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Introduction
Chronic alcoholism is a serious social problem that can cause pathological changes in the structure and function of the central nervous system (CNS), resulting in inattention, cognitive dysfunction and poor judgment, unstable walking, and even dementia (1-6). The heavy burden of alcohol has made it a global medical and social problem (7). Reactive oxygen species are thought to contribute to many neurodegenerative diseases, such as Alzheimer's or Parkinson's disease, as well as Down syndrome (8-11). However, a recent study did not find a relationship between acute ethanol-induced memory deficit and oxidative stress (12). Moreover, it has been reported that treadmill running can attenuate the adverse effects of chronic ethanol exposure on spatial memory (13). However, compliance with running training is poor. Therefore, there is still no satisfactory treatment of the nervous system impairment caused by long-term intake of alcohol.
Puerarin is one of the main active ingredients of the traditional Chinese herb, Radix Puerariae lobata, and is known chemically as 8-beta-D-grapes pyranose-4′,7 dihydroxy isoflavones (4′,7 dihydroxy-8-beta-D-glucosylisoflavone). The powder has long been used as an anti-alcoholic drug. Puerarin can easily go through the blood brain barrier into the CNS. It is reported that puerarin flavonoids improve the blood circulation in the brain and heart, counter memory disorders (14), protect nerve cells against glutamate injury, and alleviate damage of astrocytes (15,16). Puerarin has many pharmacokinetic advantages, such as a short half-life, rapid elimination, and not easily being accumulated in the body (17), all of which make it a suitable candidate for clinical use as a medication. It has been widely used to treat cardiovascular diseases of humans and animals (18-20). However, the protective role of puerarin on ethanol-induced impairment of the CNS has not been reported.
In this study, we induced chronic alcohol poisoning in a mouse model and investigated the ability of puerarin to protect against alcohol-related damage of spatial learning ability and memory. Effects of alcohol and puerarin on cerebral neurons, glial cells, and related neurotransmitters are described and a mechanism of protection proposed.
Material and Methods
Animals and treatment
All procedures were approved by the Ethics Committee of Dezhou University, China. A total of 30 healthy male C57BL/6 mice of 8-10 weeks of age and weighing 18-22 g were supplied by the Vital River Laboratory Animal Technology Co., Ltd. (China). Mice were housed in an air-conditioned facility at an ambient temperature of 22±2°C, a relative humidity of 40%-60%, and a 12-h (7:00 am to 7:00 pm) light/dark cycle. They were fed a standard laboratory diet and distilled water ad libitum. After 1 week of acclimation, the animals were randomly divided into three groups of 10 animals each. The model group was given 60% (v/v) ethanol by intragastric administration (6 mL/g daily in the first week, 12 mL/g daily in the second and third weeks, and 20 mL/g daily in the following 3 weeks) followed by intraperitoneal injection of normal saline. In the first 3 weeks, ethanol was administered once daily at 10:00 am; in the following 3 weeks, it was administered twice daily at 10:00 am and 4:00 p.m. The puerarin group received the same treatment as the model group, except that normal saline was replaced by intraperitoneal administration of puerarin solution. Mice received 100 mg/kg puerarin daily in the first week, 150 mg/kg daily in the second and third weeks, and 20 mg/kg daily in the following 3 weeks. The control group was treated with normal saline by intragastric administration and intraperitoneal injection. In all three groups, the intraperitoneal injection was administered once daily at about 10:30 am, 30 min after the intragastric treatment. Puerarin for injection was obtained from Zhejiang CONBA Pharmaceutical Co., Ltd. (approval no. H33020186, China) and anhydrous ethanol was provided by Shanghai Chemical Reagents Co., Ltd., reagent no. 2 factory (China). During the experimental procedures, all animals had free access to food and distilled water ad libitum.
Behavioral tests
The Morris water maze (MWM) and Tru Scan field activity tests were carried out on the day following the 6 weeks of treatment.
Tru Scan field activity testing
This test was performed from approximately 9:00 to 11:00 am for 3 days after completion of the experimental intervention. To adapt to the environment, mice were moved to the behavioral science laboratory 30 min before testing. Individual mice were then transferred to the middle of the Tru Scan test box and the door was closed. Under quiet experimental conditions, the distance and trajectory of spontaneous motion of the mice were recorded using the infrared detector of the Tru Scan Photobeam Activity System (Coulbourn Instruments, USA). The box was cleaned with 10% ethanol to avoid any influence of smell of the previously tested mouse.
Morris water maze
The MWM behavior test was conducted from about 14:00-16:30 p.m. as previously described (21). This test was used to assess the spatial learning and memory ability of the experimental animals. The maze consisted of a circular tank (1.50 m in diameter and 0.6 m deep) with black walls. The tank was filled with water maintained at a temperature of 25±1°C. The path-tracking and recording system was provided by Actimetrics (Actimetrics, Inc., version 3.41, USA). Testing was carried out over 6 days, with the first day regarded as screening for basic athletic ability. A small, black platform about 20 cm in diameter was placed in a quadrant of the tank so that it extended 1 cm above the water. Mice from each group were placed individually in the water maze in the same quadrant, not the one with the platform, and the times it took to find the platform were recorded and compared to evaluate vision and swimming ability. For spatial navigation training and testing, the platform was placed 1 cm underwater in a different quadrant; mice underwent the same treatment as on the first day and were required to find the hidden platform in 2 min. This test evaluated spatial learning ability by recording and analyzing the escape latency (i.e., time to reach the platform) and escape distance (i.e., length of the swimming path taken to find the platform). In the spatial probe trial that was conducted on the last day, mice underwent the same treatment as in previous days, but the platform was removed and time was controlled at 1 min. The crossing times (i.e., the number of times the mice passed through the platform position) and total swimming distance in the controlled time were recorded and compared; the escape route (i.e., the swimming path) was recorded to evaluate the escape strategy.
Immunohistochemical detection of Neu-N and Iba-1
After behavioral testing, three mice from each group were anesthetized with pentobarbital sodium (80 mg/kg) and underwent cardiac perfusion with physiological saline to obtain clean brain tissue without blood. The dissected tissue was fixed in 4% paraformaldehyde for more than 24 h, frozen, sectioned at 10 μm, and mounted on slides. After antigen retrieval and blocking of endogenous peroxidase activity, sections were incubated with monoclonal mouse anti-neuronal nuclei antibody (Neu-N, 1:500, Chemicon, USA) or rabbit-anti-mouse Iba-1 antibody (1:500, Wako Chemicals, Japan) at 4°C overnight to detect brain neurons and microglia. After that, the sections were incubated with goat anti-mouse IgG labeled with red-fluorescent Alexa Fluor 594 (Molecular Probes, USA) or goat-anti-rabbit IgG labeled with green-fluorescent Alexa Fluor 488 (Molecular Probes) secondary antibodies. At least 10 serial sections from the hippocampus and cortex of three mice were selected, viewed, and photographed using a laser confocal scanning microscope (Leica, Germany). The numbers of microglial cells and neurons in the cortex were counted during microscopic observation, and those in the hippocampal dentate gyrus were calculated from the fluorescence intensity using Image-Pro Plus 6.0 (Media Cybernetics, USA).
Assay of Glu and GABA by high-performance liquid chromatography (HPLC)
The remaining mice in each group were sacrificed, the brain cortex and hippocampus were quickly isolated, frozen separately in liquid nitrogen, and stored at -80°C until use. For the assays, the stored cortex and hippocampus were thawed and then homogenized with 4% sulfosalicylic acid. Then, the content of Glu and GABA in the homogenate was assayed by HPLC (ESA, USA) and the Glu/GABA ratio was calculated. The Glu and GABA standards were purchased from Sigma (USA).
Results
Puerarin improved movement disorders in mice with chronic alcohol poisoning
The Tru Scan results revealed that the distance covered during spontaneous movement for 30 min was shorter in the model group than in the control group. The distance covered was significantly longer in the puerarin group than in the model group, as shown in Figure 1 (P<0.05).
Figure 1 Results of the 30 min Tru Scan test. A-C, Spontaneous movement routes of mice in the control, model, and puerarin groups. D, Comparison of movement distance in 30 min among the 3 groups. *P<0.05 vs control group; #P<0.05 vs model group (one-way ANOVA).
Puerarin improved the spatial learning memory disorder of mice with chronic alcohol poisoning. In the spatial navigation test, the escape latency and escape distance of the model group were significantly longer than in the control group (P<0.05 and P<0.01, respectively; Figure 2A and B). When treated with puerarin, the escape latency on days 3, 4, and 5 and the escape distance on days 3 and 5 days in the puerarin group were significantly reduced compared with the control group (P<0.05 and P<0.01, respectively). In the spatial probe trial, the cross times and total swimming distance of the model group were significantly shorter than those in the control group, but in the puerarin group, the cross times and total swimming distance were similar to those in the control group and significantly longer than those in the model group (Figure 2C and D, P<0.05). The escape routes might reflect the escape strategy of mice. As shown in Figure 2E, the escape route of the mice in the control group was short and direct, while in the model group, the route was complex and wandering. In the puerarin group, the route was better than that in the model group, but still worse than in the control group.
Figure 2 Results of the Morris water maze test. A, Escape latency: time to reach the hidden platform. B, Escape distance: length of the swimming path taken to find the platform. C, Cross times: times the mice passed through the platform. D, Total swimming distance in controlled time (1 min). E, Typical escape route (the swimming path of mice) in the 3 groups. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs model group (one-way ANOVA).
Influence of puerarin on the number of neurons and microglial cells
As shown in Figures 3 and 4, there were significantly more microglial cells in the hippocampal dentate gyrus of the model and puerarin groups (P<0.01), as well as in the cortex (P<0.05 and P<0.01, respectively) compared with the control group. Moreover, puerarin treatment inhibited the reduction of microglial cells in both the cortex (P<0.05) and hippocampal dentate gyrus (P<0.01) that occurred in the model group. The number of neurons was reduced only in the hippocampal dentate gyrus (P<0.05, P<0.01).
Figure 3 Detection of neuron and microglial cells in cortex of mice by immunohistochemical staining. A-C, Iba-1 immunofluorescence staining in the cortex of the control, model and puerarin groups (10×). D-F, Cortical neurons of each group. G-I, Cortical microglia of each group. J, Number of neurons in the cortex of each group. K, Number of microglia in the cortex of each group. *P<0.05, **P<0.01 vs control group; #P<0.05 vs model group (one-way ANOVA).
Figure 4 Detection of neuron and microglia cells in the hippocampus of mice by immunohistochemical staining. A-C, Neu-N and Iba-1 immunofluorescence staining in the hippocampus of the control, model, and puerarin groups (10×). D-F, Hippocampal neurons of each group. G-I, Hippocampal microglia of each group. J, Number of neurons in the hippocampus of each group. K, Number of microglia cells in the hippocampus of each group. DG: dentate gyrus. **P<0.01 vs control group; #P<0.05, ##P<0.01 vs model group (one-way ANOVA).
Influence of puerarin on Glu and GABA
Compared with the control group, the Glu and GABA levels of the cortex and hippocampus were significantly reduced in the model group (Figure 5, P<0.05 and P<0.01, respectively). Puerarin treatment significantly reversed the reduction of GABA in both the cortex (P<0.05) and hippocampus (P<0.01), but reversed only the reduction of Glu in the hippocampus (P<0.05). The Glu/GABA ratio in the cortex was significantly higher in the model group than in both the control group (P<0.05) and the puerarin group (P<0.01). Puerarin treatment significantly inhibited the increase of the Glu/GABA ratio in the hippocampus compared with the model group (P<0.01).
Figure 5 Detection of glutamic acid (Glu) and gamma amino butyric acid (GABA) by high-performance liquid chromatography (HPLC). A,B, Glu content of the cortex and hippocampus. C,D, GABA content of the cortex and hippocampus. E,F, Glu/GABA ratio in the cortex and hippocampus. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs model group (one-way ANOVA).
Influence of puerarin on TNF-α and IL-1β in the cortex and hippocampus
As shown in Figure 6, TNF-α and IL-1β in the cortex and hippocampus was significantly higher in the model group than in the control group, (P<0.05 and P<0.01, respectively). However in the puerarin group, TNF-α and IL-1β were significantly increased only in the hippocampus (P<0.05 and P<0.01, respectively). Puerarin treatment reversed the increase of TNF-α and IL-1β in both the cortex and the hippocampus compared with the model group (P<0.05).
Discussion
Alcoholism is usually caused by alcohol dependence and is reported to involve about 140 million people worldwide (22,23). Animal models of alcohol poisoning are commonly established by feeding, gavage, or injection of alcoholic materials. In this study, we chose the gavage model (24), which is close to the situation in human beings. Moreover, the gavage model avoids differences caused by individual factors, such as appetite and food intake and thus more accurately reflects the impact of alcohol on the human body and the inhibitory effect of puerarin. In this study, blood alcohol concentration (BAC) reached 79.6 mg/100 mL 1 h after administration of 60% alcohol (v/v, 6 mL/kg) and 298.5 mg/100 mL after administration of 12 mL/kg. As the standard for alcohol poisoning is a BAC of 50-100 mg/100 mL, the model was successful.
Tru Scan is a versatile and simple system to monitor open field activity. The MWM test is usually used to assess spatial learning and memory ability of experimental animals, and it has been a useful laboratory tool in behavioral neuroscience (25,26). We used these tests to evaluate the effect of alcohol on the nervous system of experimental animals. A statistical analysis conducted in a large study of alcohol drinkers reported that physical activity among the excessive drinkers was significantly reduced (27). Alcohol poisoning may thus affect the CNS, which manifests as a decrease in movement function. In this study, the spontaneous movement recorded by the Tru Scan detection system confirmed the findings of Liangpunsakul et al. (27) in that ethanol poisoning led to a shorter spontaneous motion route, and that puerarin intervention inhibited the impairment by ethanol and promoted active spontaneous motion. Further, according to the results of the MWM test, chronic alcohol poisoning resulted in significant increases in the escape latency, escape distance, total swimming distance, and decrease in cross times. Therefore, chronic alcohol poisoning impaired spatial learning ability and memory. However, puerarin intervention significantly reversed these impairments as shown by decreases in escape latency, escape distance, total swimming distance, and increase in cross times. Moreover, mice in the control and puerarin groups appeared to choose a more effective route when searching for the platform. Therefore, ethanol poisoning impaired nervous system function, spatial learning ability, and memory, and puerarin intervention alleviated the damage caused by ethanol.
Microglia play a key, active role in regulating immune system functions in the CNS (28). In the absence of external stimulation, microglial cells remain in the resting state. After detecting an injury signal, their cell volume increases and the protuberances extending from their surface grow. These activated microglia, or brain macrophages, secrete neurotoxic inflammatory factors, such as TNF-α and IL-1β, which can damage or kill neurons (Figure 7) (29-31). Previous studies indicate that alcohol poisoning can cause microglial activation, release of TNF-α and IL-1β, and damage to neurons (32). In this study, the numbers of microglia in both the hippocampal dentate gyrus and the cortex significantly increased, as did the levels of TNF-α and IL-1β, and the number of neurons in the hippocampus significantly decreased. Puerarin intervention inhibited the changes in the numbers of microglia and neurons as well as secretion of TNF-α and IL-1β. Therefore, we speculate that the protective effect of puerarin against damage caused by alcohol poisoning may be related mainly to the anti-inflammatory activity of puerarin (33), which reduces the activation of microglia and subsequent damage to or death of neurons, resulting in protection of spatial learning and memory ability.
Figure 7 Diagram of changes in microglial appearance and function in the resting and activated states.
Amino acid neurotransmitters in brain tissue, especially Glu and GABA, are closely related to CNS function. Excessive Glu can have toxic effects in the CNS (34). Although the levels of Glu and GABA were decreased by alcohol poisoning, increase of the Glu/GABA ratio indicates that the model produced a neurotransmitter imbalance. Relative increase of Glu has a toxic effect on neurons in the cortex and hippocampus, influencing the structure and function of the cortex and the hippocampus and causing impairment of spontaneous movement and spatial memory (35). Puerarin intervention decreased the Glu/GABA ratio in the cortex and hippocampus, especially in the cortex, where it decreased to near the control level. Therefore, puerarin may have a regulatory effect on the balance of Glu and GABA, and further protect against the damage caused by chronic alcohol poisoning.
In conclusion, puerarin protected against damage of spatial learning and memory ability caused by chronic alcohol poisoning primarily because of anti-inflammatory activity and regulation of the balance of Glu and GABA. Further studies of its specific molecular mechanisms are needed.
