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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1938-1942, 15 July, 2012 Available online at http://www.academicjournals.org/AJPP DOI: 10.5897/AJPP12.329 ISSN 1996-0816 ©2012 <strong>Academic</strong> <strong>Journals</strong> Full Length Research Paper Effect of salidroside on Bcl-2/Bax protein levels in rats with hemisection-induced spinal cord injury Ming Wang 1 , Ning Zhang 2 , Rili Ji 3 and Jing Zhao 4 * 1 Department of Urology, The second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310000, Zhejiang Province, P. R. China. 2 Department of Orthopedics, The second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310000, Zhejiang Province, P. R. China. 3 TCM pharmacy, Zhaohui Community Health Service Center, Hangzhou 310000, Zhejiang Province, P. R. China. 4 Pharmaceutical Institute, Zhejiang Chinese Medical University, Hangzhou 310000, Zhejiang Province, P. R. China. Accepted 18 June, 2012 The effect of salidroside on Bcl-2/Bax protein levels in rats with hemisection-induced spinal cord injury (SCI) was studied. The Sprague Dawley (SD) rats were randomly divided into six groups: Sham operation group, SCI model group, methylprednisolone sodium succinate (MPSS) treatment group, salidroside-low dosage treatment group, salidroside-moderate dosage treatment group, and salidroside-high dosage treatment group. The SD rats were hemisected at the spinal cord, at the T8 vertebra to establish SCI model. 24 h after operation, different dosage of salidroside increased and reduced the protein levels. Noticeably, salidroside at the 100 mg/kg dosage exhibited similar effects as MPSS, which has been frequently used for clinical acute SCI. These results suggested that salidroside can significantly inhibit apoptosis in acute SCI. Key words: Salidroside, spinal cord injury (SCI), Bcl-2/Bax, apoptosis. INTRODUCTION Spinal cord injury (SCI) is a leading cause of permanent disability, resulting in partial or complete loss of motor and sensory function below the lesion site (Wells et al., 2003). Potentially toxic substances are activated and released in injured spinal cords, including free radicals, inflammatory cytokines, phospholipases and lipid peroxidases, which lead to oxidative stress damage, thereby resulting in neuronal necrosis or apoptosis and progressive secondary nerve tissue destruction (Bao et al., 2006). There are also numerous agents for the treatment of the SCI, such as anti-inflammatory, antioxidants, antipoptosis agents and myelin-associated growth inhibitors (Wong et al., 2011; Mallei et al., 2005; Yang and Piao, 2002; Festoff et al., 2006; Tian et al., 2007; Xie et al., 2012). Methylprednisolone sodium succinate (MPSS) is the only Food and Drug *Corresponding author. E-mail: zhaoj31@126.com. Administration (FDA) approved and clinically used agent for the treatment of acute SCI (Bracken et al., 1998; Ding et al., 2012). Nevertheless, high-dose of MPSS applications in acute SCI have high risks of serious side effects (Qian et al., 2005). Obviously, some novel and safe agents for the treatment of the SCI are urgently needed. Salidroside (p-hydroxyphenethyl-b-D-glucoside, C14H20O7, structure is shown in Figure 1) is one of the major active constituents in Rhodiola Crenulata. It has been reported to possess various pharmacological properties including resisting anti-inflammation, antioxidative, anti-apoptotic, anti-fatigue, neuroprotective, hepatoprotective and cardioprotective effects (Diaz et al., 2001; Ma et al., 2009; Nan et al., 2003; Wang et al., 2004, 2009; Zhang et al., 2007). Previous studies have shown that salidroside plays important roles in apoptosis related diseases. Therefore, we proposed that salidroside might have some effects on hemisection-induced SCI in rats.
HO CH 2OH O OH OH OCH 2CH 2 OH Figure 1. The chemical structure of salidroside. In our present work, we established rat models of SCI by hemisecting the spinal cord at the T8 vertebra and attempted to study the effects of salidroside on Bcl-2/Bax protein levels in rats with hemisection-induced SCI. The Bcl-2/Bax protein levels were evaluated by immunohistochemistry methods. MATERIALS AND METHODS Salidroside was purchased from China pharmaceutical and biological products inspection (Lot: 110818-201005). MPSS was obtained from Pharmacia and Upjohn Company (Belgium). Animals A total of 54 healthy, female, Sprague Dawley rats, aged 2 months, weighing 180 to 220 g, were purchased from the Laboratory Animal Centre of Zhejiang University, China. Animal care and experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Zhejiang University. Establishment of SCI models The SD rats were anesthetized with chloral hydrate, and placed in a prone position on a heating pad to maintain a constant body temperature. A longitudinal incision was made at the midline of the back and the paravertebral muscles were exposed. These muscles were dissected and thoracic level 7 to 11 (T7 to 11) vertebrae were exposed. A laminectomy at T7 to 11 was performed. Acute SCI was induced by hemisection at the T8 vertebra to a depth of 0.5 cm. The wound was sutured layer-by-layer (Kim et al., 2009). Rats in the Sham operation group experienced the same procedures, while without the hemisection was at the T8 vertebra. Following spinal cord hemisection, the rat tails swung spastically, and the affected hind limbs exhibited flaccid paralysis after several spastic seizures. Rats were sacrificed 24 h after administration of salidroside or 0.9% normal saline (NS). Drug treatment and sample preparation The sham operation group underwent laminectomy to expose the spinal cord without hemisection, and received 0.9% NS (2 ml/kg). The SCI model group underwent laminectomy followed by SCI, and received 0.9% NS (2 ml/kg). The MPSS treatment group, positive control group underwent laminectomy followed by SCI, and was administered 100 mg/kg single dose of MPSS (2 ml/kg, i.p.) 5 min after hemisection. The 25, 50, and 100 mg/kg salidroside groups Wang et al. 1939 underwent laminectomy followed by SCI, and were given a single dose of 25, 50 or 100 mg/kg of salidroside (dissolved in 0.9% NS, 2 ml/kg, i.p.) 5 min after hemisection. 24 h after administration, the rats were anesthetized with chloral hydrate (0.3 g/kg) transcardially perfused with 150 ml of 0.9% NS and 200 ml of 4% paraformaldehyde in 0.1 M PBS. Approximately 2 cm of spinal cord segments between the T7 and T11 levels were obtained and cryopreserved at -70°C for measurements of Bcl-2/Bax protein levels. Immunohistochemistry (ICH) The spinal cord segments were cut on a freezing microtome into six adjacent series of 4 μm-thick coronal sections. The sections were dehydrated through an alcohol series. Prior to immunohistochemical processing, sections were rinsed in 2% PBS-Triton X-100 and mounted onto gelatine-coated slides. Immunohistochemistry was performed on slide-mounted sections utilizing the following antibodies: Bax or Bcl-2 (dilution 1:100). The sections were incubated overnight at room temperature with the primary antibody diluted in PBS-bovine serum albumin (BSA). After rinsing, sections were incubated for 1 h at room temperature in biotinylated goat antimouse serum (1:500), sections were incubated for 1 h in avidin– biotin–horseradish peroxidase complex (1:200). Following rinses, sections were placed for 30 min in chromagen solution consisting of 0.05% diaminobenzidine and 0.01% H2O2. The reaction was monitored visually and stopped by rinses of 0.1 M PBS. In order to minimize variability, sections from all animals were stained simultaneously. Cell counts were performed blindly in all sections using a Nikon Eclipse E800 microscope. Counts were made in six randomly selected optical fields under 400× magnification by individuals who were blinded to diagnosis. Bcl-2 or Bax immunoreactivity were assessed semi-quantitatively using Image Pro Plus software Version 4.5.129 (Media Cybernetics). The percentage area covered by immunoreactivity was measured and the mean value taken. Statistical analysis Data were expressed as Mean ± SD, and analyzed by one-way analysis of variance followed by Least Significance Difference multiple comparison or Dunnett’s multiple comparison tests using SPSS 16.0 software. Multiple comparison tests were used when appropriate. A P-value of 0.05 was considered statistically significant. RESULTS Quantitative analysis of experimental animals Of the 72 selected adult SD rats, 60 were used to establish the model of spinal cord hemisection and were assigned to 5 groups (n=12), which were intraperitoneally injected, respectively, with 25, 50 and 100 mg/kg of salidroside, 100 mg/kg of MPSS, and 2 ml/kg of NS. The remaining 12 rats served as the Sham operation group. A total of 60 rats were finally included in the final analysis. Bcl-2 expression following SCI As shown in Figure 2 and Table 1, the protein levels of
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African Journal of Pharmacy and Pha
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Editors Sharmilah Pamela Seetulsing
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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Minimum inhibitory concentration (M
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UPCOMING CONFERENCES 6th European C
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