Harpagoside Rescues the Memory Impairments in Chronic Cerebral Hypoperfusion Rats by Inhibiting PTEN Activity

Abstract

Vascular dementia (VaD) is the second most common dementia worldwide. Unlike Alzheimer’s disease, VaD does not yet have effective therapeutic drugs. Harpagoside is the most important component extracted from Harpagophytum procumbens, a traditional Chinese medicine that has been widely used. The neuroprotective effects of harpagoside have been studied in Aβ- and MPTP-induced neurotoxicity. However, whether harpagoside is protective against VaD is not clear. In this study, with the use of chronic cerebral hypoperfusion rats, a well-known VaD model, we demonstrated that chronic administration (two months) of harpagoside was able to restore both the spatial learning/memory and fear memory impairments. Importantly, the protective effects of harpagoside were not due to alterations in the physiological conditions, metabolic parameters, or locomotor abilities of the rats. Meanwhile, we found that harpagoside suppressed the overactivation of PTEN induced by CCH by enhancing PTEN phosphorylation. Furthermore, harpagoside elevated the activity of Akt and inhibited the activity of GSK-3β, downstream effectors of PTEN. Overall, our study suggested that harpagoside treatment might be a potential therapeutic drug targeting the cognitive impairments of VaD.

Keywords

Chronic cerebral hypoperfusion GSK-3β harpagoside memory PTEN

INTRODUCTION

Dementia refers to a wide range of symptoms characterized by a decline in memory or other thinking skills sufficiently severe to reduce a person’s ability to perform everyday activities. With the inexorable aging of the population in the 21st century, dementia appears to be an intractable health problem worldwide. There are two major types of dementia, Alzheimer’s disease (AD) and vascular dementia (VaD). The former is the most common type of dementia and accounts for an estimated 60 to 80% of dementia cases, while VaD is a less common sole cause of dementia than AD and accounts for approximately 10% of dementia cases. For AD, the U.S. Food and Drug Administration (FDA) has approved two types of medications, cholinesterase inhibitors (Aricept, Exelon, Razadyne) and memantine (Namenda), to treat the cognitive symptoms. However, numerous clinical studies have revealed that the above drugs only have very limited beneficial effects on the cognitive symptoms associated with VaD and do not provide any concomitant global or clinical benefits in most cases [1]. Therefore, some new strategies should be developed for the therapy of VaD.

As we know, VaD is most frequently caused by cerebral vascular problems, hypoperfusive lesions, such as global or selective ischemia, border-zone infarcts, and incomplete white matter ischemia, or ischemic lesions involving large or small vessels, such as strategic single strokes, multi-infarct dementia, small-vessel disease, lacunes, and venous occlusions [2]. Chronic cerebral hypoperfusion (CCH) is thought to be a dominant cause of VaD. Many diseases or pre-clinical disorders can lead to CCH. For example, transient ischemic attacks, hypertension, smoking, and hyperlipidemia have been shown to be risk factors that accelerate perfusional decline and cerebral atrophy [3, 4]. The critical role of CCH in promoting neurodegeneration in dementia had been well studied, and CCH is thought to result in the overproduction of reactive oxygen species, disruption of energy generation and metabolism, and induction of inflammatory responses, in turn, impairing the white matter [5 –8]. Thus, rodent models of chronic cerebral hypoperfusion have been well established using occlusion or ligation of both common carotid arteries in rats to replicate the pathological and clinical changes in VaD [9].

Harpagoside is the natural product found in the plant Harpagophytum procumbens (devil’s claw, Pedaliaceae) [10]. Harpagoside is the active chemical constituent responsible for the medicinal properties of the plant, which has been used for centuries by the Khoisan people of southern Africa to treat diverse health disorders, including fever, diabetes, hypertension, and various blood-related diseases [11]. Harpagoside can also be extracted and purified from Scrophularia ningpoensis, another important herb commonly used in traditional Chinese medicine for the treatment of multiple diseases [12]. The application of harpagoside in neurological disorders has also been reported. Harpagoside has been reported to not only significantly ameliorate the loss of TH-positive neurons and the shortening of axonal length in MPP⁺-treated mesencephalic neurons, but also improve the loco-motor ability (rotarod test) and increase the number of TH-positive neurons in the substantia nigra pars compacta induced by MPTP [13]. Moreover, harpagoside is able to rescue the memory impairment induced by Aβ, a well-known animal model of AD [14]. However, whether harpagoside is also able to restore the learning and memory ability in a VaD animal model and the possible mechanisms are not clear as of yet.

In this study, we aimed to study the neuroprotective effect of harpagoside on a CCH rat model, especially the effect on the learning/memory ability. We reported that oral administration of harpagoside (15 mg/kg) once daily for two consecutive months improved memory retention in different tasks (Morris water maze and passive step avoidance) without affecting the motor ability. We also examined the activity of PTEN, an important phosphatase that has been proven to be crucial for CCH-induced brain pathology, and found that harpagoside inhibited the activity of PTEN and reduced the phosphorylation of PTEN, as well as the activation of the PI3K/Akt/GSK-3β signaling pathway.

MATERIALS AND METHODS

Animals

Forty-eight male Wistar rats (3-4 months old, 280–300 g) were supplied by the Experimental Animal Central of First Affiliated Hospital of Zhengzhou University. They were housed at room temperature (22±2°C) on a 12-h/12-h light/dark cycle (lights on at 8:00 a.m.) with free access to food and water. The experimental procedures were performed in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of the Medical School of Zhengzhou University.

Experimental procedures

Figure 1 shows a diagram of the experimental procedure. Rats were first acclimatized for two weeks before the experiments. The rats were randomly divided into four groups: the sham operation with vehicle treatment group (sham), 2VO surgery plus vehicle treatment group (CCH), 2VO surgery plus harpagoside treatment group (CCH+HAR), and sham operation plus harpagoside treatment group (HAR). After the surgery, harpagoside and vehicle were delivered once daily for two months. The physiological condition and metabolic parameters of all rats were monitored before the behavioral tests. Then, all rats were subjected to the open field test to evaluate motor ability. The Morris water maze and inhibitory avoidance tests were used to evaluate learning and memory. All rats were sacrificed after the behavioral tests, and half were used for biochemical examinations.

Fig.1

The diagram of the experimental procedures. 2VO, bilateral occlusion of the common carotid arteries; HAR, harpagoside.

2VO surgery and harpagoside treatment

Rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.). A ventral midline incision was made to visualize the bilateral common carotid arteries. Then, the arteries were gently separated from the carotid sheath and vagus nerve and were permanently doubly ligated with silk sutures. The sham rats received the same surgical operation without ligation of the carotid arteries. During the surgical procedure, the body temperature of the rat was kept stable at 36.5±0.5°C using a heating pad. Harpagoside with a purity of 99% determined by HPLC was supplied by Shanghai Tauto Biotechnology Co (Shanghai, China). After the surgery, all rats were given either HAR suspended in 0.5% Na-hydroxypropyl methylcellulose (HPMC-Na) (CCH+HAR groups) or vehicle (HPMC) (sham control group and CCH model group) orally by gavage once daily for 60 days according to a previous report [14].

Detection of physiological and metabolic parameters

The physiological condition of all rats, including body weight (both pre- and post-treatment), respiratory frequency, and body temperature, were monitored according to our previous report [15]^. Before the behavioral tests, 1.0 mL of blood was collected from the caudal vein of each rat under anesthesia with 10% chloral hydrate (350 mg/kg intraperitoneally (i.p.)). The metabolic parameters, including plasma glucose, triglyceride, and cholesterol levels, were measured by using the related commercial ELISA kits from Nanjing Jiancheng Biotechnology.

Open field

All rats were subject to an open field test to measure the motor behavior after the different treatments. The open-field apparatus consisted of clear Plexiglas walls covered with opaque paper around a square open arena (48×48 cm, 35 cm high). The rats were placed in the arena, along the wall, and behavior was recorded for 5 min. Movement was recorded using arrays of infrared light beam motion detectors (16×16 cm, 2.5 cm apart) controlled by the Activity Monitor program version 5.10 (Med Associates, St. Albans, VT, USA). Measures of motor activity included ambulatory distance, ambulatory time, rearing time, immobility time and average velocity, which were automatically scored by a computer using MEDPC software. To minimize odor-cued motor activity, chambers were cleaned with 70% ethanol between each session.

Morris water maze

The spatial learning and memory of rats was examined by the Morris water maze test modified according to previous studies [16]. A circular pool with a diameter of 150 cm and a height of 50 cm was divided into four imaginary quadrants and filled with water at a temperature of 20–21°C. The rats were first subjected to a visible platform test. Then, over the next 6 days, the rats were trained on a hidden platform test, followed by an additional probe test. In the visible platform test, a 12-cm platform was marked with a flag and positioned above the surface of the clear water. The position of the platform varied across trials. Rats were tested in five contiguous trials with an inter-trial interval of 30 min. In the hidden platform test, the platform was submerged 0.5 cm below the surface of opaque water in a fixed position in the center of a quadrant (defined as the target quadrant). Rats were trained for four trials with an inter-trial interval of 1 h. In each trial, rats were given a maximum of 60 s to find the platform and were allowed to remain on it for 30 sec. Escape latency, swimming speed, and path length to reach the platform were recorded as measures of spatial learning and memory. In the probe trial, the platform was removed, and the rats were allowed to swim in the pool for 60 s. The number of platform zone crossings, the total time spent in the target quadrant, and the swimming speed were analyzed.

Inhibitory avoidance tasks

The inhibitory avoidance apparatus was an acrylic box (50 cm×25 cm×25 cm), whose floor consisted of parallel stainless-steel bars (1 mm diameter) spaced 1 cm apart [17, 18]. A platform (7 cm wide×2.5 cm high) was placed in the corner. The animals were placed on the platform, and their latency to step down on the grid with the four paws was measured with an automatic device. The animals were submitted to the inhibitory avoidance task by using a protocol similar to that described previously for a double-blinded assay. During training sessions, immediately after stepping down onto the grid, the animals received a 0.4-mA, 1.0-s scrambled foot shock. During test sessions, no foot shock was administered, and the step-down latency (maximum 180 s) was used to measure the retention. The animals were submitted to a single training session. To evaluate short- and long-term memory, test sessions were performed 1.5 h and 24 h after training.

Western blot

The rats were deeply anesthetized with 10% intraperitoneal chloral hydrate (6 mL per kg) and sacrificed. The cerebral hemisphere was gently dissociated on ice under a dissection microscope. The hippocampi were quickly removed and homogenized in lysis buffer. The homogenates were centrifuged at 10000×g for 30 min, and the supernatant fractions were used for western blot analyses. The protein concentration was measured by a commercial BCA kit (Pierce, Rockford, IL, USA), and a final concentration of 10% β-mercaptoethanol and 0.05% bromophenol blue was added to the samples. After the samples were boiled in a water bath for 10 min, the samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Then, 5% fat-free milk was used to block the non-specific reaction for 1 h, and the membrane was incubated overnight at 4°C with antibodies against p-PTEN, PTEN, p-Akt, Akt, and actin for 24 h. The membranes were then rinsed three times by using 1X PBS buffer and incubated with secondary antibodies for another 2 h at room temperature. The bands were visualized by using the Odyssey Infrared Imaging System. Protein bands were quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA) [19, 20].

Activity assay for Akt and GSK-3β

The activities of Akt (Abcam, ab139436) and GSK-3β (GENMED Scientifics Inc., Arlington, MA, USA) in the hippocampal homogenates were measured by using commercial kits. All experimental procedures were performed according to the manufacturer’s instruction.

Nissl staining

The Nissl staining was used to detect the neuronal numbers in the hippocampus. Four rats in each group were anesthetized and fixed by perfusion of 4% paraformaldehyde phosphate-buffered solution. After the perfusion, the brains were harvested and post-fixed for 24 h. The hippocampal tissues were cut into 25μm slices using a vibrate microtome (VT1000 s, Leica, Germany). The slices were placed in Nissl staining fluid for 5–10 min. The cell numbers of hippocampus were counted in the six consecutive sections using a grid-installed light microscopy (BX51; Olympus, Tokyo, Japan).

Statistical analysis

All statistical analyses were conducted using SPSS version 13.0. Analyses of differences in the variables between the groups were tested by ANOVA followed by LSD post hoc test. Data were considered to be statistically significant when p < 0.05.

RESULTS

Harpagoside does not alter physiological conditions

Before the behavioral test and biochemical examinations, we first monitored the general conditions of the rats, including body weight (both pre- and post-treatment), respiratory frequency, and body temperature. As shown in Table 1, although the body weights of every group had increased by approximately 5–8% by the day the rats were sacrificed, there was no significant difference among the four groups. Meanwhile, no difference was found in the respiratory frequency or body temperature among the four groups. These results indicated that harpagoside does not affect baseline physiological function.

Table 1

Harpagoside does not alter body weight, respiratory frequency, or body temperature

	Sham	CCH	CCH+HAR	HAR
n	12	12	12	12
BW-Pre (g)	290.2±6.0	291.2±5.9	289.4±5.5	293.4±6.9
BW-Post (g)	344.5±6.6	341.2±8.2	342.3±7.7	344.7±8.2
RF (times/min)	83.1±5.1	85.1±5.3	84.1±3.7	80.4±5.4
Temp (°C)	36.7±0.6	36.7±0.4	36.8±0.5	36.6±0.5

All data are expressed as the mean±S.E.M. BW-Pre, body weight before the treatment; BW-Post, body weight after the treatment; RF, respiratory frequency; Temp, body temperature; CCH, chronic cerebral hypoperfusion; HAR, harpagoside.

Harpagoside does not alter metabolic parameters

Table 2 shows the plasma glucose, triglyceride, and cholesterol levels for each group at two different time points as indicated in Fig. 1. Obviously, harpagoside treatment did not affect the metabolic parameters.

Table 2

Harpagoside does not alter metabolic parameters

	Sham	CCH	CCH+HAR	HAR
n	12	12	12	12
Glucose (mmol/L)	5.03±0.23	4.96±0.24	5.16±0.2	5.0±0.32
Triglycerides (mmol/L)	0.82±0.19	0.78±0.2	0.83±0.2	0.77±0.15
Cholesterol (mmol/L)	1.35±0.2	1.42±0.19	1.31±0.22	1.37±0.23

All data are expressed as the mean±S.E.M. CCH, chronic cerebral hypoperfusion; HAR, harpagoside.

Harpagoside does not alter general motor ability

The general motor ability of rats exposed to the different treatments was first evaluated by the open field test. As seen in Fig. 2, we did not find any significant difference in ambulatory distance, ambulatory time, rearing time, immobility time, or average velocity in the open field test (Fig. 2). Therefore, any observed differences in behavioral and biochemical studies were not due to differences in general activity.

Fig.2

Harpagoside does not alter locomotor ability. The rats with or without the two-month harpagoside treatment were subjected to the open field test to evaluate the locomotor ability. The ambulatory distance (A), ambulatory time (B), rearing time (C), immobility time (D), and average velocity (E) were recorded and analyzed.

Harpagoside rescues CCH-induced spatial memory deficits

We then examined the spatial learning and memory abilities of the rats by using the Morris water maze task. In the visible platform test (Fig. 3A, B), the escape latencies and path lengths of the rats were not significantly different among the four groups (Two-way ANOVA, F(1,47) = 2.56, p > 0.05), suggesting that the treatments did not affect the visual ability of the rats. In the hidden platform test, CCH rats began to exhibit learning and memory deficits (Fig. 3C). The escape latencies of CCH rats on day 3 were much longer than those of the sham group (Two-way ANOVA, F(1,47) = 2.15, p > 0.05). The deficits induced by CCH were partly reversed by harpagoside treatment (Two-way ANOVA, F(1,47) = 4.33, p < 0.05). In the probe trial on the last day of testing, when the platform was removed, CCH rats showed significantly fewer platform zone crossings and a shorter duration in the target quadrant than those in the sham group (Two-way ANOVA, F(1,47) = 7.92, p < 0.01), while harpagoside treatment increased the chances of the rats passing the platform zone and the total time spent in the target quadrant (Fig. 3D, E). These data indicated that harpagoside treatment restored the spatial learning and memory abilities of the rats that were worsened by CCH. Throughout the tasks, the swimming speed of the rats showed no obvious difference across groups (Fig. 3F), which further confirmed that the differences in the Morris water maze were not due to differences in locomotor activity.

Fig.3

Harpagoside treatment ameliorates the spatial learning and memory impairments in CCH rats. A, B) Visible platform test of the Morris water maze: each group exhibited a similar latency and path length (p > 0.05). C-F) Hidden platform test of the Morris water maze: CCH impaired the learning and memory abilities of the rats, and harpagoside treatment reversed the spatial learning and memory impairments caused by CCH. C) The latencies in the learning stages. D) In the probe test, the number of platform zone crossings. E) In the probe test, the latency to the first platform zone crossing. F) The average swim speeds of rats were recorded during the test period. Data are expressed as the mean±SEM (n = 12). **p < 0.01 versus the sham group; ^# # p < 0.01 versus the CCH group.

Harpagoside rescues CCH-induced fear memory impairments

To examine whether harpagoside treatment could rescue fear memory impairments observed in CCH rats, we examined short-term fear memory (STFM) and long-term fear memory (LTFM) by using the inhibitory avoidance (IA) task 24 h after the Morris water maze tasks. According to a previous study, we measured STFM and LTFM at 1.5 h and 24 h after training, respectively. We found that CCH rats showed a significantly longer latency than controls in the LTFM test, while there were no differences in learning or STFM (Fig. 4). As expected, harpagoside treatment reduced the prolonged latency induced by CCH. No significant difference was found between the harpagoside and vehicle-treated sham rats. Thus, we concluded that harpagoside rescued the fear memory deficits observed in CCH rats.

Fig.4

Harpagoside treatment reverses the long-term fear memory impairments in CCH rats. The inhibitory avoidance task was used to evaluate the fear memory of rats. After training, short-time fear memory (STFM) and long-term fear memory (LTFM) tests were performed at 1.5 h and 24 h, respectively. The step-down latency is expressed as the mean±SEM (n = 12). **p < 0.01 versus the sham group; ^# # p < 0.01 versus the CCH group.

Harpagoside inhibits PTEN activity

We finally explored the potential mechanisms involved in the neuroprotective effects of harpagoside. As PTEN activation has been suggested to play an important role in CCH-induced neuronal injury [21], we examined the alterations in PTEN activity and the related downstream signals. By using western blot, we detected the phosphorylation of PTEN at the Ser380 site. We found that CCH induced a dramatic reduction in phospho-Ser380 but not total PTEN expression, while harpagoside treatment restored the phosphorylation of PTEN. No significant difference was found between the harpagoside-treated sham rats and vehicle-treated sham rats (Fig. 5A-C). These data suggested that harpagoside treatment inhibited PTEN activation during CCH. Because Akt and GSK-3β are well-known downstream signals of PTEN that are critical in the memory impairment of dementia [16, 22], we then examined the phosphorylation of Akt upon harpagoside treatment and found that the phosphorylation of Akt at Ser473 was reduced in CCH rats compared to that in sham rats. However, harpagoside treatment rescued the phosphorylation of Akt (Fig. 5D, E). We also measured the activity of Akt and GSK-3β. We found that CCH rats displayed lower Akt activity and higher GSK-3β activity than sham rats, while harpagoside treatment increased Akt activity and attenuated GSK-3β activity (Fig. 5C, D). These data further confirmed that harpagoside treatment suppressed PTEN activity and the downstream signals. Finally, we examined the number of neurons in the different groups and found that CCH induced a dramatic reduction in the number of neurons, as we previously reported. Importantly, harpagoside treatment significantly preserved the number of neurons (Fig. 6A, B).

Fig.5

Harpagoside treatment inhibits the PTEN signaling pathway. A-C) Western blots to examine the phosphorylation of PTEN in the four groups. Samples were prepared from the hippocampi of rats exposed to different treatments. A) The original blots showing p-PTEN, PTEN, and actin as indicated. B) The quantitative analysis of p-PTEN expression. C) The quantitative analysis of PTEN expression. **p < 0.01 versus the sham group; ^# # p < 0.01 versus the CCH group. D, E) Western blots to examine the phosphorylation of Akt in the four groups. Samples were prepared from the hippocampi of rats exposed to different treatments. D) The original blots for p-Akt, Akt and actin as indicated. E) The quantitative analysis of p-AKT expression. **p < 0.01 versus the sham group; ^# # p < 0.01 versus the CCH group. F, G) Commercial ELISA kits were used to detect the activities of Akt and GSK-3. **p < 0.01 versus the sham group; ^# # p < 0.01 versus the CCH group.

Fig.6

Comparison of the number of neurons in the hippocampus. A) Nissl staining of hippocampal neurons of rats in each group (×400). B) Changes in hippocampal neuron numbers of rats in each group. **p < 0.05 versus the sham group; ^# # p < 0.05 versus the CCH group. Bar = 100μm.

DISCUSSION

In this study, we found that two-month harpagoside (15 mg/kg) treatment via gavage in rats restored both the spatial and fear memory impairments caused by CCH. This restoration was specific because harpagoside did not affect the physiological conditions, metabolic parameters, locomotor ability, or visual discrimination of the rats. We also reported that harpagoside treatment significantly suppressed the activity of PTEN and the downstream Akt/GSK-3β signal, which were activated by CCH.

As a well-established rat model of chronic cerebral hypoperfusion, the 2VO surgery has been widely used [23, 24]. Although human studies have shown a moderate association between CCH and cognitive impairment at best [25], animal CCH models, especially the 2VO rat model, have provided compelling evidence that CCH could result in memory decline as shown by the Morris water maze and radial arm maze tasks used to evaluate spatial reference learning and spatial working memory [9]. In line with those studies, our data here demonstrated that CCH not only induced a spatial learning memory impairment but also resulted in fear memory dysfunction. Oral administration of harpagoside was able to rescue the memory impairments induced by CCH. As an important component of the plant Harpagophytum procumbens, harpagoside has been reported to exert a neuroprotective effect in primary cortical neurons against glutamate-induced neurotoxicity [26], which has been recognized as an important mechanism for ischemic brain injury [27]. Meanwhile, harpagoside exerts cognitive-enhancing and antioxidant activities to alleviate the memory deficit induced by scopolamine in mice [28]. Our studies here further support the neuroprotective roles of harpagoside on memory deficits. Furthermore, we also reported that the motor activity in the open field test in CCH rats was not different from that in the vehicle or HAR-treated rats, implying that motor function was not affected by HAR as reported by previous studies [14].

PTEN was first identified as a tumor suppressor gene and plays important roles in cell proliferation and apoptosis [29]. PTEN is also highly expressed in the brain and negatively regulates the PI3K/Akt pathway to inactivate Akt by dephosphorylating PIP3 to PIP2 [30, 31]. The activity of PTEN is regulated by its phosphorylation at Ser380, which inhibits the activation of PTEN via a post-translational modification [32]. A higher level of p-PTEN was found in the surviving neurons in the ischemic area than in those in the normal area, suggesting that hyperphosphorylation of PTEN is beneficial for neuroprotection against ischemic injury [30, 33]. In the current study, the phosphorylation of PTEN was reduced in CCH rats compared to that in sham rats, while harpagoside treatment restored the p-PTEN level, indicating that harpagoside treatment inhibited the aberrant activation of PTEN signals. Correspondingly, Akt was inhibited and GSK-3β was activated in CCH rats according to the ELISA results, but harpagoside treatment increased the activity of Akt and suppressed the activity of GSK-3β, both of which are downstream molecules of PTEN. Previous studies have revealed that down-regulation of PTEN is neuroprotective in cerebral ischemia and traumatic CNS injury [34 –36]. The neuroprotective role of PTEN depression on ischemic neuronal death was thought to be through enhanced Akt activation, which is also verified by our study here. In addition, the protective effects of PTEN depression against ischemic neuronal death have also been reported to be induced through inhibition of NR2B-containing NMDA receptors and preservation of GABA_A receptors [35, 37]. PTEN suppression also prevents excitotoxicity-induced neuronal death via the up-regulation of nuclear TDP-43 [38]. Thus, the neuroprotective effects of PTEN inhibition might be multi-functional and deserve further study.

In summary, our study is the first to demonstrate that oral administration of harpagoside has beneficial therapeutic effects on chronic brain hypoperfusion rats, an animal model of vascular dementia.

Footnotes

ACKNOWLEDGMENTS

We thank Dr. Na Wei for her help during the experiments and manuscript preparation.

Authors’ disclosures available online ().

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