1 in the mitochondria, CDP (Cistanche deserticola polysaccharides) treatment increased cell survival, stabilized Ca2+ homeostasis, ameliorated MMP dissipation, and prevents mitochondrial-related apoptosis [43]. Overall, our results indicated that CDP exerts a novel neuroprotective mechanism in the treatment of ischemic stroke.

5. Conclusion

Our results indicated that CDP (Cistanche deserticola polysaccharides) protects PC12 cells against OGD/RP-induced injury through its antioxidant effects, which are partially attributed to the DJ-1 mediated pathway. These effects include a decrease in the rate of cell membrane damage, preservation of [Ca2+] I homeostasis, amelioration of MMP dissipation, inhibition of cell apoptosis, attenuation of intracellular ROS, and modulation of DJ-1 levels. The results also indicated that the interactions between CDP and DJ-1 amplify neuroprotection and maintain mitochondrial integrity. Therefore, vulnerable neurons can be protected by CDP (Cistanche deserticola polysaccharides) via the enhancement of DJ-1 expression during ischemia stroke.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant 81360649), the National Science and Technology Key Program (grant 2015BAK45B01), and the Ningxia Hui Autonomous Region Science and Technology Support Program (grant 2016BZ07).

Cistanche deserticola polysaccharides have a Neuroprotective effect

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