J. Exp. Biomed. Sci. 14 (2008) 19 26 Antidiabetic, Antioxidative and Renoprotective Effects of Rehmanniae Radix preparata Extract in Streptozotocin-induced Diabetic Rats Hye-Jeong Kim 1, In-Sook Yoon 2 and Young-Chul Kim 1, 1 Department of Public Health, Keimyung University, Daegu 704-701, Korea. 2 Department of Clinical Pathology, Daegu Health College, Daegu 702-722, Korea This study investigated the effect of Rehmanniae Radix preparata extract on the antioxidant enzymes of kidney and renal function in streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were divided into three groups including normal control (NC), diabetic control (DC), and diabetic treatment with Rehmanniae Radix preparata (DRR). Over a 4-week study period, Rehmanniae Radix preparata aqueous extract was administered orally at 1124 mg/kg BW/day. The serum glucose level in the DRR group was significantly lower (P<0.05) than the DC group. The serum blood urea nitrogen in diabetic groups was significantly higher (P<0.001) than the NC group. The urinary total protein level in the DRR group was significantly lower (P<0.05) than the DC group. The renal xanthine oxidase activity in the DRR group was significantly lower (P<0.01) than the DC group. The renal catalase activity in the DC group was significantly lower (P<0.05) compared to the NC group and that was significantly higher (P<0.05) in the DRR group than the DC group. In conclusion, these results indicated that Rehmanniae Radix preparata can prevent or retard the development of diabetic nephropathy via its beneficial effects for correcting the hyperglycemia and favorable effects on antioxidant enzyme system. Key Words: Rehmanniae Radix preparata, Streptozotocin, Diabetic rat, Antioxidant effect, Renoprotective effect 서 현대과학의큰발전에도불구하고당뇨병의발병률은증가하고있으며그예방과치료에대한관심이집중되고있다. 당뇨병은췌장에있는 Langerhans 섬의 β-cell 에서분비되는 insulin의생리작용이저조하거나 insulin receptor의수가적어 insulin의생리적기능이불충분할때나타나는혈당증이다 (Rayfield and Ishimura, 1987; Nepom, 1990). 당뇨병은만성퇴행성질환으로신증, 신경변증망막증의미세혈관합병증과동맥경화, 고혈압, 심근경색등의대혈관합병증 (West et al., 1983) 과같은여러합병증을유발한다. 당뇨합병증의유발요인은고혈당의지속화와만성화로여러가지자유라디칼 (free radical) 의생산이증가되 * 논문접수 : 2008 년 2 월 15 일수정재접수 : 2008 년 3 월 15 일 교신저자 : 김영철, ( 우 ) 704-701, 대구광역시달서구신당동 1000, 계명대학교자연과학대학공중보건학과 Tel: +82-53-580-5931, Fax: +82-53-588-5233 e-mail: yckim@kmu.ac.kr 론 고, 반응성이높은이들에의해세포막인지질의산화에의한조직의과산화적손상으로혈관내피세포가손상되면서각종혈관성합병증이발생된다고보고 (Hammers et al., 1991) 되고있다. 인체는정상적인생리상태에서는자유라디칼의생산과항산화방어체계 (antioxidant defense system) 의활성이균형을이루고있으며체내에서자유라디칼이과다생성되거나혹은항산화방어계의기능이감소되어균형이깨지면서산화적스트레스가일어난다 (Lawrence et al., 2001). 고혈당은신장조직에산화스트레스를크게유발시키게되는데이는신장에서고혈당으로인해촉진되는단백질과지질의비효소적당화로유리기가과다하게생성되기때문이다 (Mohamed et al., 1999). 당뇨병환자에서신증의합병증발생률은인슐린의존형당뇨병환자의경우는 30% 정도이며인슐린비의존형당뇨병환자의경우는 15~60% 로알려져있다 (Krolewski et al., 1985). 당뇨병성신장병변은말기신부전의주요한원인으로신장의사구체여과계를손상시켜과여과가발생하며 (Jensen et al., 1981) 과여과는사구체상피세포의변화를일으켜단백뇨와신피질간질에단백질침착을유도하고간질의 - 19 -
비대, 사구체경화등으로진행되어간다 (O'Donnell et al., 1988). 고혈당을조절함과더불어과산화적스트레스를억제시키는것이당뇨및합병증을예방하기위해서필수적이며현대의학에서인슐린에의한당뇨병치료는부작용이수반되기때문에최근에는천연물이나한약재로부터당뇨병의예방과억제를위한생리활성물질을찾으려는연구들이시도되고있다. 한약에서보약으로알려진지황 (Rehmannia glutinosa) 은현삼과에속하는다년초로써그뿌리를약으로사용한다. 채취후파지에저장한생지황, 외피를죽도로벗겨서양건한건지황, 생지황을구증구폭한숙지황 (Rehmanniae Radix preparata) 등이있으며그중약재로는숙지황이가장잘알려져있으며효능도높은것으로나타나있다. 숙지황은補腎長壽, 生精血, 補血등에효과가있을뿐만아니라신혈관확장과강심작용및이뇨작용이있으며장기투여시혈압하강에효과가있다고보고되었다 (Lee et al., 1981). 또한숙지황이독성약물에의해손상당한신장세포의시험관내실험에서항산화효과가있다고보고되었으며 (Cho, 2003), STZ로유도한당뇨쥐의신장조직에서지질과산화물 (TBARS) 함량을낮춘연구보고 (Yokozawa et al., 2004) 는있었으나신장조직에서의유해산소해독계효소의활성과신기능에대한보고는없었다. 이연구에서는 streptozotocin (STZ) 을투여한인슐린의존성당뇨병실험모델동물에숙지황추출물을투여하여식이효율, 혈당, 신장기능및신장조직의항산화효소의변화를관찰함으로서숙지황의당뇨병개선및신장보호효과에대한과학적인근거자료를제공하고자하였다. 재료및방법 1. 시약및기기혈청의 glucose, BUN, creatinine, urinary creatinine는 commercial kit (EIKEN, Japan) 를이용하여 Konelab 20XT Analysis System (Thermo, Finland) 으로측정하였고 insulin 농도는 ELISA kit (Mercodai, USA) 를이용하여 microplate reader (Molecular devices, USA) 로측정하였다. 뇨중 total protein은 commercial kit (Auto kit Micro TP, Wako Pure Chemicals, Japan) 를이용하여 ADVIA 1650 Analysis System (BAYER, USA) 으로측정하였다. 실험기기는 UV spectrophotometer (UV-1601, Shimazu, Japan), refrigerated centrifuge (Beckman Coulter, Beckman, USA), glass teflon homogenizer (T25 basic, Ultra Turrax, Japan), 혈당계 SUPERGLUCOCARD TM II (ARKRAY, Japan) 를사용하였다. 2. 시료의추출및투여전북군산에서유기농재배된숙지황 500 g을구입하여 3차증류수 6 l를가하여초고속진공저온추출기 (COSMOS-660, 경서기계산업, 인천 ) 로 100 의온도에서 2시간 30분열수추출하였다. 열수추출물을진공농축용기에담아 110 온도에서 1.5 l로농축하고 1일투여할용량씩파우치처리하여냉장보관하여실험에사용하였다. 수율은동결건조로측정한결과 33.7% 였다. 추출물의경구투여는위존데를사용하여 10 ml/kg BW/day (1,124 mg/kg BW/day) 로주 6일 4주간투여하였고대조군은동량의증류수를투여하였다. 3. 실험동물실험동물은 6주령 Sprague-Dawley 웅성 SPF 랫드를 ( 주 ) 바이오코리아로부터구입하여사육실 ( 온도 22±1, 상대습도 50±5%) 에서고형사료를먹여 1주일적응시킨후난괴법에의해정상군 (Normal Control; NC), 당뇨대조군 (Diabetic Control; DC), 당뇨숙지황군 (Diabetic Rehmanniae Radix preparata; DRR) 으로나누어각군당 7 마리씩 4주간사육하여실험하였다. 사료및물섭취량, 체중은주 1회, 혈당은주 1회아침 9:00~10:00시에측정하였다. 4. 당뇨유발인슐린의존성당뇨병과유사한실험동물을만들기위하여 streptozotocin (STZ, Sigma Co., USA) 을 0.4 M citrate buffer (ph 4.5) 용액에용해시켜 70 mg/kg (0.5 ml/100 g BW) 으로복강내주사하였다. 당뇨유발확인은 STZ 주사 72시간후측미정맥에서채혈하여혈당량이 300 mg/ dl 이상이면당뇨병이유발된것으로간주하였다. 5. 시료채취및실험동물처치실험추출물투여전안와정맥에서채혈하여혈청분리후 glucose 측정에사용하였고실험추출물투여 26일후실험쥐를대사케이지로옮겨 24시간동안뇨를수집하여 total volume, total protein과 urine creatinine 측정에사용하였다. 실험추출물투여 28일째날에실험사육한랫드를 - 20 -
14시간동안절식시킨후 ether 마취하에복부대동맥에서혈액을채취하여 3000 rpm에서 30분간원심분리하여혈청을분리한후 glucose, BUN, creatinine, insulin 측정에사용하였다. 장기는채혈후즉시적출하여생리식염수로씻어내고수분을여과지로제거한후무게를칭량하였다. 적출한신장조직을칭량한후 4배량의 0.25M sucrose 용액을넣고 glass teflon homogenizer를이용하여 20% (w/v) 마쇄균질액을만들었다. 이균질액을 600 g에서 10분간원심분리하여핵및마쇄부분을제거한상층액을 10,000 g 에서 20분간원심분리한후 pellet인 mitochondria 분획과그상층액인 post-mitochondria 분획을얻어효소활성측정시료로사용하였다. 6. 신장조직의효소활성도측정 1) Xanthine Oxidase (XO) XO의활성도는 xanthine을기질로사용하여 30 에서 20분간반응시켜생성된요산을 292 nm에서흡광도를측정하는 Stripe와 Della (1969) 의방법에준하여측정하였다. 활성도단위는효소액중에함유된단백질 1 mg이 1분동안반응하여기질인 xanthine으로부터생성된 uric acid의양을 nmole로표시하였다. 2) Superoxide dismutase (SOD) SOD의활성도는 hematoxylin 자동산화의억제정도를관찰하는 Martin 등 (1987) 의방법에따라 0.1 mm EDTA가함유된 50 mm 인산완충액 (ph 7.5) 에 10 µm hematoxylin 및효소액을가해 25 에서반응시켜생성된 hematein을 560 nm에서측정하여효소의활성도를산정하였다. 활성도단위는효소액을넣지않은반응액중의 hematoxylin 자동산화를 50% 억제하는정도를 1 unit로하여단백질 1 mg이 1분동안반응한 unit로표시하였다. 3) Catalase (CAT) CAT 활성도는 hydrogen peroxide를기질로하여환원되는정도를파장 240 nm에서흡광도를읽고분자흡광계수 (E=0.04 mm -1 cm -1 ) 를이용하여활성을산출하는 Aebi (1974) 의방법에준하였다. 활성도단위는간조직중에함유된단백질 1 mg이 1분동안반응하여감소되는 hydrogen peroxide의양을 nmole로표시하였다. 4) Glutathione S-transferase (GST) GST의활성도는 Habig 등 (1974) 의방법에따라측정하였다. 1-chloro-2,4-dinitrobenzene과 glutathione을기질로하여 25 에서 10분간반응시켜생성된 2,4-dinitrobenzeneglutathione conjugate 양을 340 nm에서측정하였다. 활성도단위는효소반응액중에함유된단백질 1 mg이 1분간반응하여생성시킨 conjugate의양을 nmole로나타내었다. 5) 신장조직의단백질함량측정신장조직중단백질함량은 Lowry 등 (1951) 의방법에따라 bovine serum albumin을표준품으로사용하여측정하였다. 7. 자료분석 SPSSWIN (v12.0) 통계프로그램을이용하여 NC군, DC 군, DRR군간의분석자료의비교를위하여 t-test로분석하였다. 통계적유의수준은 0.05이하로하였다. 결과 1. 물, 식이섭취량및식이효율음수량은 NC군에비해 DC군이약 6배유의하게 (P< 0.001) 높았고식이섭취량은약 2배유의하게 (P<0.001) 높았으며음수량은 DRR군이 DC군에비해유의하게 (P<0.01) 낮았고식이섭취량은 DRR군과 DC군간에유의한차이가없었다. 일일체중변동량은 NC군이증가추세를보였고 DC군이감소하는현상을보였으나 DRR 군은 NC군에비해증가폭은적었으나증가추세를보였으며 DC군에비해 230% 유의하게 (P<0.05) 높게나타났다. 식이효율또한 NC군에비해 DC군이유의하게 (P<0.001) 낮았으나 DC군에비해 DRR군은 230% 유의하게높게나타났다 (P<0.05) (Table 1). 2. 혈당및혈청 glucose, insulin, BUN, creatinine 농도혈중포도당농도변화는 Fig. 1과같다. 혈당치는전실험기간동안 NC군에비하여 DC 및 DRR군은 5~6배현저하게높았으며, 실험 4주후 DC군에비하여 DRR군은 14% 유의하게낮았다 (P<0.05). 혈청중 glucose 농도는당뇨군의경우초기혈당치에비하여유의하게증가하였으며 DC군에비하여 DRR군은 20% 유의하게낮았다 (P<0.05) (Fig. 2). Insulin 농도는 NC군에비하여 DC군이 88% 유의하게 (P<0.001) 낮았으며 DC군에비하여 DRR군은 30% 높았으나통계적으로유의하지않았다. BUN 농도는 NC군에비하여 DC군이 135% 유의하게 (P<0.001) 높았으며 DC군에비하여 DRR군은다소감소하는경향을보였다. Creatinine 농도 - 21 -
Table 1. Water intake, food intake, body weight gain and food efficiency ratio of normal and diabetic rats fed with Rehmannia Radix preparata extract for 4 weeks Items Groups a) Water intake (ml/day) 49.40±3.85 c) 294.29±20.64 ### 260.71±16.63 ###** Food intake (g/day) 27.40±2.30 51.00±3.65 ### 50.71±1.60 ### Body weight gain (g/day) 3.15±0.29-0.34±0.29 ### 0.45±0.52 ###* Food efficiency ratio b) (%) 11.56±1.49-0.67±0.61 ### 0.89±1.04 ###* a) NC: Normal control, DC: Diabetic control, DRR: Diabetic Rehmannia Radix preparata. b) Food efficiency ratio=(body weight gain/ food intake) 100. c) Values are mean ± SD of 7 rats. The value with a sharp-note is significantly different from NC group by t-test ( ### ; P<0.001). The value with an asterisk is significantly different from DC group by t-test ( * ; P<0.05, ** ; P<0.01). Fig. 1. Change in blood glucose of diabetic rats fed with Rehmannia Radix preparata extract for 4 weeks. Values are mean ± SD of 7 rats. The value with an asterisk is significantly different from DC group by t-test ( * ; P<0.05)., NC;, DC;, DRR. 는각군간에별다른차이를보이지않았다 (Table 2). 3. 뇨량및뇨중 total protein, creatinine clearance 24시간뇨량은 NC군에비하여 DC군이약 13배유의하게 (P<0.001) 높았으며 DRR군과 DC군간에는별다른차이를보이지않았다. 뇨중 total protein은 NC군에비하여 DC군은 150% 유의하게 (P<0.01) 높았으나 DC 군에비하여 DRR군은 24% 유의하게낮았다 (P<0.05). Creatinine clearance는 NC군에비하여 DC군이 15% 낮았으며 DC군에비하여 DRR군은 17% 높았으나통계적으로유의하지않았다 (Table 3). 4. 장기무게 간과신장의상대적무게는 NC군에비하여 DC군이 1.5~2배유의하게 (P<0.001) 높았으며 DC군과 DRR군간에는유의한차이가없었다. 비장 (P<0.001) 과심장 (P<0.01) 의절대무게는 NC군에비하여 DC군이유의하게낮았으며상대무게에서비장은유의한차이가없었으나심장은 DC군이 NC군에비해유의하게높았다 Fig. 2. Change in serum glucose of normal and diabetic rats fed with Rehmannia Radix preparata extract for 4 weeks. Values are mean ± SD of 7 rats. The value with a sharp-note is significantly different from NC group by t-test ( ### ; P<0.001). The value with an asterisk is significantly different from DC group by t-test ( * ; P< 0.05)., 0 week;, 4 weeks. Table 2. Change in serum glucose, insulin, BUN, creatinine of normal and diabetic rats fed with Rehmannia Radix preparata extract for 4 weeks Weeks (P<0.05) (Table 4). Groups Glucose 167.00±7.04 668.40±41.29 ### 538.00±103.78 ###* Insulin 156.41±80.80 18.14±1.24 ### 23.49±7.93 ### BUN 17.98±2.20 42.25±8.14 ### 40.51±11.69 ## Creatinine 0.58±0.08 0.55±0.08 0.53±0.05 Unit: mg/dl, BUN: Blood Urea Nitrogen. Values are mean ± SD of 7 rats. The value with a sharp-note is significantly different from NC group by t-test ( ## ; P<0.01, ### ; P<0.001). The value with an asterisk is significantly different from DC group by t-test ( * ; P<0.05). 5. 신장조직의유해산소대사효소활성도변동 XO 활성은 NC 군에비하여 DC 군이 27% 높았으며 DC - 22 -
Table 3. Urine volume, urine protein and creatinine clearance of normal and diabetic rats fed with Rehmannia Radix preparata extract for 4 weeks Items Groups Urine volume (ml/kg/day) 64.13±12.76 815.52±75.17 ### 859.30±90.83 ### Urine total protein (mg/day) 16.04±3.88 40.35±5.38 ## 30.54±3.72 ##* Creatinine clearance a) (ml/min) 3.00±0.80 2.54±0.75 2.98±0.30 a) Creatinine clearance: (urine creatinine/serum creatinine) (total volume/24h 60 min). Values are mean ± SD of 7 rats. The value with a sharp-note is significantly different from NC group by t-test ( ## ; P<0.01, ### ; P<0.001). The value with an asterisk is significantly different from DC group by t-test ( * ; P<0.05). Table 4. Organ weight of normal and diabetic rats fed with Rehmannia Radix preparata extract for 4 weeks Organs Liver 군에비하여 DRR군은 33% 유의하게낮았다 (P<0.01). SOD 활성은 NC군에비하여 DC군이 200% 유의하게 (P<0.001) 높았으며 DC군과 DRR군간에는유의한차이가없었다. CAT 활성은 NC군에비하여 DC군이 27% 유의하게 (P<0.05) 낮았으며 DC군에비하여 DRR군은 48% 유의하게높았다 (P<0.05). GST 활성은 NC군에비하여 DC군이 28% 유의하게 (P<0.05) 높았으며 DC군과 DRR 군간에는유의한차이가없었다 (Table 5). 고 Groups 10.796±1.118 a) 9.267±0.955 # 9.883±1.400 2.999±0.228 b) 4.463±0.341 ### 4.491±0.377 ### Spleen 0.722±0.073 0.424±0.108 ### 0.496±0.152 # 0.201±0.015 0.201±0.027 0.223±0.051 Kidney 1.206±0.105 1.310±0.131 1.396±0.156 # (right) 0.336±0.033 0.631±0.052 ### 0.637±0.055 ### Kidney 1.210±0.106 1.387±0.121 # 1.424±0.140 # (left) 0.337±0.032 0.668±0.042 ### 0.650±0.049 ### 1.094±0.145 0.726±0.126 ## 0.761±0.091 ## Heart 0.304±0.036 0.347±0.031 # 0.346±0.021 # a) Absolute organ weight, Unit: g. b) Relative organ weight, Unit: g/100 g body weight. Values are mean ± SD of 7 rats. The value with a sharp-note is significantly different from NC group by t-test ( # ; P<0.05, ## ; P<0.01, ### ; P<0.001). 생명과학의큰발전에도불구하고당뇨병의발병율과당뇨합병증으로인한사망률이증가함에따라그예방과치료에대한관심이집중되고있으나현대의학에서도아직근원적인치료법이개발되지못한실정이다. 다만혈당유지를위하여약물요법, 운동요법, 식사조절방법등이주로시행되어지고있다 (Koivisto, 1993). 주된치료법 찰 Table 5. Effect of Rehmannia Radix preparata on kidney SOD, CAT, XO, GST activities in STZ-induced diabetic rats Enzymes Groups XO a) 1.64±0.35 2.08±0.52 1.39±0.17 ** SOD b) 2.18±1.34 6.59±0.49 ### 6.48±1.54 ## CAT c) 4.21±0.59 3.08±0.90 # 4.55±1.40 * GST d) 21.48±3.52 27.55±3.72 # 27.90±1.89 # a) Unit: nmole uric acid formed/mg protein/min. b) Unit: U (50% inhibition of autoxidation of hematoxylin)/mg protein/min. c) Unit: nmole H 2 O 2 reduced/mg protein/min. d) Unit: nmole 2,4-dinitrobenzene-glutathione conjugate/mg protein/ min. Values are mean ± SD of 7 rats. The value with a sharp-note is significantly different from NC group by t-test ( # ; P<0.05, ## ; P< 0.01, ### ; P<0.001). The value with an asterisk is significantly different from DC group by t-test ( * ; P <0.05, ** ; P<0.01). 인인슐린투여는근원적으로당뇨를치료하는데한계가있고부작용의위험도수반하고있어최근에는전통적인천연물인한약재나천연기능성식품을이용한연구가활발히진행되고있다. 이러한연구동향에맞추어이연구에서는신을보하고소갈치료처방에주로사용되는한약재숙지황을열수추출하여 STZ (70 mg/kg BW, i.p.) 로유발된당뇨쥐에서혈당과신기능계에미치는영향을살펴보고자 SD랫드 (6주령) 수컷을 7마리씩 3군 (NC, DC, DRR) 으로나누어추출물을 1124 mg/kg BW/day 용량으로 4주간경구투여하였다. 4주간의실험을통하여얻어진연구결과에서음수량, 식이섭취량이정상군에비해당뇨군에서현저하게높게나타났으며숙지황추출물로인한음수량과식이섭취량변화는관찰되지않았다. 혈당이올라가면소변으로걸러진포도당의일부는재흡수가일어나지않으며이렇게빠져나온포도당은체내수분을같이끌고체외로배출되기때문에당뇨에서는다뇨와다갈의증상이나타나게된다. 식이효율과체중증가율은정상군에비하여당뇨군에서현저하게감소하였으나당뇨대조군에비해 - 23 -
숙지황투여군에서유의하게높게나타났다 (P<0.05). 당뇨군에서식이섭취량이많음에도불구하고지속적인체중감소가나타났는데이는당뇨에의한체내대사의퇴행적인변화때문인것으로보여지며 STZ에의해췌장의 β-cell이파괴되어당대사의불균형을초래한당뇨쥐는체중이쉽게회복되지않는다는연구보고 (Lee et al., 1994) 가이를뒷받침해준다. 당뇨대조군에비해숙지황투여군에서유의하게식이효율과체중이증가를보여숙지황이당뇨로인한체중감소를개선하는효과가있음을시사해준다. 현재인슐린의존성당뇨병의연구에사용되는 streptozotocin (STZ) 약물은췌장 Langerhans' islet의 β-cell만을선택적으로파괴하여인슐린결핍을초래함으로써포도당에대한 β-cell의예민도를저하시켜고혈당을초래하는것으로알려져있다 (Matkovics et al., 1998). 이연구에서혈중포도당농도를측정한결과 STZ로유도된당뇨군은정상군에비하여 5~6배유의한증가를보였으며실험 4주후당뇨대조군에비하여숙지황투여군은 20% 유의하게감소를보였다 (P<0.05). 혈청중 glucose 농도또한숙지황투여군에서당뇨대조군에비하여 24% 유의하게감소한것으로나타나 (P<0.05) 숙지황이혈당강하효과가있는것으로관찰된다. 혈청인슐린농도를측정한결과 STZ로유도된당뇨군은정상군에비하여유의한감소를보였으며숙지황투여군에서다소증가를보였으나유의한차이는없었다. 혈중 creatinine은사구체여과율과 creatinine 배설률즉 creatinine clearance와직접적인상관관계를가지고있으며신피질간질의섬유화에비례하여높아진다. BUN (Blood urea nitrogen) 은단백질대사의주된최종산물이며간에서암모니아로부터합성되어신장으로배설되므로단백질의섭취와신장의배설능력을반영하는것으로 creatinine과 BUN은신장기능지표로사용된다 (Grover et al., 2003). 고혈당으로인한사구체상피세포의손상과밀도의변화는여과기능의장애를초래하며선택적투과성이없어져당뇨병성신증의진행과단백뇨를증가시킨다 (Dalla Vestra et al., 2003). 이연구에서정상군에비하여당뇨군의혈중 BUN 농도는 135% (P<0.001), 24시간 urine volume은약 13배 (P<0.001), urine total protein은 150% (P<0.01) 각각유의하게증가하여신장에문제가있음을시사하였다. BUN과 creatinine은당뇨대조군에비하여숙지황투여군에서다소감소를보였으나유의한차이는없었다. 1일소변으로배출된 total protein 량은 당뇨대조군에비하여숙지황투여군에서 24% 유의하게감소하였으며 creatinine clearance는유의하지는않았으나당뇨대조군에비하여 17% 증가하여숙지황이신기능을개선시킬것으로기대된다. 장기의상대적중량비교에서간과신장의경우정상군에비해당뇨군이유의하게 (P<0.001) 증가하였으며당뇨대조군과숙지황투여군간에는유의한차이가없었다. 당뇨군에서간무게증가는체내인슐린저하로인해당대사가정상적으로진행되지않아간내에지질이축적됨으로써간이비대해지는것으로보고되어져있다 (Dai et al., 1994). 신장무게의증가는고혈당으로인해 RNA 및 DAN의합성을증가시킴으로써신장의세포분열을촉진시켜신장이비대해진다고보고 (Dai et al., 1994) 되어져있으며사구체여과율의증가와함께크기와용적이증가하기때문이라는연구보고도있다 (Gallaher et al., 1993). 당뇨병상태에서생긴산화적스트레스는생체대사과정중에 oxygen free radical을생성하여세포에상해를주며특히세포막의다가불포화지방산에작용하여지질과산화물을끊임없이생성하고이로인해세포의기능손상을초래한다 (Morel and Chisolm, 1989). XO는생체조직에존재하면서산소자유라디칼 (oxygen free radical) 생성계효소로알려져있으며이효소에의해생성된 oxygen free radical 중세포내호흡부산물로생성되는 superoxide anion radical (O 2 -) 은세포상해의직접적인원인으로작용하는것으로알려져있다. Superoxide anion radical은 SOD 에의해 hydrogen peroxide (H 2 O 2 ) 로전환되며이것은다시 CAT 및 GPX에의해서 H 2 O로전환되어무독화되고 (Deisseroth and Dounce, 1970), GST도유해산소해독에관여하는것으로알려져있다. 신장조직의 XO, SOD, GST 활성이정상군에비해당뇨대조군에서높게나타난이연구결과는 STZ로유도된당뇨쥐에서 oxygen free radical 의생성이증가하여이로인한지질과산화물의증가로산화적손상이가속화되고이를방어하기위해생체내유해산소해독계효소들의활성이높아진것으로해석된다. 숙지황투여군에서당뇨대조군에비하여 XO 활성이유의하게 (P<0.01) 낮게나타남으로써숙지황이 oxygen free radical 생성을낮추었음을가늠케한다. CAT 활성이정상군에비하여당뇨대조군에서유의하게 (P<0.05) 감소한연구결과는당뇨로인한 oxygen free radical과지질과산화물제거를위해 SOD의활성이높아지고세포소기관들의산화적손상이가속화되어유해대사산물의제거하기위해 CAT 효소가소모된결과로해석된다. 숙지황 - 24 -
투여군에서당뇨대조군에비하여 CAT 활성이유의하게 (P<0.05) 높게나타남으로써숙지황이산소자유라디칼생성을낮추고이로인해해독계방어효소의활성도가높게나타난것으로보아진다. 정확한기전규명을위해서는시간경과에따른추가적인실험이필요하리라생각된다. 이상의실험결과를통해숙지황은혈당강하효과를보여당대사를개선할뿐아니라당뇨로인한체중감소를방지하고신장의항산화계에긍정적인영향을미침으로써단백뇨를감소시켜당뇨병개선및신장보호효과에유용성이있을것으로기대된다. REFERENCES Aebi H. Methods of Enzymatic Analysis. 1974. Vol 2. Academic Press. New York, USA. Cho SI. Anti-oxidative Effects of RADIX REHMANNIAE PREPARATA on Toxic Agent Induced Kidney Cell Injury. Kor J Herbol. 2003. 18: 119-126. Dai S, Thompson K, Mcneill JH. One-year treatment of streptozotocin-induced diabetic rats with vanadyl sulphate. Pharmacol Toxicol. 1994. 74: 99-107. Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P. Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes. Diabetes 2003. 52: 1031-1035. Deisseroth A, DounceA L. Catalase physical and chemical properties, mechanism of catalysis and physiological role. Physiol Rev. 1970. 50: 3-24. Gallaher DD, Casallany AS, Shoeman DW, Olson JM. Diabetes increase excretion of urinary malonaldehyde conjugates in rats. Lipids 1993. 28: 663-666. Grover JK, Yadav SP, Vats V. Effect of feeding Murraya koeingii and Brassica juncea diet kidney functions and glucose levels in streptozotocin diabetic mice. J Ethnopharmacol. 2003. 85: 1-5. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferase: The first enzymatic step in mercapturic acid and formation. J Biol Chem. 1974. 249: 7130-7139. Hammers HP, Martin S, Federlin K, Geisen K, Brownlee M. Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proc Nat Acad Sci. USA. 1991. 88: 11555-11558. Jensen PK, Christiansen JS, Steven K, Parving HH. Renal function in streptozotocin-diabetic rats. Diabetologia 1981. 21: 409-414. Koivisto VA. Insulin therapy in type II diabetes. Diabetes Care 1993. 16: 29-39. Krolewski AS, Warram JH, Christlieb AR, Busick EJ, Kahn CR. The changing natural history of nephropathy in type I diabetes. Am J Med. 1985. 78: 758-794. Lawrence JC, Jill SG, Eric PD, Joyce AD, Donald DL, Mark AY. Effect of antioxidant treatment of streptozotocin-induced diabetic rats on endoneurial blood flow, motor nerve conduction velocity and vascular reactivity of epineurial arterioles of the sciatic nerve. Diabetes 2001. 50: 1927-1937. Lee JS, Son HS, Maeng YS, Chang YK, Ju JS. Effects of buckwheat on organ weight, glucose and lipid metabolism in streptozotocin-induced diabetic rats. Korean J Nutrition. 1994. 27: 819-827. Lee HS, Km ST, Cho KC. Effects of Rhemanniae Radix water extract on the renal function and renin secretion rate in unanesthetized rabbits. Am J Chin Med. 1981. 21: 179-186. Lowry OH, Rosenbrough NJ, Far AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951. 193: 265-275. Martin JP, Dailey M, Sugarman E. Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation. Arch Biochem Biophys. 1987. 255: 329-336. Matkovics B, Kotorman M, Varga IS, Hai DQ, Varga C. Oxidative stress in experimental diabetes induced by streptozoticin. Acta physiol Hung. 1998. 85: 29-38. Mohamed AK, Bierhaus A, Schiekofer S, Tritschler H, Ziegler R, Nawroth PP. The role of oxidative stress and NF-kappaB activation in late diabetic complications. Biofactors 1999. 10: 157-167. Morel DW, Chisolm GM. Antioxidative treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity. J Lipid Res. 1989. 30: 1827-1834. Nepom GT. A unified hypothesis for the complex genetics of HLA association with IDDM. Diabetes 1990. 39: 1153-1157. O'Donnell MP, Kasiske BL, Keane WF. Glomerular hemodynamic and structural alterations in experimental diabetes mellitus. FASEB J. 1988. 2: 2339-2347. Rayfield EJ, Ishimura K. Environmental factors and insulindependent diabetes mellitus. Diabetes Metab Rev. 1987. 3: 925-931. Stirpe F, Della CE. The regulation of rat liver xanthine oxidase: Conversion in vitro of the enzyme activity from dehydro- - 25 -
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