Kami-untan-to (KUT) improves cholinergic deficits in aged rats

Phytomedicine Vol. 2 (3), pp. 253-258,1996 © 1996 by Gustav Fischer Verlag, Stuttgart· Jena . New York

Kami-untan-to (KUT) improves cholinergic deficits in aged rats 1. VASE, K. TORIIZUKA and H. YAMADA* Oriental Medicine Research Centerof the Kitasato institute, 5-9-1, Shirokane, Minato-ku,Tokyo 108, Japan

Summary The Effects of the kampo (Japanese herbal) medicine ,kami-untan-to (KUT)' on the central cholinergic system and passive avoidance behavior in aged rats (2 years old) were examined. When KUT was orally administered to aged rats, mean latency was significantly increased on passive avoidance test of step through type and choline acetyltransferase (ChAT) activity was increased in the frontoparietal cortex. Oral administration of KUT also increased ChAT mRNA levels in the basal forebrain and nerve growth factor (NGF) mRNA levels in the frontoparietal cortex as demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. These results suggest that oral administration of KUT improves age-related disturbance of memory via increasing effects of ChAT and NGF. KUT may be useful as a potential therapeutic agent in diseases caused by cholinergic deficit such as senile dementia of the Alzheimer type. Key words: aged rats, choline acetyltransferase, Kami-untan-to (KUT), nerve growth factor, Kampo medicine

Introduction Alzheimer's disease is the most popular dementia in elderly people and has been characterized by memory loss and a progressive global impairment of intellect. It has been reported that senile dementia of the Alzheimer type (SDAT) associates with decreased levels of choline acetyltransferase (ChAT: EC 2.3.1.6) activity in the central nervous system (Davis and Maloney 1976; Whitehouse et aI., 1982). Cholinergic neurons in this area innervates to the cortex and hippocampus, and may closely concerned with the cognitive function and memory. The drop out and degeneration of neuronal cells in this area have been considered to be responsible for the several types of dementia including SDAT. We recently reported that a kampo (Japanese herbal) medicine "kami-untan-to" (KUT; Jia-wei-wen-dan-tang in Chinese) was screened as a neurotrophic-like agent by the ChAT activity of rat embryo septal cultures (Yabe et aI., 1995 a). KUT induced the ChAT activity of basal forebrain cultured cells and cerebral cortex of basal forebrain lesioned rats (Yabe et aI., 1995 b). When KUT was orally administered to the ibotenic acid-induced basal forebrain le-

sioned rats, mean latency was significantly increased on passive avoidance test of Step through type (Yabe et aI., 1995 b). Therefore KUT was suggested to be an effective agent for the cholinergic nervous system. The present paper deals with the effect of KUT on memory related behavior and expression of cholinergic-related factors (ChAT and NGF) in aged rats in order to estimate the possible therapeutic efficacy of KUT for the treatment of SDAT.

Materials and Methods

Materials Fetal bovine serum (FBS) was purchased from Bioserum., Australia and horse serum was purchased from cell culture laboratories, U. S. A. [1-14C]Acetyl CoA (0.15 GBq/mmol) was purchased from NEN, U. S. A. Nerve growth factor 2.5 S was purchased from Biomedical Technologies Inc. U. S. A. Ibotenic acid was purchased from Sigma, U. S.A. Medicinal plants used for preparation of KUT were purchased from Uchida Wakan-Yako Co. Ltd. (Tokyo, Japan).

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Preparation of Kami-Untan-To Karni-Untan-To (KUT: ji a-wei-wen-dan-tang in Chinese) con sists of 13 kind s of dried medicinal herbs as follows with dail y dosage given in parenth eses: Pinelliae Tuber (6.0 g, tub er of Pinellia ternata Breit. ), Phyllostachys caul is in Taeniam (3.0 g, sta lk of Phy//ostachys nigra Munro. ), Auranti i Fructus Immature (3.0 g, immature fruit of Citrus aurantium L.), Hoel en (3.0 g, fungus of Poria cocos Wolf.), Auranti i nobilis Pericarp ium. (3.0 g, peel of Citrus unshiu M ark ov.), Glycyrrhizae Radi x (2.0 g, root of Glycyrrhiza glabra L.), Polygalae Radi x (2.0 g, root of Polygala tenuifolia Willd), Scro phulariae Radi x (2.0 g, root of Scrophularia ningpoensis Hemsl.), Ginseng Rad ix (2 .0 g, root of Panax ginseng C. A. M eyer ), Rehm ann iae Radi x (2 .0 g, root of Rehmannia glutinosa Lib.), Z izyphi Fructus. (2.0 g, fruit of Zizyphus jujuba M ill.), Ziz yphi spinosi Semen (2.0 g, seed of Zizyphus jujuba M ill.), and Zingiberis Rhizoma (0.5 g, rh izome of Zingiber officinale Rose.). One day's do sage for human adults consisted of the formulation being mixed with 60 0 ml of distilled wa ter, and the whole mixture was then decocted until the volume wa s reduced to half volume. For anima l administra tion, th e daily dose was controlled corres po nding to a 3-4 fold dose per Kg body weight in human adults by regulating its concentration in relation to wat er consumptio n. For measuring th e ChAT activity several KUT dru g mixtures with drugs of different origin (batches) were prepar ed and ad ministered. The ChAT activities found sho wed no sta tistical significant differenc es.

to the acq uisition test, except that an electric shock was not applied. Two kind s of lat ency were recorded, according to wh ether the body entered the dark room from the head to th e first lumb er level (upper bod y lat ency) or from the head to th e hind limb s (wh ole body lat ency). If the latency per iod was longer th an 300 sec., it was recorded, the exp eriment was sto pped and a value of 390 sec. wa s recorded.

Determination of ChAT activity in cerebral cortex At one month after the passive avoidance test, all rats were sacrificed by decapitation. Th e brain was quickly removed and placed int o liquid nitrogen and kept at -80 °C unt il use. Th e cerebral cor tex was dissected and solubil ized in 25 mM Tris-H CI buff er (pH 7.6) containing 1% TritonX 100 and 5 mM EDTA. Th e solubilizate was taken for the determination of ChAT activity according to the method of Fonnum (1975) .

Analysis of ChAT and N GF mRNA by RT-PCR method

Total RNA was extracted by using an ISOGEN (Nippon Gean , Co . Ltd., Jap an). Th e expressions of ChAT mRNA in basal forebr ain and NGF mRNA in frontopa rietal cortex were detected by RT-PCR method. One microgra m of Tota l RNA was tr an scribed into cDNA using a First Stra nd cDNA synthesis kit (Pha rmacia Biotech, Sweden ) according to the supplier's instructions, and then eDNA was precipitat ed with eth anol. The pellet was resuspended in wat er (50 ul) an d frozen at -20°C until Animals used. Th e result ing eDNA was subjected to peR. Briefly, ~l of the cDNA was amplified in 50 ul of a PCR mixfive M ale Wistar rats (Nih on SLC, Japan) were obtained as ture contai ning 20 mM Tris-H CI (pH 8.0), 25 mM KCI, retired breeders at 6 months of age and were housed for an 1.5 mM MgCI2 , 0.0 5% Tween 20 , 0.2 mM deoxynucleadditio na l 16 months before th e beginning of the experiat es, 1 ~M of specific-primer, and 2.5 unit s oside triphosph ment . Young male control rat s were 6 weeks of age and of Vent DN A polymerase (New England Lab., U. S. A.). hou sed for another 4 weeks before th e beginning of th e tests. All rat s were housed in a lighting sched ule (14 hr of Each 50 ul sample was overla id with 40 ul of mineral oil lighten ing; 10 hr of darkness) an d controlled temperature (Sigma, U. S. A.) and incub ated in a DNA thermal cycler, type PC-700 (ASTEC, Jap an ) for a total of 35 cycles for (24°C) was used in th is experiment . NGF, 40 cycles for ChAT or 33 cycles for cyclophilin. Each cycle consisted of 0.5 min at 94 °C for denaturation, Passive avoidance test (Step-through type) for aged rats 1 min at 60°C for annea ling, and 2 min at 72 °C for exAged rat s (22 months old ) were divided in two groups, tension. A sence prim er an d an antisense primer were synone gro up received drinking wat er, and the other received thesized using the publi shed eDNA sequences for NGF KUT instea d of dr ink ing wat er. Young (2 months old ) rats (Ulrich et aI., 1983), ChAT (Berrard et aI., 198 7) ~-actin received the dr inkin g wa te r as control group. At one month (Alonso et al., 1986) and cyclo philin (Da nielsonet et aI., after the administratio n of KUT, the passive avoidance test 1988) , and were defining a 756 -base for NGF, 206 -base (Step thr ough type) was sta rted. Tra ining commenced with for ChAT, 540-base for ~-actin and 372-base for cyclophithe rat being placed in a sma ll lighted room (11 x 26 x lin tar get seq uences, respectively. The pr imers used for 29 em); the guillotine door was raised. Immediately after amplificatio n were synt hesized on a Model 394 DNA the anima l moved int o a large dark room (31 x 31 x 34 em ), snythesizer (Applied Biosystems). Primer sequences were an elect ric shock (5 rnA) was given via the metal grid floor chosen from separate exo ns of the genes so that the prodfor 3 sec. (acqu isition test). After four acquisition tests wer e uct from eDNA could be distinguished from the product performed, a retent ion test was given nine times during 20 - derived from any contaminat ing genomic DNA. The actudays. Retenti on lat ency was measured in a manner similar al sequences of the oligonucl eot ides were as follows:

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N GF (sense) 5' -GTAATGTCCATGTIGTICTACACTCTG -3' NGF (antise nse) 5'-GGCAAGTCAGC CT CTICTTGTTAGCCTTCC-3' ChAT (sense) 5'-GGAGCCACCT GAGATGTTCATGGAT-3' ChAT (ant isense) 5'-CACAGACGAGGCTCTTTGGCAGCT-3' cyciophilin (sense) 5'-GGTCAACCCCACCGTGTC TTCGA-3 ' cyciophilin (antisense) 5'-AACGGTTAGGTCGGTAAGTCAGAAC-3' ~-actin (sense) 5' -GTGGGCCGCTCTAGGCACCAA-3' ~-a ctin (antis ense) 5' -CTCTTT GATGTCACGCACGATTTC-3' All amp lified cDNA's were stained with ethidium-bromide after agarose gel electropho resis and photograp hed using Polaro id film, type 667 (Polaro id, U. S. A.).

T he digitized band images were processed using an Image processing program (N IH Image, public domain pr ogram, Version 1.47) and normalized against cyciophilin values. Th e an alyzed data were expre ssed as pixel values.

Protein assay Protein concen tration was measured according to th e methods of Brad for d (1976) wit h bovine seru m albumin as a stan dard.

Statistics Statistical significance of difference of means were per forme d by ANOVA followed by Dunn ett's post hoc procedure . The step throu gh latencies were analyzed with th e Kruskal-Wallis test followed by Scheffe's F test.

Results

Analysis of PCR products Relative intensi ties of ethidi um-bromide stained PCR produ cts representing tra nscri pts from each gene in the brain of each rat were ana lyzed by using the optical scann er and th e Image program . Briefly, the image of stained agarose gel was captured using the op tical scanner (EPSON GT-6000, Japan ) with a resolution of 36 0 dots per inch by assigning one of 256 gray level to each dot in the image.

Effect of K UT on passive avoidance test in aged rats In th e first acqu isition trial, th e mean latent period was short in th ree groups; young control rats, aged rats and KUT administered aged rat s. After four acquisition tri als, nine retent ion trials wer e given during 20 days. No significant statistical difference was observed among the th ree gro ups in the acqui sition tria ls. Any cha nges in gross behavior were also not observed . In th e retention trials,

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however, aged rats significantly decreased mean latency (Fig. 1 A) and numbers of rats staying in the light room over 150 sec (data not shown). At the 12th trials, the mean latencies of mature rats and aged rats were 241.3 ± 39.1 sec. and 98.7 ± 34.1 sec., respectively but that of KUT administrated aged rats was 215.5 ± 42.7 sec. (Fig. 1 B).

Effect of KUT on ChAT activity in frontoparietal cortex and ChAT mRNA expression in basalforebrain At two weeks after the end of the passive avoidance test, ChAT activity in the frontoparietal cortex was measured (Fig. 2). Aged rats significantly decreased the ChAT activity compared with mature control rats. However, when KUT was administrated to aged rats orally, enhanced ChAT activity was observed in the frontoparietal cortex of the rats. In order to confirm the induction of ChAT by KUT administration, the expression of ChAT mRNA was determined using RT-PCR method. The ChAT mRNA expression in basal forebrain of aged rats was decreased compared with mature control rats (data not shown). However the ChAT mRNA expression in basal forebrain of the KUT administered aged rats had a tendency to increase compared with aged rats (Fig. 3 A). This induction was measured by an Image processing program (NIH image), and was estimated to be about 1.8-fold higher than the level of aged rats (Fig. 3 B).

The acetylcholine mediated neuronal system is known to play an important role in higher cognitive functions, especially memory and learning. SDATand aging are associated with a degeneration of cholinergic neurons (Bartus et al., 1982). Ogawa et al. (1987) have already reported that the ChAT activity in the brain is decreased in aged rats and this decrease was correlated with reduced learning ability in aged rats. In the present study, impaired memory acquisition in the passive avoidance test and the reduction of ChAT activity in the frontoparietal cortex (Fig. 2) were also demonstrated in aged rats. Therefore, if induction and/or enhancement of ChAT activity in central nervous neurons are possible, there may be improvment of damaged cognitive function in SDAT and/or aging. Recently, it has been reported that some Kampo medicines and herbal extracts improve the lesion of cognitive function in the dementia animal model (Kishikawa et al., 1993; Fujiwara and Iwasaki, 1993; Zhou et al., 1992). Moreover, the clinical application of some Kampo medicines for patients with DAT improves their memory related behavior and intellectual function (Mizushima et al., 1989; Yamamoto et al., 1989; Yamamoto, 1991). These observations suggest that some Kampo medicines and herbal extracts have therapeutic efficacies for SDAT and/or senile forgetfulness. In order to find an effective drug for dementia, we recently screened for ChAT-enhancing activity in 52 kinds of Kampo formulas on septal cultured neurons using ChAT activity as a marker. Of the formulas tested, only one of the kampo formulas (KUT) increased ChAT activity in septal cultures significantly (Yabe et al., 1995 a). Furthermore, we have already reported that oral administration of KUT increased ChAT activity in the frontoparietal cortex and attenuated memory disturbance in basal forebrain-lesioned rats (Yabe et al., 1995 b). When several KUT preparations, which were prepared independently, were examined on ChAT activity, significant enhanced activities without great differences were observed in all KUT preparations (data not shown). The present results demonstrate that oral administration

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of KUT was increased ChAT activity in the frontoparietal cortex and ChAT mRNA expression in the basal forebrain of aged rats. These results are compatible with our previous observations (Yabe et al., 1995 a and b). KUT had no direct effect on the ChAT enzyme reaction (Yabe et al., 1995 b). These results suggest that ChAT enzyme level in frontoparietal cortex of aged rats was increased after oral administration of KUT. The positive effect of KUT on memory-related behavior of aged rats may be due to an increased level of synthesis of acetylcholine in the frontal cortex by induction of ChAT enzyme. Since NGF has been shown to increase ChAT enzyme levels, induction of NGF has generally been considered to be an essential step of ChAT induction. NGF is a targetderived neurotrophic factor which affects differentiation, survival and function of cholinergic neurons in basal fore-

brain (Thoenen., 1991). Therefore, it has been suggested that the administration of NGF improves some of the biological abnormalities that occur in disorders such as SDAT. However, there is a problem that oral administration of NGF has no effect in the central nervous system neuron, because NGF is a high molecular weight protein that can not pass through the blood-brain barrier. Therefore, an agent with neurotrophic-like activity, or stimulator of NGF-synthesis in the central nervous system, which can pass through the blood brain barrier, may have a possibility of becoming a therapeutic agent for dementia. The present results indicate that oral administration of KUT increased NGF mRNA levels in the frontoparietal cortex. It was also suggested that the effects of KUT on ChAT activity and passive avoidance behavior of aged rats was due to a stimulatory effect of NGF synthesis in the fron-

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toparietal cortex. In order to clarify the precise mechanism of KUT for the cholinergic system and the active ingredients for a neurotrophic-like effect, further experiments are in progress by this research group. In conclusion, the present results show that KUT improves age-related memory deficits and increases ChAT activity and NGF mRNA in aged rats. These results suggest that KUT is a neurotrophic-like agent which is directly and/or indirectly effective on the cholinergic nervous system. KUT may be useful as a potential remedy for treatment of the patients with SDAT and senile forgetfulness. Acknowledgement This work was supported in part by a research fund from Japan foundation for Aging and Health. References Alonso, S., Minity, A., Bourlet, Y. and Buckingham, M.: Comparison of three actin-coding sequences in the mouse; Evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23: 11-12, 1986. Bartus, R. T., Dean III, R. L., Beer, B. and Lippa, A. S.: The cholinergic hypothesis of geriatric memory dysfunction. Science 217: 408-417,1982 Berrard, S., Brice, A., Lottspeich, E, Braun, A., Barde, Y.A. and Mallet, ].: cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein. Proc. Natl. Acad. Sci. USA. 84: 9280-9284, 1987 Bradford, M.: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of proteindye binding. Anal. Biochem. 72: 248-254,1976. Davis, P. and Maloney, A.]. E: Selectiveloss of central cholinergic neurons in Alzheimer's disease. Lancet 2: 1403, 1976 Danielson, P.E., Forss-Petter, S., Brow, M. A., Calavetta, L., Douglass, ]., Milner, R.]. and Sutcliffe,]. G.:p IBI5: a cDNA clone of the rat mRNA encoding cyclophilin. DNA, 7: 261-277,1988 Fonnum, E: A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24: 407-409, 1975. Fujiwara, M. and Iwasaki, K.: Toki-Syakuyaku-san and Orengedokuto improve the disruption of spatial cognition induced by ce-

rebral ischaemia and central cholinergic dysfunction in rats. Phytotherapy Res. 7: S60-S 62, 1993. Kishikawa, M., Nishimura, M.,Sakae, M. and Iseki, M.: The learning ability and mobility of senescence accelerated mice (SAM-P/1) treated with Toki-Syakuyaku-san. Phytotherapy Res. 7: S 63-S 66, 1993. Mizushima, N. and Ikeshita, T.: The effect of Toki-Shakuyaku-san on the senile dementia. ]pn. J. Med. Pharm. Soc. Wakan-Yaku 6: 456-457, 1989. Ogawa, N., Mizukawa, K. and Sora, 1.: Chronic dihydroergotoxine administration increases muscarnic cholinergic receptor binding in aged-rat brain. Res. Commun. Pharmacal., 57: 149-159,1987. Thoenen, H.: The changing scene of neurotrophic factors. Trends Neurosci., 14: 165-170, 1991. Ulrich, A., Gray, A., Berman, C. and Dull, T.].: Human beta-nerve growth factor gene sequence is highly homologous to that of mouse. Nature, 303: 821-825, 1983. Whitehouse, P.j, Price, D. L., Strube, R. G., Clark, A. w., Coyle,]. T. and De long, M. R.: Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science, 215: 1237-1239, 1982. Yabe, T., Toriizuka, K. and Yamada, H.: Effects of Kampo medicines on choline acetyltransferase activity in rat embryo septal cultures. J. Trad. Med. (former name: ]pn. Med. Pharm. Soc. Wkan- Yaku), 12: 54-60, 1995 a. Yabe, T., Toriizuka, K. and Yamada, H.: Choline acetyltransferase enhancing effects of kami-untan-to (KUT) on basal forebrain cultured neurons and lesioned rats. Phytomedicine, 2: 41-46, 1995b. Yamamoto, T. and Kawano, K.: Kampo-treatment for dementia of Alzheimer's type. ]pn.]. Pharm. Soc. Wakan-Yaku 6: 454-455, 1989. Yamamoto, T.: Kampo therapy for dementia. ]pn. ]. Med. Pharm. Soc. Wakan-Yaku 8: 478-479,1991. Zhou, Y., Saito, H. and Nishiyama, N.: Effects of Acarus gramineus Soland on leaning and memory performances in mice. Shoyakugaku Zasshi 46: 103-108, 1992.

Address Haruki Yamada, Ph. D., Oriental Medicine Research Center, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan.