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. 2009 Jun 11;29(8):1211–1221. doi: 10.1007/s10571-009-9417-z

Neuroprotective and Antioxidative Effect of Cactus Polysaccharides In Vivo and In Vitro

Xianju Huang 1,2, Qin Li 1, Huige Li 3, Lianjun Guo 1,✉
PMCID: PMC11506276  PMID: 19517228

Abstract

Cactus polysaccharides (CP), some of the active components in Opuntia dillenii Haw have been reported to display neuroprotective effects in rat brain slices. In the present study, we investigated the neuroprotective properties of CP and their potential mechanisms on brain ischemia-reperfusion injury in rats, and on oxidative stress-induced damage in PC12 cells. Male Sprague–Dawley rats with ischemia following middle cerebral artery occlusion and reperfusion were investigated. CP (200 mg/kg) significantly decreased the neurological deficit score, reduced infarct volume, decreased neuronal loss in cerebral cortex, and remarkably reduced the protein synthesis of inducible nitric oxide synthase which were induced by ischemia and reperfusion. Otherwise, the protective effect of CP was confirmed in in vitro study. CP protected PC12 cells against hydrogen peroxide (H2O2) insult. Pretreatment with CP prior to H2O2 exposure significantly elevated cell viability, reduced H2O2-induced apoptosis, and decreased both intracellular and total accumulation of reactive oxygen species (ROS) production. Furthermore, CP also reversed the upregulation of Bax/Bcl-2 mRNA ratio, the downstream cascade following ROS. These results suggest that CP may be a candidate compound for the treatment of ischemia and oxidative stress-induced neurodegenerative disease.

Keywords: Cactus polysaccharides, MCAO, iNOS, Oxidative stress, PC12 cell, Apoptosis

Introduction

Opuntia dillenii Haw was introduced from Milpa region in Mexico into China in 1997. The adaptable cultivation and breed screening in China have been successful. Its tender leaf can be used as both vegetable and medicine. Since its rhizome is an excellent herb medicine, it’s being recognized and utilized by Chinese gradually.

Although Opuntia species are currently consumed for their nutritional properties (Stintzing and Carle 2005; Feugang et al. 2006), fruits and stems of many Opuntia species have been used in folk medicine for burns, wounds, edema, bronchial asthma, hypertension, indigestion, and type II diabetes (Morton 1990; Lopez 1995). Extracts of fruits and stems from Opuntia species have been reported to exhibit antioxidant (Lee et al. 2002), anti-inflammatory (Park et al. 2001), and immunomodulatory (Schepetkin et al. 2008) activities. Indeed, the main substance produced by these plants is mucilage composed primarily of water and polysaccharides, which helps prevent dehydration and freezing of their tissues. While little is known regarding the medicinal properties of polysaccharides from Opuntia, our previous studies had found that cactus polysaccharides (CP) could protect rat brain slices from H2O2-induced oxidative stress injury (Huang et al. 2008a) and oxygen/glucose deprivation (OGD)-induced damage (Huang et al. 2008b). However, the neuroprotective effect of CP in ischemic animal models and the molecular mechanisms underlying these effects are poorly understood.

Oxidative stress which occurs in situations in which there is an imbalance toward high pro-oxidative states with the accumulation of free radicals (reactive oxygen and nitrogen species) and/or low antioxidant defense. It has been implicated as a major cause of cellular injuries in a vast variety of clinical abnormalities including neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease (Finkel and Holbrook 2000; Barnham et al. 2004). It’s mediated mainly by reactive oxygen species (ROS), including free radical such as superoxide ions (O2 −) and hydroxyl radicals as well as non-free radical species such as hydrogen peroxide (H2O2) (Halliwell and Gutteridge 1999). Another important free radical is the nitric oxide (NO), a reactive nitrogen species (RNS). It is well known that NO at physiological concentrations regulates the vascular system and modulates neuronal excitability (Colasanti and Suzuki 2000). On the contrary, significant increase in NO concentration, commonly due to iNOS activity, causes detrimental effect on cell functions. Therefore, it is likely that the beneficial or harmful role of NO in the ischemia could depend on its concentration in damaged tissue and, thereby, on the NOS isoform(s) mainly activated.

Here, the present study was designed to examine the effects of CP in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion, and in cultured PC12 cells with hydrogen peroxide (H2O2) insult. Moreover, the influence of CP on iNOS expression in the brain tissues of ischemic rats and the antioxidative and anti-apoptotic properties in PC12 cells were studied to further discuss their mechanism.

Materials and Methods

Materials

Cactus polysaccharides were extracted and prepared as we previously described (Huang et al. 2008b). The content of total sugar was detected as 42.9%. Filter paper chromatography detection showed that it included glucose, galactose, arabinose, beechwood sugar, and rhamnose.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Biotech (USA). Acridine orange (AO) and nitroblue tetrazolium (NBT) were obtained from Amerisco (USA). Ethidium bromide (EB) and 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) were purchased from Sigma Chemicals Co (St Louis, Missouri, USA). Lactate dehydrogenase (LDH) assay was from Jiancheng-Bioeng Institute (China). Dulbecco’s modified Eagle’s medium (DMEM) was from Hyclone (Logan, UT). Fetal bovine serum (FBS) was from GIBCO Life Technologies. 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma (St Louis, Missouri, USA). Rabbit anti-rat iNOS polyclonal IgG was from Santa Cruz Biotechnology (Santa Cruz, USA). All chemicals were of the highest purity commercially available.

Animals and Drug Application

Adult male Sprague-Dawley rats (200–250 g) were obtained from Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). All procedures used in this study comply with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). The rats were housed in a temperature and humidity-controlled room (temperature: 22 ± 1°C, humidity: 60%) with free access to food and water. They were kept on a 12-h light/dark cycle and adapted to these conditions for at least 7 days before experiments. CP was dissolved in normal saline (NS) and administered intraperitoneally (i.p.). Rats were randomly divided into different groups, namely, sham-operated, vehicle, CP-treated groups. For the sham group, the rats were only anesthetized and their carotid arteries were separated but not occluded. The CP-treated group was administered with CP (200 mg/kg, i.p.) at 72, 48, and 24 h before MCAO and 15 min after MCAO, whereas the vehicle group was given the same amount of saline.

MCAO Surgery and Neurological Deficit Evaluation

Rats were anesthetized using 10% chloral hydrate (350 mg/kg, i.p.). The middle cerebral artery was occluded as described before (Longa et al. 1989; Li et al. 2009). Reperfusion was induced after 2 h MCAO by filament withdrawal. The rat rectal temperature was maintained at 37°C throughout the anesthetic period. After revival from anesthesia, animals were housed back at room temperature 22 ± 1°C with free access to food and water.

Neurological behavioral assessment was performed 6 and 26 h after ischemia, and scored on a 6-point scale (Longa et al. 1989): 0, no neurological deficit; 1, failure to extend left forepaw fully; 2, circling to the left; 3, inability to bear weight on the left; 4, no spontaneous walking with depressed level of consciousness; and 5, death.

Cerebral Infarct Size Measurement

Cerebral infarct size was assessed with TTC staining method (O’Donnell et al. 2004; Li et al. 2009). After 2 h MCAO and 24 h reperfusion, all animals were anesthetized and the brains quickly isolated and sectioned into consecutive 2-mm-thick coronal slices using a Vibratome (Campden Instruments, USA). Slices were immediately immersed in 2% TTC medium at 37°C for 30 min. Stained slices were washed in phosphate buffer saline (PBS) for 5 min and fixed in buffered formaldehyde solution for 24 h. After the end of staining and fixation, color image of these slices was captured using a video camera (Olympus, Japan). All brain slices of each experimental group were analyzed for the infarct size using the Image-Pro plus 5.0 analysis software. Percentage infarct size was calculated as described (Swanson et al. 1990; Li et al. 2009): [(V C − V L)/V C] × 100%, V C is the volume of control hemisphere and V L the volume of non-infarcted tissue in the lesioned hemisphere.

Morphological Observation

Rats were anesthetized with chloral hydrate and then perfused transcardially with normal saline followed by 4% paraformaldehyde. All brains were then fixed in the same fixative at 4°C, dehydrated and then embedded in paraffin blocks. Coronal sections of 5 μm were stained with hematoxylin-eosin (H & E).

Immunohistochemistry

Tissue sections prepared as earlier were deparaffinized and wet through graded alcohol. Endogenous peroxidase activity was blocked by incubation in 10% hydrogen peroxide for 10 min. After rinsed with PBS three times, the sections were blocked with 1:10 normal goat serum to minimize nonspecific background staining. After rabbit anti-rat iNOS polyclonal antibody (Santa Cruz, USA) (dilution 1:100) was applied to the samples and incubated for 24 h at 4°C, the sections were incubated with biotinylated goat anti-rabbit IgG (1:100) for 1 h at 37°C, followed by the steps according to SABC (HRP) kit protocol. The sections were subsequently incubated with diaminobenzidine (DAB) 0.5 g/l and observed under light microscope (Li et al. 2009).

Western-blot Analysis of iNOs

For the immunoblot analysis, all procedures were done keeping the brain on ice. After the rats were decapitated, the infarct side of cortex (1–5 mm posterior to the bregma and 1–5 mm beside the sagittal suture) was harvested for assay of protein expression of iNOS. The brain tissue was homogenized and centrifuged as described earlier (Li et al. 2009). The supernatants were then collected as total proteins, which were electrophoresed through sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) and electrically transferred to a nitrocellulose membrane. The membrane was then incubated at 4°C overnight in tris-(hydroxymethyl)-aminomethane buffered saline (TBS) containing 5% milk and the primary rabbit polyclonal antibodies against iNOS of rat (1:500 dilution, Santa Cruz, USA). After washing with TBST, the membranes were incubated with the secondary antibody (goat anti-rabbit IgG) at room temperature for 1 h. The protein band intensities were quantified using Scion Image (Scion, Maryland, USA), and the amount was normalized with β-actin values in the same lane.

Cell Culture and Drug Treatment

The rat pheochromocytoma cell line PC12 was originally obtained from Chinese Type Culture Collection. All cells were cultured in poly-l-lysine coated culture dishes. The cells were maintained in Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a water-saturated atmosphere of 5% CO2 at 37°C. The medium was changed every other day and cells were subcultured about once a week. All experiments were carried out 12 h after cells were seeded at an appropriate density according to each experimental scale.

The PC12 cells were preincubated with indicated concentration of CP or edaravone before the hydrogen peroxide was added to the medium. In a pilot investigation, cells were treated with H2O2 at concentrations ranging from 100 to 500 μM for various intervals and then examined for cell viability. Concentration of 300 μM H2O2 as control was used in an extensive study of the markers of cell death after 12 h exposure. CP (0.1, 0.25, 0.5 mg/l) or edaravone (60 μM) was added 30 min before H2O2 was added.

Analysis of Cell Viability

Cell survival was observed with phase-contrast microscope (Suzhou Matsushita Communication Industrial Co., Ltd. Suzhou, China). At the same time, cell viability was evaluated by the reduction of MTT and LDH release. Briefly, PC12 cells (1 × 105 cells/ml) were treated with 0.3 mM H2O2 in the presence of CP for 12 h at 37°C. After 3 h incubation with MTT (0.5 mg/ml), cells were lysed in dimethyl sulfoxide (DMSO) and the amount of MTT formazan was qualified by determining the absorbance at 570 nm using a microplate reader (TECAN A-5082, megllan, Austria). Cell viability was expressed as a percent of the control culture value.

As for LDH detection, PC12 cells were washed with ice-cold PBS, harvested by centrifugation at 1000×g for 5 min, pooled in 0.5 ml of 0.1 M phosphate buffer (pH 7.4) and homogenized. The homogenate was centrifuged at 3000×g for 20 min at 4°C, and the supernatant was used for the activity assay according to the manufacturer instructions. The cell pellet and the cells remaining on the multiwell were lysed in 0.5 ml of lysis buffer (0.5% Triton X-100 in 0.1 M potassium phosphate buffer, pH 7.0). LDH release (% of total) was calculated as the percentage of LDH in the medium versus total LDH activity in the cells (Suuronen et al. 2000; Benedi et al. 2004). The absorbance was measured at 440 nm using a microplate reader as described earlier.

Measurement of Apoptosis by AO/EB Double Staining

Evaluation of apoptosis was also done by examining the differential uptake of two fluorescent DNA-binding dyes, AO and EB. AO is taken up by all cells and stains the nuclei bright green, whereas EB is taken up only by cells that has lost membrane integrity and stains the nuclei bright orange (Hiroia, et al. 2005). Four types of cells can be distinguished according to the fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei. (1) Normal cells (VN) have uniform bright green nuclei with organized structure. (2) Early apoptotic cells (VA) (which still have intact membranes but have started to undergo DNA cleavage) have green nuclei, but perinuclear chromatin condensation is visible as bright green patches or fragments. (3) Late apoptotic cells (NVA) have orange to red nuclei with condensed or fragmented chromatin. (4) Necrotic cells (NVN) have a uniformly orange to red nuclei with organized structure. 1 μl of AO (1 mg/ml) and EB (1 mg/ml) were added to 1 ml of cell suspension and incubated for 30 min at 37°C in a well of 6-well plates. After the cells were washed with PBS, AO/EB fluorescence was examined with fluorescence microscope fitted with a camera (Olympus made in Japan.) at 400× magnification. A minimum of 300 cells was counted in every sample. Apoptotic rate was calculated as below: apoptotic rate = (VA + NVA)/(VN + VA + NVA + NVN) × 100%.

Real-time RT-PCR of Apoptosis–Related Gene Expression

Briefly, Total RNA was isolated from PC12 using Trizol Reagent according to the manufacture’s instructions. One microgram of RNA was reverse transcribed to cDNA with the use of ReverTra Ace™ First Strand cDNA Synthesis Kit. The total volume of reverse transcription reaction was 20 and 2 μl cDNA was used for each amplification. cDNA was amplified by quantitative real-time PCR (ABI Prism 7700 Sequence Detector, Applied Foster City, CA, USA) using SYBR Green Realtime PCR Master Mix Reagent (Toyoba, Japan). Data from the reaction were collected and analyzed by the complementary computer software. Relative quantization of gene expression was calculated using 2−△△Ct data analysis method (Kenneth and Thomas 2001) and normalized to β-actin in each sample.

Primers used in this study were as follows:

  • Bcl-2 Forward: 5′-TGAACCGGCATCTGCACAC-3′;

  • Reverse: 5′-CGTCTTCAGAGACAGCCAGGAG-3′;

  • Bax Forward: 5′-AGACACCTGAGCTGACCTTGGAG-3′;

  • Reverse: 5′-GTTGAAGTTGCCATCAGCAAACA-3′;

  • β-actin Forward: 5′-GGAGATTACTGCCCTGGCTCCTA-3′;

  • Reverse: 5′-GACTCATCGTACTCCTGCTTGCTG-3′

β-actin was chosen as housekeeping gene.

Measurement of Intracellular ROS by DCFH-DA Staining and Total ROS by NBT Method

Determination of intracellular oxidant production in PC12 was based on the oxidation of DCFH-DA by intracellular ROS, resulting in the formation of the fluorescent compound 2′,7′-dichlorodihydrofluorescin (DCF) (Wang and Joseph 1999). ROS prober dye 2′,7′-DCFH-DA (10 μM) was added and incubated for 1 h at 37°C. After the cells were washed with PBS, DCF fluorescence was examined with fluorescence microscope (Olympus, made in Japan.) fitted with a camera. The fluorescence intensity (relative fluorescence units) was measured at 485 nm excitation and 530 nm emission in a Fluorescence Spectrometer (Perkin Eimer precisely LS55, made in UK). The results were normalized according to the intensity of the cells.

NBT reduction can be facilitated by superoxide or can be catalyzed by reductase enzymes in an oxygen-independent manner. A quantitative NBT test was used to measure both intra- and extracellular ROS generation (Rook et al. 1985; Banfi et al. 2001). The PC12 cells (1 × 105 cells/well) were plated on 96-well plates and incubated in PBS solution containing 0.5 g/l NBT for 2.5 h. The cells were washed with methanol to remove the non-reduced NBT. The reduced formazan was then dissolved in 80 μl of 2 M potassium hydroxide (KOH) and 120 μl of dimethylsulfoxide reaction product. The absorption was measured at 630 nm for total generation of ROS in cells.

Statistical Analysis

Data are presented as mean ± SD. One-way ANOVA followed by Student’s t-test, Fisher’s PLSD test, Dunnett’s test, or the Mann–Whitney U-test was used for the statistical analysis by employing SPSS. The statistical significance of P < 0.05 was indicated in the figure legends.

Results

Effects of Cactus Polysaccharides on Cerebral Infarct Size and Neurological Deficit Score In Vivo

Representative consecutive 2-mm-thick coronal slices stained with 2% TTC from one sample of sham, vehicle and CP-treated groups, respectively were presented in Fig. 1A. CP (200 mg/kg, i.p.) treatment resulted in a significant decrease in cerebral infarct size after 2 h MCAO and 24 h reperfusion (Fig. 1B) (P < 0.01). Neurological deficit score was measured at 6 and 26 h after MCAO, and inhibited at both time points by CP treatment (Fig. 1C) (P < 0.01).

Fig. 1.

Fig. 1

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Effects of cactus polysaccharides (CP) on cerebral infarct size and neurological score after 2 h MCAO and 24 h reperfusion (n = 10). A Representative coronal brain sections stained with 2% TTC from sham, vehicle, CP-treated groups, respectively. B Quantitative analysis of cerebral infarct size from vehicle, CP-treated groups (# P < 0.01 vs. vehicle group). C Neurological deficit score was measured at 6 and 26 h after MCAO, respectively. (# P < 0.01 vs. vehicle group). Sham group includes rats without MCAO, vehicle group was NS-treated rats with MCAO, and the CP-treated group was administrated with CP (200 mg/kg i.p.) before and after MCAO, respectively

Effects of Cactus Polysaccharides on Morphological Changes of Rat Cortex In Vivo

In the frontal cortex marked morphological changes were visualized in ischemic region of vehicle group: neuronal cell loss, nuclei shrinkage, and dark staining of neurons. CP treatment (200 mg/kg i.p.) markedly attenuated these pathological changes (Fig. 2).

Fig. 2.

Fig. 2

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Effects of cactus polysaccharides (CP) on morphologic changes in ischemic regions of rat cortex induced by 2 h MCAO and 24 h reperfusion. The different treatments of animals are shown in A the cortex tissues from the rats without MCAO (sham group); B the tissues from NS-treated rats with MCAO (vehicle group); C the tissues from CP-treated rats with MCAO. CP treatment (200 mg/kg i.p.) markedly attenuated these pathological and morphological changes as compared with vehicle group

Effects of Cactus Polysaccharides on Expression of iNOS in Cortex

The iNOS protein expression was examined by Western blot and immunohistochemistry. The photomicrographs of immunohistochemical localization of iNOS in ischemic brain tissues were illustrated in Fig. 3. The rats with MCAO and reperfusion (vehicle group) exhibited a remarkable increase in iNOS reactivity. However, CP treatment at 200 mg/kg attenuated these changes (Fig. 3A), indicating that the CP-treated group altered iNOS protein expression caused by 2 h MCAO and 24 h reperfusion.

Fig. 3.

Fig. 3

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Effects of cactus polysaccharides (CP) on the expression of iNOS in ischemic regions of rat cortex after 2 h MCAO and 24 h reperfusion. A Immunohistochemistry graphs for iNOS: a is sham group for rats without MCAO; b is vehicle group treated with NS in rats with MCAO; c is CP-treated rats with MCAO (CP 200 mg/kg i.p.). Arrows represent iNOS positive cells, respectively (X 400). B Western analysis showing protein bands of rat cortex from each group; the corresponding β-actin bands as controls shown in the same blot (upper lane). C After densitometric quantification, the data represent mean ± SEM from three independent experiments (* P < 0.01 vs. sham; # P < 0.05 vs. vehicle). Each group designated as sham, vehicle, and CP-treated, was described as in Fig 3

In western blot, compared with sham group, there was a marked increase in iNOS expression in ischemic brain tissues of vehicle group (P < 0.01) (Fig. 3C). CP treatment (200 mg/kg i.p.) significantly decreased iNOS expression in cortex of rats with MCAO and reperfusion (P < 0.05) (Fig. 3C).

Effects of Cactus Polysaccharides on Viability of H2O2-treated PC12 Cells

As shown in Fig. 4A, PC12 cells exhibited a marked decrease in cell number with most cells darkened or shrinked, and some of the treated cells were even lyzed to yield debris after 12 h exposure to 0.3 mM H2O2. Meanwhile, cell survival rate as determined by MTT method was significantly decreased (Fig. 4B) as well as LDH release (% of total) increased (Fig. 4C), suggesting that PC12 cells were very sensitive to H2O2. However, CP (0.1, 0.25, 0.5 mg/l) could attenuate H2O2-induced cell toxicity in a dose-dependent manner, which was similar to the effect of edaravone (60 μM).

Fig. 4.

Fig. 4

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Effects of cactus polysaccharides (CP) on morphological characteristics (A) and cell viability (B and C) of H2O2-treated PC12 cells. (a) is control group for PC 12 cells without treatment; (b) is injury group for PC12 cells exposed to 0.3 mM H2O2 for 12 h; (c) is CP group for PC12 cells treated with CP at 0.5 mg/l for 30 min before exposure to 0.3 mM H2O2 for 12 h; (d) is edaravone group for PC12 cells treated with edaravone at 60 μM for 30 min before exposure to 0.3 mM H2O2 for 12 h. Cell survival rate was assayed by MTT method. The data was expressed as percent of control value. LDH release (% of total) was calculated as the percentage of LDH in the medium versus total LDH activity in the cells. The data represent mean ± SEM from eight independent experiments (* P < 0.01 vs. control; # P < 0.01 vs. injury)

Effects of Cactus Polysaccharides on H2O2-Induced Apoptotic PC12 Cells

As shown in Fig. 5, AO/EB double staining divided the cells into four categories as normal, early apoptosis, late apoptosis, and necrosis. A significant increase in the apoptosis rate (from 7.4 ± 2.7 to 37.4 ± 10.1%) was seen after PC12 cells were treated with 0.3 mM H2O2 for 12 h. When they were pretreated with CP (0.5 mg/l) or edaravone (60 μM), the percentage of apoptotic cells decreased to 23.8 ± 3.6 and 20.3 ± 4.2%, respectively (compared with injury, P < 0.01), which indicated a significant protection against the toxicity caused by H2O2.

Fig. 5.

Fig. 5

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Effects of cactus polysaccharides (CP) on H2O2-induced apoptotic PC12 cells. A AO/EB double staining of PC12 cells (400× magnification). B Apoptotic rate of the cells. A minimum of 300 cells was counted in every sample. Values represent mean ± SME (n = 5). Apoptotic rate was calculated as below: apoptotic rate = (VA + NVA)/(VN + VA + NVA + NVN) × 100%. VN normal cells, VA early apoptotic cells, NVA late apoptotic cells, NVN necrotic cells (* P < 0.01 vs. control; # P < 0.01 vs. injury)

Effects of Cactus Polysaccharides on mRNA Changes Associated with Apoptosis

The mRNA levels of Bcl-2 and Bax were expressed as a ratio of control values, respectively by real-time RT-PCR analysis. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease of Bcl-2 mRNA and increase of Bax mRNA. As shown in Fig. 6, There was an approximate 3.6-fold increase in the ratio of Bax/Bcl-2 expression in H2O2 treatment compared with the control. CP treatment could down-regulate the ratio of Bax/Bcl-2 mRNA in a dose-dependent manner. Since Bax is a kind of pro-apoptosis related gene but Bcl-2 anti-apoptosis related (Spanos et al. 2002), these results indicated that the anti-apoptotic effect of CP may be mediated by mechanism(s) involving Bax/Bcl-2 transcription in injured PC12 cells (Aravind et al. 1999; Spanos et al. 2002).

Fig. 6.

Fig. 6

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Effects of cactus polysaccharides (CP) on Bax/Bcl-2 mRNA changes. The mRNA levels of Bcl-2 and Bax were expressed as a percentage of control values, respectively by real-time RT-PCR analysis and the ratio of Bax/Bcl-2 mRNA in each group were then calculated. Data are expressed as mean ± SEM (n = 5). (* P < 0.05, **P < 0.01 vs. injury)

Effects of Cactus Polysaccharides on Intracellular and Total ROS in H2O2-Induced PC12 Cells

As shown in Fig. 7A–C, the level of intracellular and total ROS was up-regulated dramatically in H2O2-induced PC12 cells (Compared with control, P < 0.01). Pre-incubated with CP (0.5 mg/l) or edaravone (60 μM) for 30 min before H2O2 treatment could decrease the fluorescence intensity of the cells and inhibit A630 value (Compared with injury, P < 0.01). These results suggested that CP could decrease both intracellular and total ROS in H2O2-induced PC12 cells.

Fig. 7.

Fig. 7

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Effects of cactus polysaccharides (CP) on intra- and total ROS in H2O2-induced PC12 cells. (A) Measurement of intracellular ROS by DCFH-DA staining (400× magnification); B DCFH-DA florescence intensity of the cells; C Measurement of total ROS by NBT method. Data are expressed as mean ± SD (n = 5). (* P < 0.01 vs. control; # P < 0.01 vs. injury)

Discussion

Recent studies have showed that a number of plant products including polyphenols, flavonoids, and terpenes and various plant extracts exerted an antioxidant action (Benedi et al. 2004). One of the most promising groups of antioxidative compounds is thought to be polysaccharides such as those extracted from plants, animals, and fungi (Hou et al. 2002; Qin et al. 2002; Qian et al. 2008). Our previous study revealed that cactus polysaccharides (CP) have the ability to protect brain tissues from oxidative damage and improve the antioxidant defense (Huang et al. 2008a, b). The role of oxidative stress in the neuroprotective effects of CP has been further determined in current study. Since PC12 is a rat pheochromocytoma clonal cell line which has become a very suitable model to study neuronal function and differentiation and to explore neuroprotective drugs (Vaudry et al. 2002; Ravni et al. 2006), the current study demonstrates that CP possess a significant neuroprotective effect in vivo and in vitro. Moreover, our study showed that several mechanisms, separately or in association, may be involved in the neuroprotective effects of CP. These studies are an extension of previous work from our group studying the effects of these polysaccharides in various models of neural damage.

Oxidative stress plays a major role in cell death and neurodegeneration (Boldogh and Kruzel 2008; Moon et al. 2008). ROS, which were predominantly produced in the mitochondria, led to the free radical attack of membrane phospholipids and loss of mitochondrial membrane potential, which caused the intermembrane protein to be released out of the mitochondria and ultimately led to DNA breakage, nuclear chromatin condensation, and cell apoptosis (Bras et al. 2005). Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a ROS scavenger which was used as positive control in this study, has protective effects against cobalt chloride-induced apoptosis in PC12 cells (Chen et al. 2009). It is also expected to play an integral role in the treatment of many oxidative stress-related diseases (Watanabe et al. 2008; Rajesh et al. 2003). The fluorescent probes used in this study suggested that edaravone prevented the generation of ROS and the apoptosis of PC 12 cells caused by H2O2. Even though the neuroprotective effect of CP was weaker than edaravone, they have a similar mode of action. CP could reduce H2O2-induced elevation of apoptotic-like cells and Bax/Bcl-2 mRNA Ratio, the downstream of cascade following ROS (Bras et al. 2005).

Animal models of focal cerebral ischemia, for which MCAO and reperfusion are usually used, reproduced the pattern of ischemia brain damage observed in many human ischemic stroke patients (Ginsberg and Busto, 1989). It was reported that the in vivo effects of intra-peritoneal treatment with polysaccharides have been shown in previous studies (Shang et al. 2003; Schepetkin et al. 2008). Moreover, the brain protective effect of intra-peritoneal treatment with ganoderma lucidum polysaccharides on AD has also been reported by some investigators (Guo et al. 2008). So it is possible that these big molecules can be transported across cell membrane (Rodrigues et al. 2007) and distributed into brain (Lu et al. 2008). The pharmacokinetics of CP will be studied in our future study. Edaravone is an established oxygen free radical scavenger and used in our in vitro oxidative stress study as a control to compare the pharmacodynamic action of Edaravone with CP. Our findings suggest that the ability of CP (0.5 mg/l) on scavenging oxygen free radical was similar to that of Edaravone (60 μM). Although edaravone (on the market since 2001) seems to be well-tolerated in patients, various adverse reactions were observed (Watanabe et al. 2008). In contrast as naturally occurring polysaccharides from plant origins, CP might be associated with fewer side effects.

The cascade of events leading to neuronal injury and death in ischemia includes the release of cytokines and free radicals, platelet activation, and apoptosis (Kuroda and Siesjo 1997). The brain is especially vulnerable to free radical-induced damage because of its high oxygen consumption, abundant lipid content, and relative paucity of antioxidant enzymes (Olanow 1992). Reperfusion of ischemic area can exacerbate ischemic brain damage through the generation of ROS and by excessive production of NO through the induction of iNOS (Iadecola et al. 1996). NO neurotoxicity is likely mediated by its free radical character, which makes NO react with certain proteins containing heme-iron prosthetic groups, iron-sulfur clusters, or reactive thiols. In addition to directly reacting with protein prosthetic groups, NO also reacts readily with superoxide (O2 −) to produce peroxynitrite (ONOO−), which may mediate much of the NO neurotoxicity (Christopherson and Bredt 1997). The present study showed that iNOS protein expression in the cortex was increased after MCAO and reperfusion, and treatment of CP significantly reduced iNOS expression. It’s obvious that the inhibition of iNOS activation may mediate CP’s neuroprotective effects.

Our findings are in agreement with previous studies in which treatment with CP was shown to protect rat brain slices from H2O2-induced oxidative stress injury (Huang et al. 2008a) and oxygen/glucose deprivation (OGD)-induced damage (Huang et al. 2008b). These findings, taken together, support that neuroprotection mediated by CP is partly due to inhibition of the oxidative stress and reducing neuronal apoptosis. Our findings suggest that this extract may be a candidate chemical for the treatment of stroke and oxidative stress-induced neurodegenerative disease. Clarification of the effect of CP on PC12 cells involved in apoptosis may provide a new insight into the mechanism of neuroprotection. Considering the activities already identified and the traditional use of this medicinal species, data presented in this report may be valuable to the development of this plant-based drug.

Acknowledgments

This work was supported by the national Foundation of Nature and Science of China (No. 30171082 and No. 30772559).

Footnotes

X.-J. Huang and Q. Li contribute equally to the paper.

References

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