Bisbenzimide was obtained from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Thermo Fischer Scientific (Waltham, MA, USA).

Rheum officinale was collected between August and September of 2012 from a local site in Guangzhou, China. The identity of the plant material was confirmed by an experienced taxonomist. The rhizomes of Rheum officinale were thoroughly washed with tap water, shade dried and cut into small sections. Ethanol (95%) was used for hot extraction, which was conducted over 3 h using a soxhlet extraction apparatus (Jianhu Shendi Glass Instruments Co., Ltd., Yancheng, China). The extract was then concentrated under reduced pressure in a rotary evaporator (Hangzhou Greatcool Refrigeration Equipment Co., Ltd., Hangzhou, China) at 40°C and maintained in a refrigerator at 4°C prior to use. Emodin and aloemodin were obtained from Sigma-Aldrich.

Female Sprague-Dawley rats were obtained from the Experimental Animal Centre of Sichuan University (Chengdu, China). Animals were treated in accordance with the Guide for Animal Care and Use of Laboratory Animals (National Institute of Health, 1996). All procedures were approved by the Ethics Committee of the General Hospital of Chengdu Military Region (Chengdu, China).

Primary neuronal cultures were obtained from the cortex of rat embryos aged 20 days. Pregnant rats were sacrificed by decapitation and the embryos were removed aseptically. Cortices were removed from the embryos using a dissection microscope (Ningbo Zhanjing Optical Instruments Co., Ltd., Ningbo, China). Tissues were ground and trypsinized using 0.21% trypsin-EDTA in 0.2 M PBS for 10 min at 37°C. Following centrifugation, tissues were suspended in modified Eagle’s medium (MEM; Hangzhou Sijiqing Biological Products Co., Ltd., Hangzhou, China) containing 10% FBS and triturated with a Pasteur pipette (Runlab Labware Manufacturing Co., Ltd., Taizhou, China). The resulting single-cell suspension was collected and the cell density of the suspension was measured using a hemocytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were cultured onto poly-D-lysine coated 96-well plates (1×10⁶ cells/well). Cell cultures were maintained at 37°C in a CO₂ incubator. The resultant culture comprised 95% neuronal cells, 3.5% astrocytes, 0.5% oligodendrocytes, 0.6% microglia and 0.4% ependymal cells.

Following culture for seven days, cells were treated with 20 μg/ml rhubarb extract, 10 μM emodin and 10 μM aloemodin in MEM and 10% FBS. The primary cultures were then subjected to irradiation in a ¹³⁷Cs irradiator (J.L. Shepherd and Associates, San Fernando, CA, USA) with a 2 Gy γ-ray dose. Following irradiation, cultures were maintained for 24 h at 37°C in a 7.5% CO₂ incubator prior to analysis.

ROS generation was measured using a 2′,7′-dichlorofluorescein (DCFH-DA) probe as described previously (18). DCFH-DA enters cells where it is cleaved by cellular esterases and becomes impermeable. The probe becomes fluorescent on oxidization by ROS. Cell cultures were washed with PBS, supplemented with 0.15 g/l CaCl₂ and 0.2 g/l MgCl₂, incubated with 10 μM DCFH-DA for 30 min, washed with PBS to remove any excess probe and incubated with 10 μg/ml rhubarb extract. After 3 h, the cells were irradiated in a ¹³⁷Cs irradiator with a single dose of 2 Gy γ-rays. At 1 h following irradiation, ROS generation was measured using a FACS BD Calibur (BD Biosciences, Bedford, MA, USA), and BD Cell Quest Pro 6.0 software was used to analyze the data.

At 24 h post-irradiation, the cell culture medium was harvested and rendered cell-free using centrifugation (15,000 x g for 5 min at 4°C). LDH Cytotoxicity Assay kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) was used to measure the release of LDH from the cells. After a 40 min incubation at room temperature using the LDH kit, the LDH release was measured at a wavelength of 495 nm using a microplate system enzyme-linked immunospot assay reader (MHM-96B; Changchun MH Medical Co., Ltd., Changchun, China). Three different concentrations (2, 5 and 10 μg/ml) of the rhubarb extract were used to evaluate its effect on lactate dehydrogenase release in neuronal cell cultures.

Bisbenzimide nuclear staining was used to detect DNA fragmentation. Cells were plated onto 96-well plates coated with poly-D-lysine for bisbenzimide staining. Cells were fixed with 5% paraformaldehyde in 0.1 M PBS, washed with 0.1 mM PBS and stained with bisbenzimide (10 μg/ml), which is a fluorescent DNA-binding dye, for 10 min at 25°C. Three different concentrations (2, 5 and 10 μg/ml) of the rhubarb extract were used to investigate its effect on neuronal apoptosis. Cells were examined under an optical fluorescence microscope (Ningbo Sunny Instruments Co. Ltd., Zhejiang, China). The number of cells with apoptotic bodies per total cell number was calculated from 8–10 random fields of 6×10³ cells/well. Three wells were assessed per treatment.

LC-MS equipment consisted of a chromatographic system (LC-MS QqQ-6410B Agilent, 1260 Infinity series (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent Triple Quad mass spectrometer fitted with an electrospray ionization source, using the following MS conditions: MS range, 100–1,200 Da; MS spectra obtained using positive and negative modes; nebulizer gas, 45 ψ; and capillary voltage, 4000 V.

HPLC analysis was conducted using an Agilent 1260 infinity series (Agilent Technologies). A chromolith RP-18e column (4.6 mm ID, 50 mm length) was used. The mobile phase consisted of (A) aqueous acetic acid (0.5%) and (B) 70% methanol. The mobile phase gradient was as follows: 0–8 min, linear gradient from 10 to 25% of B; 8–12 min, isocratic conditions at 25% of B; 12–16 min, linear gradient from 25 to 40% of B; 16–40 min, linear gradient from 40 to 50% of B; and 40–50 min, linear gradient from 50 to 100% of B. The flow rate used was 1 ml/min.

All data are expressed as the mean ± standard error of the mean. One way analysis of variance was used. All statistical analyses were conducted using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). P≤0.05 was considered to indicate a statistically significant difference.

The present study used the oxidation-sensitive probe DCFH-DA to determine ROS generation in neuronal cells that had been incubated with the rhubarb extract prior to irradiation with a single dose of 2 Gy γ-rays. As hypothesized, irradiating the cells resulted in enhanced ROS production even at 1 h post-irradiation. This increased ROS generation was inhibited in cells treated with rhubarb extract at a dose of 10 μg/ml. This result was confirmed by incubating the cells with N-acetylcysteine, a known ROS scavenger. This also inhibited the radiation-induced increase in DCF fluorescence, which corresponded to decreased ROS generation (Fig. 1).

Significant cell death, as measured by lactate dehydrogenase release, was observed following irradiation at a dose of 2 Gy. Irradiation reduced the numbers of neuronal cells by 50–55% and apoptosis was observed at 24 h post-irradiation. Using bisbenzimide staining, condensed and fragmented DNA was detected in the apoptotic cells. The extract exhibited a dose-dependent inhibition of radiation-induced apoptotic neuronal cell death, with the 10 μg/ml dose of the extract resulting in the greatest degree of inhibition (P<0.05; Figs. 2 and 3). Furthermore, in the bisbenzimide stained cultures, the rhubarb extract significantly reduced the number of neurons with condensed and fragmented DNA, particularly at the higher concentration (10 μg/ml). As shown in Fig. 2, healthy surviving neurons have a large, round and intact nucleus without any deformation, whereas in apoptotic neurons the chromatin is condensed and fragmented. As shown in Fig. 2, in the control cell cultures no apoptotic neurons were observed. In the cell cultures treated with rhubarb extract, few apoptotic neurons were visible. Administration of the extract also prevented the increase in lactate dehydrogenase release induced by irradiation (P<0.05). Three different concentrations (2, 5 and 10 μg/ml) of the extract were used in the experiments that observed lactate dehydrogenase release and apoptosis. The extract exhibited a dose-dependent inhibition of lactate dehydrogenase release. The 10 μg/ml dose inhibited lactate dehydrogenase release by >85% (Fig. 4).

The phytochemical analysis of the Rheum officinale extract was conducted by LC-ESI-MS and HPLC with diode-array detection techniques. The extract was run under positive and negative ESI-MS conditions and it showed several major and minor ionic species. Fragmentation of the major peaks was used for the identification of compounds present in the extract. The identification of the chemical compounds was also conducted by comparing the molecular ion peaks, along with the MS fragmentation patterns, with those in the literature. The five chemical constituents identified were emodin, aloe-emodin, chrysophanol, physcion and rhein (Fig. 4). Extraction of these phytoconstituents has previously been reported from this and other species of Rheum. These constituents have also been reported to possess a spectrum of biological properties, including antioxidant, antitumor and anti-inflammatory effects.