Effect of Ginseng Radix on Cell Proliferation in Dentate Gyrus of Streptozotocin-induced Diabetic Rats

INTRODUCTION

The hippocampal formation generates new neu-

rons throughout life in the mammals including humans (Kuhn et al., 1996; Eriksson et al., 1998;

Gage et al., 1998; Gould et al., 1998). Hippocam- pus is known to be implicated in memory acquisi- tion and learning ability (Milner et al., 1998). Pre- vious studies have shown that cell proliferation in the hippocampus is increased by spatial learning, serotonin, estrogen, N-methyl-D-aspartate (NMDA)

Effect of Ginseng Radix on Cell Proliferation in Dentate Gyrus of Streptozotocin-induced Diabetic Rats

*Department of Health and Sports, Dongseo University, Busan, Korea,

†Department of Physiology, College of Medicine, Kyung Hee University, Seoul, Korea

Jin-Kook Park*, Baek-Vin Lim*, Chang-Ju Kim

ABSTRACT

인삼이 Streptozotocin에 의하여 유발된 당뇨 흰쥐의 치상회에서 신경세포 생성에 미치는 영향

*동서대학교 레포츠과학부 건강스포츠학과, ^†경희대학교 의과대학 생리학교실

박 진 국*․임 백 빈*․김 창 주

인삼이 streptozotocin에 의하여 당뇨가 유발된 흰쥐의 치상회에서 신경세포 생성에 미치는 영향을 면역조 직화학법으로 실험하였다. 인삼은 정상 흰쥐의 체중에는 아무런 영향을 미치지 않은 반면, 당뇨가 유발된 흰쥐의 체중감소는 의미 있게 억제하였다. 당뇨가 유발된 흰쥐의 해마 치상회에서 신경세포 생성은 정상 흰 쥐보다 감소되었다. 인삼은 정상 흰쥐의 해마 치상회에서 신경세포의 생성에 영향을 미치지 않은 반면, 당뇨 가 유발되어 신경세포 생성이 억제된 흰쥐에서는 신경세포 생성이 인삼에 의하여 증가되었다. 본 실험의 결 과 인삼은 당뇨에 의한 신경계 후유증을 감소시키는 작용이 있음을 알 수 있었다.

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key words : Ginseng radix, Streptozotocin-induced diabetes, Cell proliferation, Dentate gyrus

책임저자: 김창주, 서울시 동대문구 회기동 1번지 ꂕ 130-701, 경희대학교 의과대학 생리학교실 Tel: 02-961-0407, Fax: 02-964-2195

E-mail: [email protected]

접수일：2004년 11월 29일, 게재승인일: 2004년 12월 15일

receptor antagonists, ischemia, seizure, and physical exercise, and inhibited by adrenal steroids, stress, and aging (Kempermann et al., 1998; Gould et al., 1999; van Praag et al., 1999; Fuchs and Gould, 2000; Kim et al., 2001; Trejo et al., 2001).

Diabetes mellitus is one of the most serious metabolic disorders in humans. In addition to the diabetic condition itself, secondary complications involving several organs, the including brain, occur. Increasing evidences point towards an association between diabetes mellitus and deficits in learning and memory (Biessels et al., 1994;

Jackson-Guilford et al., 2000). Previous studies have suggested that diabetes mellitus might induce functional and structural changes in the brain (Kamal et al., 2000). Streptozotocin (STZ)-induced diabetic rats have been used as an animal model for the study diabetic neuropathy and have been shown to accrue deficits in memory retention and retrieval (Biessels et al., 1996). Recently, it has been demonstrated that STZ-induced diabetic rats undergo a significant reduction in the number of proliferating cells in the dentate gyrus (Jackson- Guilford et al., 2000; Kamal et al., 2000).

Ginseng radix is the root of Panax Ginseng C.A.

Meyer (Araliaceae; Ginseng radix). Ginseng radix is one of the most famous herbs in Oriental medi- cine, and it contains several triterpene glycosides, referred to as ginsenosides (or panaxosides) (Bara- nove, 1982; Chong and Oberholzer, 1988). The aqueous extract of Ginseng radix has been used to treat a wide variety of diseases including anemia, diabetes mellitus, insomnia with neurasthenia, gastritis, abnormalities in blood pressure, dyspepsia, overstrain, and fatigue (Braranov, 1986; Wesnes et al., 2000). Ginseng radix has also been used in Oriental medicine to restore and enhance well- being, and these effects are described as increasing resistance against noxious or stressful influences

without impairing physiological functions (Chong and Oberholzer, 1988). In addition, it has been reported that Ginseng radix improves the learning capacities of animals (Petkov and Mosharrof, 1987;

Petkov et al., 1993).

In the present study, the effect of aqueous ex- tracts of Ginseng radix on cell proliferation in the dentate gyrus of STZ-induced diabetic rats was investigated via immunohistochemistry.

MATERIALS AND METHODS

Adult male Sprague-Dawley rats weighing 250±

10 g (8 weeks of age) were obtained from a com- mercial breeder (Daehan Biolink Co., Chungbuk, Korea). The experimental procedures were per- formed in accordance with the animal care guide- lines of the National Institute of Health (NIH) and the Korean Academy of Medical Sciences. Each animal was housed under controlled temperature (20±2^oC) and lighting (08:00∼20:00 h) condi- tions with food and water made available ad libitum. The animals were equally divided into four groups: the control group, the non-diabetic and Ginseng radix-treated group, the STZ-in- duced-diabetes group, and the STZ-induced-dia- betes and Ginseng radix-treated group (n=5 in each group). To induce diabetes in the experi- mental animals, a single intraperitioneal injection of STZ (50 mg/Kg; Sigma Chemical Co., St. Louis, MO, USA) was given to each animal, while animals of the control group and the non-diabetic and Ginseng radix-treated group received equivalent dose of normal saline. Blood glucose levels were determined from blood drawn from the tail vein 2 days after STZ injection using a blood glucose

analyzer (Arkray, Kyoto, Japan). Only the animals with blood glucose levels of 300 mg/dl or higher were used in this study.

Ginseng radix used in this experiment was obtained from Kyung-Dong market in Seoul. After washing, Ginseng radix was immersed in cold water for 12 h and aqueous extracts was made by using a rotary evaporator. Rats of the control group and the STZ-induced-diabetes group were injected intraperitoneally with 5-bromo-2'-deoxyu- ridine (BrdU) (50 mg/kg in saline; Sigma, St.

Louis, MO, USA) once a day for 3 consecutive days starting on the second day after STZ injection, while animals of the non-diabetic and Ginseng radix-treated group and the STZ-induced-diabetes and Ginseng radix-treated group were injected with an equivalent dose of BrdU and 30 mg/kg of Ginseng radix extracts once a day for the same duration of time. Animals were sacrificed on the fifth day after commencement of the experiment.

2. Tissue preparation

Animals were fully anesthetized using Zoletil 50^Ⓡ (10 mg/kg, i.p.; Vibac, Carros, France), transcardially perfused with 50 mM phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in 100 mM phosphate buffer (PB) at pH 7.4. Brains were removed, postfixed in the same fixative over- night, and transferred into a 30% sucrose solution for cryoprotection. Coronal sections of 40 μm thickness were made using a freezing microtome (Leica, Nussloch, Germany). Five sections on aver- age were selected in each brain from the region spanning from Bregma -3.30 mm to -4.16 mm.

3. Immunohistochemistry

For detection of cell birth in the dentate gyrus,

BrdU-specific immunohistochemistry was performed according to a previously described method (Gage et al., 1998; Kim et al., 2001). In brief, sections were first pretreated by immersing in 50% for- mamide-2 x standard saline citrate (SSC) at 65^oC for 2 h, denaturing in 2 N HCl at 37^oC for 30 min, and rinsing twice in 100 mM sodium borate (pH 8.5). After the pretreatment, the sections were incubated overnight at room temperature with BrdU-specific mouse monoclonal antibody (1：

600; Boehringer Mannheim, Mannheim, Germany).

The sections were then washed three times with PBS and incubated for 1 h with biotinylated mouse secondary antibody (1：200; Vector Laboratories, Burlingame, CA, USA). The sections were incu- bated for another 1 h with avidin-biotin-horseradish peroxidase complex (1：100; Vector Laboratories, Burlingame, CA, USA). For staining, the sections were reacted with 0.02% 3,3'diaminobenzidine containing nickel chloride (40 mg/ml; Nickel-DAB) and 0.03% hydrogen peroxide in 50 mM Tris-HCl (pH 7.6) for 5 min and were mounted onto gelati- nized glass slides.

4. Data Analysis

The mean number of BrdU-positive cells in each group was obtained according to a previously described stereological method (Fuchs and Gould, 2000; Kim et al., 2001). The area of the dentate gyrus region was measured hemilaterally in each of the selected sections after immunostaining with mouse anti-neuronal nuclei (NeuN) antibody using an image analyzer (Multiscan, Fullerton, CA, USA). Results were obtained as cells per mm² of cross sectional area of the granular layer of the dentate gyrus. Data was analyzed using SPSS by one-way analysis of variance (ANOVA) followed by Duncan's post-hoc test, and results are expressed

as mean±standard error mean (S.E.M.). Differ- ences were considered significant for p＜0.05.

RESULTS

The average weight was 250.00±4.08 g, 259.25

±12.10 g, and 282.50±6.29 g in the control group, 240±6.33 g, 250.00±4.08 g, and 280±

4.08 g in the non-diabetic and Ginseng radix- treated group, 230.00±5.11 g, 231.80±9.35 g, and 210.00±10.00 g in the STZ-induced-diabetes group, and 240.00±5.93 g, 248.00±4.57 g, and 242.00±6.63 g in the STZ-induced-diabetes and Ginseng radix-treated group on the 1st, 3rd, and 5th day of experiment, respectively. In the STZ- induced-diabetes group, significant weight loss was

observed; in contrast, time-dependent weight gain was seen in rats of the control group and the non-diabetic and Ginseng radix-treated group.

The STZ-induced-diabetes and Ginseng radix- treated group showed no significant change in weight. Application of aqueous extracts of Ginseng radix exerted no significant effect on weight gain in normal rats, while it prevented weight loss seen in diabetic rats (Fig. 1).

2. Blood glucose level in each group

The blood glucose level was 89.25±2.33 mg/dl, 90.00±3.53 mg/dl, and 87.00±3.31 mg/dl in the control group, 87.11±3.52 mg/dl, 86.00±3.03 mg/dl, and 89.25±2.13 mg/dl in the non-diabetic and Ginseng radix-treated group, 88.25±3.53 mg/

dl, 436.80±36.68 mg/dl, and 469.00±24.98 mg/dl in the STZ-induced-diabetes group, and 90.25±

3.30 mg/dl, 457.80±30.40 mg/dl, and 493.40±

Fig. 1. Weight change in each group. Significant weight loss was observed in rats with streptozocin- induced diabetes; in contrast, significant weight gain was observed in the control group and the non- diabetic and Ginseng radix-treated group. Ginseng radix treatment appears to have inhibited diabetes- induced weight loss. (A) Control group, (B) non- diabetic and Ginseng radix-treated group, (C) strep- tozotocin (STZ)-induced-diabetes group, (D) STZ-in- duced-diabetes and Ginseng radix-treated group. * p

＜0.05 compared to the 1st day.

Fig. 2. Blood glucose level in each group. Blood glucose levels were increased significantly 2 days after streptozotocin injection. Ginseng radix treatment had no significant effect on blood glucose levels under both normal and diabetic conditions. (A) Control group, (B) non-diabetic and Ginseng radix- treated group, (C) streptozotocin (STZ)-induced-dia- betes group, (D) STZ-induced-diabetes and Ginseng radix- treated group.

10.45 mg/dl in the STZ-induced-diabetes and Gin- seng radix-treated group on the 1st, 3rd, and 5th day of experiment, respectively. Blood glucose levels were seen to be increased significantly on the second day after STZ-injection. Ginseng radix had no significant effect on blood glucose levels under both normal and diabetic conditions (Fig. 2).

3. BrdU-positive cells in dentate gyrus of each group

Photomicrography of BrdU-positive cells in the

dentate gyrus of each group is presented in Fig. 3.

The mean number of BrdU-positive cells in the dentate gyrus was 87.00±3.88/mm² in the control group, 88.30±4.28/mm² in the non-diabetic and Ginseng radix-treated group, 64.00±4.07/mm² in the STZ-induced-diabetes group, and 81.00±4.52/

mm² in the STZ-induced-diabetes and Ginseng radix-treated group. In the STZ-induced-diabetes group, the number of BrdU-positive cells was significantly decreased compared to the control group. There was no significant difference in the number of BrdU-positive cells between the control

Fig. 3. Photomicrograph of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in each group. (A) Control group, (B) non- diabetic and Ginseng radix-treated group, (C) streptozotocin (STZ)-induced-diabetes group, (D) STZ-induced-diabetes and Ginseng radix- treated group. Sections were stained for BrdU (black) and neuronal nuclei (brown). The scale bar represents 100μm.

A

C

B

D

group and the non-diabetic and Ginseng radix- treated group. Significant increase in the BrdU- positive cell population in the dentate gyrus was observed in the STZ-induced-diabetes and Gin- seng radix-treated group compared to the STZ-in- duced-diabetes group. These observations indicate that Ginseng radix increases cell proliferation in the dentate gyrus of diabetic rats (Fig. 4).

DISCUSSION

In the present study, significant weight loss was observed in the STZ-induced-diabetes group.

Previous studies have also reported that body weight was reduced in STZ-induced diabetes (Wu and Lin, 1999; Chaillous et al., 2000). There was no significant difference in weight between the control group and the non-diabetic and Ginseng

radix-treated group. On the other hand, Ginseng radix treatment prevented diabetes-induced weight loss. Chung et al., (2001) reported that application of Ginseng radix to KKAy mice, with type II diabetes mellitus, prevents significant weight loss, which is consistent with the results of the present study.

As for blood glucose levels, Ginseng radix exerted no significant effect under both normal and diabetic conditions. Many studies reported that Ginseng radix reduces blood glucose levels in type II diabetes mellitus (Sotaniemi et al., 1995;

Vuksan et al., 2000a; Vuksan et al., 2000b; Chung et al., 2001), but the results presented in this study showed that Ginseng radix has no signif- icant effect on blood glucose levels in STZ-induced type I diabetes mellitus.

In rats of the STZ-induced-diabetes group, significant reduction in the number of BrdU- positive cells in the dentate gyrus was observed.

Streptozotocin-induced suppression in the number of BrdU-positive cells in the dentate gyrus might be of particular hazard to the hippocampus, a region that has been identified as an important integration center in learning and memory. Dis- crete cognitive impairments as well as subtle structural and neurophysiological changes in brain have been observed in diabetes patients (Araki et al., 1994; Di Mario et al., 1995; Strachan et al., 1997; Kamal et al., 1999). Recent evidences have pointed towards an association between diabetes mellitus and deficits in learning and memory, and it has been reported that STZ-induced diabetes produces a significant reduction in the number of proliferating cells within the dentate gyrus (Jackson-Guliford et al., 2000; Kamal et al., 2000).

In the present results, Ginseng radix had no significant effect on cell proliferation under nor- mal conditions while it enhanced new cell for- Fig. 4. The mean number of BrdU-positive cells in

the hippocampal dentate gyrus. New cell formation was decreased in diabetic rats, and treatment with Ginseng radix counteracted the streptozotocin (STZ)- induced suppression of cell proliferation in the den- tate gyrus. (A) Control group; (B) non- diabetic and Ginseng radix-treated group; (C) STZ- induced-dia- betes group; (D) STZ-induced-diabetes and Ginseng radix-treated group. Values are represented as the mean±S.E.M. * p＜0.05 compared to the control group, ^†p＜0.05 compared to the STZ-induced-dia- betes group.

mation significantly under diabetic conditions.

Saponins are key pharmacological components of Ginseng and they can be classified into three major groups according to their chemical struc- tures: protopanaxadiol (PD; ginsenoside Rb1, Rb2, Rc, and Rd), protopanaxatriol (PT; ginsenoside Re, Rf, Rg1, and Rg2), and oleanolic acid sapo- nins. Ginseng saponin is known to prevent memo- ry impairments in mice follwoing electroconvulsive shocks (Lasarova et al., 1987). Previous studies have reported that ginsenoside Rb1 increases the release of acetylcholine and the number of choline uptake sites in the rat hippocampus (Benishin et al., 1991; Benishin, 1992; Jin et al., 1999) and that ginsenoside Rg1 increases the expression of cho- line acetyltransferase in the basal forebrain (Salim et al., 1997). It is possible that Ginseng saponin exerts its memory-enhancing effects via up-regu- lating the cholinergic system (Jin et al., 1999).

Based on the results presented in the study, it may be suggested that Ginseng radix helps in recovery from the CNS complications of diabetes mellitus. Further studies are needed to clarify the details of the mechanisms of CNS impairments from diabetes mellitus and the possibility of Gin- seng radix-induced functional recovery.

CONCLUSION

The effect of Ginseng radix on cell proliferation in the dentate gyrus of rats with streptozotocin- induced diabetes was investigated via immunohis- tochemistry, in the present study. Aqueous extracts of Ginseng radix was shown to exert no significant effect on weight in the normal rats, while Ginseng radix prevented weight loss in rats with strepto- zotocin-induced diabetes. Cell proliferation in the dentate gyrus of the diabetic rats was increased by

Ginseng radix treatment, but it had no effect on cell proliferation in normal rats. These results suggest that Ginseng radix may help in improve- ment from the central nervous system complica- tions of diabetes mellitus.

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