Lithospermate B 가당뇨백서 사구체에미치는영향 연세대학교대학원 의학과최훈영 1

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1 Lithospermate B 가당뇨백서 사구체에미치는영향 연세대학교대학원 의학과최훈영 1

2 Lithospermate B 가당뇨백서 사구체에미치는영향 지도한대석교수 이논문을박사학위논문으로제출함 2004 년 12 월일 연세대학교대학원 의학과최훈영 2

3 최훈영의박사학위논문을인준함 심사위원 인 심사위원 인 심사위원 인 심사위원 인 심사위원 인 연세대학교대학원 2004 년 12 월일 3

4 감사의글 본논문이완성되기까지세심한지도와사랑으로이끌어주신은사한대석교수님께진심으로감사드립니다. 또한실험의모든것을계획하시고시작부터끝까지같이고민하시며문제점을지적하여주신강신욱교수님과허종호선생님께감사드립니다. 실험의기반을제공하여주신이현철교수님과차봉수교수님, 긴기간동안너그럽고도자상한지도를아끼지않으신김덕희, 정현주교수님께도깊은감사를드립니다. 실험을진행하는데있어많은힘이되어주신신장내과교수님들과동료들, 그리고이수현, 정동섭, 김진주, 곽승재선생님에게도감사드립니다. 항상저를위해걱정해주시는부모님과동생, 그리고늘부족한며느리를너그럽게이해해주시는시부모님께깊은사랑과감사를드리며모든것을이해해주고늘힘이되어주고있는사랑하는남편과더할수없는기쁨을안겨주는지인, 진표에게이논문을바칩니다. 저자씀 4

5 차 례 국문요약 1 I. 서론 4 II. 재료및방법 7 1. 실험동물 7 가. 약물투여 7 나. 신장적출및사구체분리 8 2. RT-PCR 분석 8 가. 총 RNA 추출 9 나. 역전사 (reverse transcription, RT) 9 다. 중합효소연쇄반응 (polymerase chain reaction) 단백추출및 Western blot 분석 면역조직화학염색 (immunohistochemical staining) 통계및분석 15 III. 결과 동물자료 LAB가사구체 TGF-β1 mrna와단백의발현에미치는영향 LAB가사구체 fibronectin mrna의발현에미치는영향 대조군, 당뇨군, LAB 투여당뇨군에서 TGF-β1과 fibronectin에대한면역조직화학염색 22 IV. 고찰 25 V. 결론 32 참고문헌 34 영문요약 45 5

6 그림차례 Figure 1. Glomerular TGF-β1 mrna expression in control, DM, and DM+LAB rats assessed by RT-PCR 19 Figure 2. Glomerular TGF-β1 protein expression in control, DM, and DM+LAB rats assessed by Western blot 20 Figure 3. Glomerular fibronectin mrna expression in control, DM, and DM+LAB rats assessed by RT-PCR 21 Figure 4. Immunohistochemical staining for TGF-β1 in control, DM, and DM+LAB glomeruli after 12 weeks 23 Figure 5. Immunohistochemical staining for fibronectin in control, DM, and DM+LAB glomeruli after 12 weeks 24 표차례 Table 1. Primers of rat TGF-β1, fibronectin, and GAPDH 12 Table 2. Glucose, body weight, kidney weight/body weight, and 24hr-urinary protein excretion after 12 weeks 17 6

7 국문요약 Lithospermate B 가당뇨백서사구체에미치는영향 당뇨병성신병증은전세계적으로말기신부전증의원인질환중가장많은빈도를차지하고있으며, 이의발생및진행기전은수많은노력에도불구하고아직명확히정립되어있지않다. 대규모의임상연구들을통해고혈당뿐만아니라전신적인고혈압역시당뇨병성신병증의발생및진행에중요한역할을하는것으로되어있으나, 혈당을엄격하게조절하고안지오텐신전환효소 (angiotensin-converting enzyme) 억제제나안지오텐신 II 수용체길항제와같은항고혈압제를사용하여혈압을철저히조절함에도불구하고당뇨병성신병증은점차증가하고있는추세이다. 따라서, 당뇨병성신병증의발생및진행에관한치료방법에대한새로운시각이요구되고있는실정이다. Magnesium lithospermate B (LAB) 는당뇨병성합병증의치료에쓰여온민간약제인 Salvia miltiorrhizae Radix에서새롭게추출해낸합성제로서신절제로유발된신부전백서에서신기능및형태학적소견의호전효과를나타내며, 활성산소족생성을감소시키는것으로보고되고있다. 이에본연구에서는 LAB 투여가 1

8 당뇨병성신병증에서신비대및요단백배설등에미치는영향에대해조사함과동시에당뇨병성신병증의중요매개인자인 transforming growth factor-β1 (TGF-β1) 과세포외기질의주요성분인 fibronectin의사구체내발현에미치는영향을알아보고자 streptozotocin (STZ) 으로당뇨가유발된백서에서 LAB를 12주간투여한후사구체내 TGF-β1과 fibronectin의발현을관찰하여다음과같은결과를얻었다. 1. 체중당신장무게는당뇨군에비해 LAB 투여당뇨군에서유의하게적었으며 (1.79 ± 0.11% vs ± 0.06%, p<0.05), 24시간요단백배설량도 LAB 투여로의의있게감소되었다 (90.6 ± 11.3 mg/day vs ± 5.1 mg/day, p<0.05). 2. 사구체내 TGF-β1 mrna와단백발현은대조군에비해당뇨군에서각각 190% 와 140% 증가되었으며 (p<0.05), LAB 투여로당뇨사구체내 TGF-β1 mrna와단백발현증가가각각 89% 와 73% 억제되었다 (p<0.05). 3. Fibronectin mrna 발현은대조군에비해당뇨군에서 180% 증가되었으며 (p<0.05), LAB 투여당뇨군에서는당뇨군에비해사구체내 fibronectin mrna의발현증가가 61% 억제되었다. 2

9 4. 면역조직화학염색을시행한결과, 당뇨군에서대조군에비해사구체내 TGF-β1과 fibronectin 단백의발현이유의하게증가되어있었으며 (2.1 ± 0.3 vs. 0.7 ± 0.1; 2.3 ± 0.2 vs. 0.8 ± 0.1, p<0.05), LAB 투여당뇨군에서는당뇨군에비해 TGFβ1과 fibronectin 단백발현의증가가의의있게감소된소견을보였다. 이상의결과로, STZ로당뇨가유발된백서에서관찰되는사구체내 TGF-β1과 fibronectin mrna와단백의발현증가가 LAB 투여로유의하게억제됨을확인하였다. 따라서 LAB가당뇨병성신병증의발생및진행예방에유용한약제로사용될수있을것으로생각된다. 핵심되는말 : 당뇨병성신병증, lithospermate B, transforming growth factor-β1, fibronectin 3

10 Lithospermate B 가당뇨백서사구체에미치는영향 < 지도교수한대석 > 연세대학교대학원의학과 최훈영 I. 서론 당뇨병성신병증은말기신부전증의원인질환중가장많은빈도를차지하고있으며, 이의발생및진행기전은수많은노력에도불구하고아직명확히정립되어있지않다. 기존의보고들에의하면당뇨병성신병증은병리학적으로신비대, 기저막비후, 사구체내점진적세포외기질축적등이특징적인소견으로알려져있는데 1, 2, 초기에는주로사구체와세뇨관의비후로인한신비대가주요한구조적변화로나타나고, 진행된당뇨병성신병증에서는전반적인사구체경화가동반되면서때로는사구체경화의결절성형태가나타나기도한다고알려지고있으나 2, 3, 그확실한기전은아직밝혀져있지않은실정이다. 4

11 대부분의당뇨병환자는진단당시이미신장크기의증가를동반하는데이는사구체와세뇨관의비후가주요한요인이며, 사구체의비후는주로메산지움의팽창에의해나타나게된다 1, 2. 이러한신비대와메산지움의팽창, 그리고세포외기질축적에는고혈당뿐만아니라사구체내고혈압이관여하는것으로되어있으며, 중요한매개인자로는 transforming growth factor-β1 (TGF-β1) 과안지오텐신 II가널리알려져있다 4-9. 이러한이유에서당뇨병성신병증의예방및치료로철저한고혈당조절과함께안지오텐신전환효소억제제 (angiotensin-converting enzyme inhibitor, 이하 ACEi) 나안지오텐신 II 수용체차단제를이용한고혈압치료가근간을이루게되었다. 그러나정상수준의혈당과혈압에도불구하고당뇨병성신병증은점차증가되고있는추세이다 따라서당뇨병성신병증의예방및치료에대한새로운대책이요구되고있는실정이다. Magnesium lithospermate B ( 이하 LAB) 는당뇨병성합병증의치료에사용되어온민간약제인 Salvia miltiorrhizae Radix로부터새롭게추출한물질로서신기능호전의효과를나타내며, 활성산소족생성을감소시키는것으로보고되고있다 Yokozawa 등은 LAB를백서에투여한결과신혈류량이증가하고소변내 prostaglandin E2의배설과 kallikrein 활성도가증가하는등 5

12 신장의혈액순환이개선되었다고보고하였으며 16, 신부전동물모델에서는 LAB에의해혈압이의미있게감소되었다고하였다 17. Lee 등은 streptozotocin (STZ) 으로당뇨가유발된백서에당뇨유발 8주후부터 LAB를경구로 8주간투여한결과비당뇨병대조군에비해신장내 malondialdehyde (MDA) 농도, 미세알부민뇨, 그리고사구체비대및메산지움팽창이감소되었으며, 신장조직내 TGF-β1, fibronectin 및 collagen 단백의발현증가가억제되었다고보고하였다. 또한메산지움세포에서고농도포도당으로유도된세포내활성산소족생성과 protein kinase C (PKC) 의활성이 LAB에의해억제되는결과를보여당뇨병성신병증의새로운치료약제로서의가능성을제시하였다 18. 그러나 Lee 등은신장전체를이용한연구를하였으며, TGF-β1과 fibronectin mrna의발현변화에대한실험을시행하지않았다. 최근까지도 LAB가당뇨병에의한사구체병변에미치는영향을따로분석한보고는없는실정이다. 이에본연구자는 LAB가당뇨병성신병증, 특히사구체병변의발생에미치는영향을알아보고자당뇨유발과동시에 LAB를투여하였으며, LAB에의한사구체내 TGF-β1과 fibronectin mrna와단백의발현변화를중심으로 LAB의효과를알아보고자하였다. 6

13 II. 재료및방법 1. 실험동물 가. 약물투여 모든동물실험은승인된기안서에따라시행되었다. 실험동물로는무게 g의웅성 Sprague-Dawley 백서 18마리를당뇨군 12마리와비당뇨대조군 (control) 6마리로나누어사용하였다. 당뇨군은다시 2군으로나누어 6마리는표준식이를투여하였고 (DM군), 나머지 6마리는표준식이를투여하면서경관으로 LAB를 10 mg/kg/day 용량으로 12주간투여하였다 (DM+LAB군). 당뇨는 STZ 65 mg/kg를백서복강내에주사하여유발시켰으며, 대조군은같은부피의위약을투여하였다. 당뇨유발은 STZ 투여 72시간후 commercial enzymatic test strip을이용한 glucometer로혈당을측정하여확인하였다. 모든백서는자동온도조절시설에서사육되었으며, 실험기간동안물과표준실험식이를자유롭게섭취할수있도록하였다. 모든백서는당뇨유발 12주후에희생시켰으며, 희생시키기직전에체중, 혈당, 그리고 7

14 24시간요단백배설량을측정하였다. 혈당은 glucometer로측정하였으며, 24시간요단백배설량은 Bio-Rad Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) 를이용한 Bradford 방법으로측정하였다. 나. 신장적출및사구체분리 백서를 thiopental sodium 50 mg/kg 마취하에단두한후신장을적출하여무게를측정하였다. 형태학적검사를위하여일부신장조직은 10% neutral-buffered formalin으로고정한후표준적방법으로처리하고 5 μm 절편으로잘랐다. 사구체는 sieve를이용하여분리하였다. 적출된신장을면도날로장축을따라반으로절개한후가위로피질만을분리하여 Hanks' balanced salt solution (HBSS) 완충용액에옮긴다음, 체공이 200, 150 및 75 μm인 stainless sieve에차례로통과시켜 75 μm 소공에걸린사구체를실험에이용하였다. 2. RT-PCR 분석 가. 총 RNA 추출 8

15 총 RNA를추출하기위해 RNA STAT-60 reagent (Tel-Test, Inc., Friendswood, TX, USA) 를사용하였다. RNA STAT-60 reagent 100 μl로사구체를처리하고, 동결과해동을 3회반복하여용해시켰다. 여기에 700 μl의 RNA STAT-60 reagent를첨가하고 vortex한후상온에서 5분간두었다. 400 μl의 chloroform을첨가한후 30초간손으로세게섞어주고, 4 C에서 12,000rpm으로 15분간원심분리한후상층액을취한다음 400 μl의 isopropanol을첨가하고, 다시 4 C에서 12,000rpm으로 30분간원심분리하였다. 침전물을 70% ethanol로처리한후건조시키고, diethyl pyrocarbonate로처리한증류수로침전물을적절한농도로희석시킨다음 spectrophotemeter를이용하여 RNA 농도를측정하였다. 나. 역전사 (reverse transcription, RT) RNA 1 μg, 8 mmol/l MgCl 2, 1 mmol/l dntp, 25U RNase inhibitor, 40U AMV reverse transcriptase, 10 μmol/l의 random hexamer (Roche Diagnostics GmbH, Mannheim, Germany) 를함유한 20 μl 반응용액으로부터 Eppendorf Mastercycler 9

16 gradient (Eppendorf Scientific, Inc., Westbury, NY, USA) 를이용하여 first-strand c-dna를합성하였다. 총 20 μl의반응혼합물을 30 C에서 10분, 그리고 42 C에서 60분간 incubation 한후 99 C에서 5분간가열하여효소를비활성화시켰다. 역전사가끝난검체는 PCR 전까지 -70 C에보관하였다. 다. 중합효소연쇄반응 (polymerase chain reaction, PCR) 백서의 GAPDH, TGF-β1 및 fibronectin에대한 primer를설계한후 invitrogentm Life Technologies사 (Rockville, MD, USA) 에의뢰하여제작하였다 (Table 1). GAPDH와 fibronectin의 PCR은 10 ng RNA에해당하는 cdna, Taq DNA polymerase 1.25 U, dntp 0.2 mmol/l, sense 및 antisense primers 각각 25 pmol/l, 1.5 mmol/l MgCl 2 를함유한 50 μl 반응혼합물을이용하여시행하였으며, TGF-β1의경우 Taq-Gold DNA polymerase 2.5U와 sense 및 antisense primer 각각 30 pmol/l 외다른조성은동일하게함유한반응혼합물을사용하였다. PCR은 95 C에서 3분간가열한후 94 C에서 30초, annealing 온도인 62 C에서 30초, 그리고 72 C에서 1분간 36 cycles 시행하였으며, 이후 72 C에서 7분간더반응시켰다. 10

17 mrna의발현은역전사효율의차이를보정하기위해모든검체의 PCR 산물의 optical density (O.D.) 를 GAPDH의 O.D. 로보정하였다. 이를위해실험에서얻은 PCR 산물과 GAPDH 비의예민도가가장높은 cdna의양과 PCR cycle의수를결정한후본실험을시행하였다. House keeping 유전자인 GAPDH는다른시험관에서동시에시행하였다. PCR이끝난후개개의증폭된 PCR 산물중 30μL를취하여 0.05 μg/ml의 ethidium bromide를함유한 2% agarose gel 에서전기영동하였다. PCR 산물의 O.D. 는 TINA image software (Raytest, Straubenhardt, Germany) 를이용하여측정하였으며, GAPDH의 O.D. 로보정하였다. 11

18 Table 1. Primers of rat TGF-β1, fibronectin, and GAPDH TGF-β1 Sequence (5' 3') Sense Antisense CGA GGT GAC CTG GGC ACC ATC C GCT CCA CCT TGG GCT TGC GAC C Fibronectin Sense Antisense GCA AGC CTA AAC CTG AAG AGA CC CCT GGT GTC CTG ATC ATT GCA TC GAPDH Sense Antisense GAC AAG ATG GTG AAG GTC GG CAT GGA CTG TGG TCA TGA GC 3. 단백추출및 Western blot 분석 200 μl의 lysis buffer (50 mm Tris-HCL ph 7.5, 100 mm NaCl, 1 mm EDTA, 2% SDS, 0.1 mm phenylmethylsulfonyl fluoride, 2μg/mL leupeptin) 를이용하여세포균질액을만들고 4 C에서 12000rpm으로 10분간원심분리한후상층액을취하여분석에 12

19 이용하였다. Bio-Rad kit (Bio-Rad Laboratories, Inc., CA, USA) 를이용한 Bradford 방법으로단백을정량한후동량의단백을 SDS와 β-mercaptoethanol이포함된 sample buffer와혼합하여 95 C에서 5분간끓이고 10-15% polyacrylamide gel에서전기영동한후 Hoeffer semidry blotting apparatus (Hoeffer Instruments, San Francisco, CA, USA) 를이용하여 Hybond-ECL 흡착지 (American Life Science, Inc., Arlington Heights, IL, USA) 에이전시켰다. Blocking buffer A (1 PBS, 0.1% Tween-20, 8% nonfat milk) 로 1시간동안처리한후, 1:1000으로희석한 TGF-β1과 fibronectin에대한일차항체 ( 다중클론 rabbit anti-tgf-β1 항체와단일클론 mouse anti-fibronectin 항체 ; Santa Cruz Biotechnology, Santa Cruz, CA, USA) 로 4 C에서하룻밤동안반응시킨다음, 세척하고이차항체를이용하여반응시켰다. 세척을반복한후발현은 enhanced chemiluminescence 시약 (ECL; Amersham Life Science, Inc., Arlington Heights, IL, USA) 을이용하여가시화하였으며, densitometer로 O.D. 를측정하였다. 13

20 4. 면역조직화학염색 (immunohistochemical staining) 5 μm 신장조직절편의파라핀을제거한후에탄올로적시고세척하였다. Black and Decker vegetable steamer를이용하여 10 mm/l sodium citrate buffer에서 20분간 antigen retrieval을시행한후 3% H 2 O 2 로처리하여 endogenous peroxide 활성을제거하였다. TGF-β1과 fibronectin 염색을위해각각다중클론 rabbit anti-tgf-β1 항체와단일클론 mouse anti-fibronectin 항체를 1:100으로희석하여상온에서하룻밤동안반응시켰다. 세척후 biotinylated linked anti-rabbit and anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) 로 30분간처리한다음세척하고 streptoavidin-hrp (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) 로 30분간처리하였다. Diaminobenzidine으로처리하고세척한후표본을 hematoxylin으로대조염색하였다. 절편내혈관극 (vascular pole) 이포함된사구체 30개에서 TGF-β1과 fibronectin의면역조직화학염색정도는 0~+4까지의점수로반정량화하였다. 14

21 5. 통계및분석 모든결과는평균 ± 표준오차 (SEM) 로표시하였다. 통계분석은개인용컴퓨터통계프로그램 SPSS 윈도우용 11.0판 (SPSS, Inc., Chicago, IL, USA) 을이용하였다. 결과들은 Analysis of variance (ANOVA) 검정또는 Kruskal-Wallis 다중비교용비모수검정을사용하여분석하였으며, ANOVA 검정상의미있는차이는 Students t-test로, Kruskal-Wallis 검정상의의있는차이는 Mann-Whitney U-test로확인하였다. P 값이 0.05 미만인경우에의미있는것으로간주하였다. 15

22 III. 결과 1. 동물자료 모든군에서실험 12주후체중증가를보였으나, 대조군에비해당뇨군과 LAB 투여당뇨군에서통계적으로유의하게체중이적었다 (p<0.01). 실험 12주후백서를희생시킬때신장의무게를측정하였으며, 체중당신장무게는대조군에비해당뇨군과 LAB 투여당뇨군에서의의있게높았으며, LAB 투여당뇨군에서는당뇨군에비해유의하게낮았다 (p<0.01) (Table 2). 12주후평균혈당은대조군에서 ± 8.5 mg/dl로당뇨군과 LAB 투여당뇨군에비해의미있게낮았으며 ( 각각 ± 7.1와 420 ± 9.4 mg/dl, p<0.01), 24시간요단백배설량은대조군에비해당뇨군에서유의하게증가되어있었고, LAB 투여당뇨군에서당뇨군에비해요단백배설량이의의있게감소하였다 (Table 2). 16

23 Table 2. Glucose, body weight, kidney weight/body weight, and 24hr-urinary protein excretion after 12 weeks Control DM DM + LAB Glucose (mg/dl) ± ± 7.1 * ± 9.4 * Body weight (g) ± ± 7.9 * ± 5.5 * Kidney/body weight ( 10-2 ) 0.58 ± ± 0.11 * 1.14 ± 0.06 * Proteinuria (mg/day) 19.8 ± ± 11.3 * 31.6 ± 5.1 * 1 Data are expressed as mean ± SEM. * p<0.01 vs. Control p<0.05 vs. DM 2. LAB 가사구체 TGF-β1 mrna 와단백의발현에미치는영향 당뇨유발 12주후추출한사구체내 TGF-β1 mrna의발현은당뇨군에서대조군에비해 1.9배증가되었으며 (p<0.05), 당뇨유발직후부터 12주간 LAB를투여한군에서의 TGF-β1 mrna 발현은당뇨군에비해 73% 억제되었다 (p<0.05) (Figure 1). TGF-β1 단백의발현도대조군에비해당뇨군에서 1.4배증가되었으며 (p<0.05), LAB 투여로 TGF-β1 단백발현의증가가 89% 억제되었다 (p<0.05) (Figure 2). 17

24 3. LAB 가사구체 fibronectin mrna 의발현에미치는영향 Fibronectin mrna 의발현은당뇨군에서대조군에비해 1.8 배 증가되었으며 (p<0.05), LAB 투여당뇨군의 fibronectin mrna 발현은당뇨군에비해 61% 억제되었다 (p<0.05) (Figure 3). 18

25 Control DM DM + LAB TGF-β1 GAPDH 오류! Figure 1. Glomerular TGF-β1 mrna expression in control, DM, and DM+LAB rats assessed by RT-PCR * p<0.05 vs. Control p<0.05 vs. DM 19

26 Control DM DM + LAB TGF-β1 β-actin Control DM DM + LAB Figure 2. Glomerular TGF-β1 protein expression in control, DM, and DM+LAB rats assessed by Western blot * p<0.05 vs. Control p<0.05 vs. DM 20

27 Control DM DM + LAB Fibronectin GAPDH Control DM DM + LAB Figure 3. Glomerular fibronectin mrna expression in control, DM, and DM+LAB rats assessed by RT-PCR * p<0.05 vs. Control p<0.05 vs. DM 21

28 4. 대조군, 당뇨군, LAB 투여당뇨군에서 TGF-β1 과 fibronectin 에 대한면역조직화학염색 각군에서 TGF-β1에대한면역조직화학염색을시행한결과, 당뇨군에서대조군에비해사구체내 TGF-β1 단백발현이유의하게증가되어있었으며, LAB 투여당뇨군에서는투여하지않은당뇨군에비해의의있게감소된소견을보였다 (Figure 4). TGF-β1에대한면역조직화학염색의정도를 0~+4까지의점수로반정량화하였을때, 당뇨군에서대조군에비해유의하게높았으며 (2.1 ± 0.3 vs. 0.7 ± 0.1, p<0.05), LAB 투여당뇨군에서는당뇨군에비해의미있게점수가낮았다 (1.1 ± 0.2, p<0.05). Fibronectin에대한면역조직화학염색결과역시대조군에비해당뇨군에서사구체내 fibronectin 단백발현이의미있게증가되어있었으며, LAB 투여당뇨군에서당뇨군에비해유의하게감소되었다 (Figure 5). 면역조직화학염색의정도를 0~+4까지반정량적점수로점수화하였을때, 당뇨군에서대조군에비해유의하게높았으며 (2.3 ± 0.2 vs. 0.8 ± 0.1, p<0.05), LAB 투여당뇨군에서는당뇨군에비해의미있게점수가낮았다 (1.0 ± 0.1, p<0.05). 22

29 D Figure 4. Immunohistochemical staining for TGF-β1 in control (A), DM (B), and DM+LAB (C) glomeruli after 12 weeks. Expression of TGF-β1 protein (arrows) in DM glomeruli was increased compared to that of control and this increment was ameliorated by LAB treatment. Semiquantitative immunohistochemical score of control, DM and DM+LAB glomeruli (D) * p<0.05 vs. Control p<0.05 vs. DM 23

30 D Figure 5. Immunohistochemical staining for fibronectin in control (A), DM (B), and DM+LAB (C) glomeruli after 12 weeks. Expression of fibronectin protein (arrows) in DM glomeruli was increased compared to that of control and this increment was ameliorated by LAB treatment. Semiquantitative immunohistochemical score of control, DM and DM+LAB glomeruli (D) * p<0.05 vs. Control 24

31 p<0.05 vs. DM IV. 고찰 당뇨병의중요한합병증중하나인당뇨병성신병증은말기신부전증의가장많은원인으로서전세계적으로투석치료가필요한환자의약 40% 를차지하는것으로보고되고있다 22. 당뇨병성신병증의병인을밝히고병태생리적기전뿐만아니라분자생물학적인기전까지알아내고자하는수많은연구가있어왔으며, 임상적으로도대규모의연구들이활발히이루어지고있고, 새로운치료법을제시하고자하는많은연구들이진행중이다. 당뇨병성신병증의발생및진행에있어사구체경화와세뇨관- 간질섬유화의중요성이대두되면서세포외기질의생성증가또는분해감소의기전을규명하고자하는연구가활발히이루어지고있다. 당뇨병성신병증과연관된이러한병태생리학적변화를매개하는것으로알려진여러인자중가장중요한인자가 TGFβ1이다. TGF-β1의활성은고혈당 23-25, 단백질의 nonenzymatic glycation 증가 26, 27, diacylglycerol 생성및 PKC의활성증가 28, 안지오텐신 II 29, 30, 그리고 thromboxane 31 등에의해활성화되는 것으로보고되고있다. 이렇게활성화된 TGF-β1 은세포외기질을 구성하는성분중 type I collagen, type IV collagen, fibronectin, 25

32 그리고 laminin 등의 mrna와단백발현을증가시키며 32, 기질을분해하는각종효소중 plasminogen activator, collagenase, 그리고 stromelysin등의생성은억제하고, 기질분해효소억제제인 plasminogen activator inihibitor를활성화시켜세포외기질의축적을촉진한다 33. TGF-β1은세포외기질수용체인 integrin의발현을조절하여특이기질단백질과의작용을강화시키기도하며 34, 강력한화학주성성질을보유하고있어섬유모세포의작용을증강시키는역할을하기도한다 35. 당뇨병성신병증에서이와같은 TGF-β1의역할을증명한연구들을보면, 고농도포도당이함유된배지로배양한메산지움세포와근위세뇨관세포에서 TGF-β1의발현이정상포도당이함유된배지에비해증가되었다는보고가 있으며 23, 24, Kolm-Litty 등은고농도포도당배지에서배양된 메산지움세포에서포도당대사로부터생성되는세포내 glucosamine이 TGF-β1의발현을증가시켰다고보고하였다 25. 안지오텐신 II, endothelin, thromboxane과같은여러혈관작용인자들도 TGF-β1의이차적유도를통해당뇨병성신병증의병태생리로알려져있는성장자극과 profibrogenic action에관여하는것으로보고되고있으며 29-31, 사구체모세혈관압상승으로인한신장내혈역학적변화도 TGF-β1 생성을자극한다는연구결과들도있다

33 이외에도 TGF-β에대한중화항체 (neutralizing anti-tgf-β antibody) 가메산지움세포와사구체상피세포에서각각고농도포도당으로유도된 collagen 합성과 fibronectin 생성을감소시켰다는연구결과들도있다 37, 38. Oh 등은고농도포도당 (30 mm) 이 fibronectin의생성에앞서매우초기에 TGF-β1을자극할뿐만아니라고농도포도당으로유도된 fibronectin 생성이 TGFβ1에의해매개됨을보고하여 TGF-β1이고농도포도당조건하의메산지움세포에의한세포외기질축적을매개한다는사실을증명하였다 39. 이러한연구들을통해당뇨병성신병증에서 TGFβ1이매우중요한매개인자임을알수있다. Fibronectin (; high molecular weight α 2 glycoprotein) 은사구체내세포외기질의내인성구성인자로서주로간에서생산되지만혈관내피세포와혈소판으로부터도생성이되는것으로알려져있다 40. Fibronectin은혈관내피세포와적혈구의부착을강화시키고당뇨병환자에서는적혈구의변성과여과감소를일으키는등당뇨병성말초혈관변성의병인에도중요한역할을한다. Davies 등은처음으로당뇨병환자에서혈장 fibronectin 농도가증가되었다고보고하였으며 41, Ozata 등은혈장 fibronectin 농도가당뇨병성신병증의지표로이용될수있다는가능성을제시하기도하였다 42. 당뇨병성신병증의경우사구체내 TGF-β1의 27

34 발현이초기부터증가하는반면에 fibronectin 발현은주로현저한단백뇨가동반되는시기에증가하는것으로보고되고있다. 따라서 TGF-β1이나 fibronectin과같은인자들의생성을억제시키는물질을사용할경우당뇨병성신병증의발생및진행이예방될수있을것으로생각되어많은연구가진행되어왔으며, 그중에서도 ACEi가당뇨병성신병증에서 TGF-β1이나 fibronectin의발현을억제시켰다는연구가보고되어왔다 44. Magnesium lithospermate B (LAB) 는전통적으로당뇨병의합병증에사용되어온한약제로알려진 Salvia miltiorrhizae Radix로부터추출한물질로서신장에미치는영향에대한몇몇연구결과들이보고되었다 Yokozawa 등은총신장부피의 5/6를절제한백서에서 70일동안 LAB를경구투여한결과신절제에의한메산지움증식, 세뇨관-간질조직의병변, 그리고사구체경화등이호전되었다고하였다 19. 또한 Yokozawa 등은총신장부피의 2/3를절제한백서에서나타나는혈중 superoxide dismutase (SOD) 와 catalase의활동성감소와 hydroxyl radical의증가가 30일간 LAB를경구로투여한후거의정상으로회복되는결과를나타내어 scavenging radical에의한신부전증으로의진행을예방하는데 LAB가도움이될수있을것이라고하였다

35 Jung 등은 Salvia miltiorrhizae Radix로부터효과적인추출방법을이용해 magnesium lithospermate B를합성하였으며, LAB가메산지움세포에서 aldose reductase 활성도를억제시킬뿐만아니라 fibronectin 축적도용량의존적으로억제시키는효과가있었다고보고함으로써당뇨병성신병증에서새로운치료약제로서의가능성을제시하였다 21. 본연구에서도 in vivo 실험을통해당뇨사구체에비해 LAB를투여한당뇨백서사구체에서 fibronectin mrna의발현과단백축적이의미있게억제되었음을확인하였다. Lee 등은 STZ 투여로당뇨가유발된백서에서 LAB를당뇨유발 8주후부터 8주간투여한결과신조직내 TGF-β1과 fibronectin 단백의발현이의미있게감소되었다고보고하였다 18. 이들은대조군과 LAB를투여한대조군, 당뇨군, 그리고 LAB를투여한당뇨군등 4군으로나누어여러항목을비교하였는데, 대조군과 LAB를투여한대조군에비해당뇨군과 LAB를투여한당뇨군에서혈당, 체중당신장무게, 사구체부피, 그리고 24시간요알부민배설량이의미있게증가되었으며, LAB를투여한당뇨군에서는당뇨군에비해체중당신장무게와 24시간요알부민배설량이의의있게감소되었다. 또한 TGF-β1, fibronectin, 그리고 collagen에대한면역조직화학염색을시행한결과당뇨군의신장 29

36 피질내사구체와세뇨관-간질에증가되었던 TGF-β1, fibronectin, 그리고 collagen의단백발현이 LAB 투여로억제되었으며, TGFβ1과 fibronectin의변화는 ELISA와 Western 분석을통해재확인되었다. 본연구에서도 Lee 등의보고에서와같이 LAB 투여당뇨군에서당뇨군에비해체중당신장무게와요단백배설량이유의하게감소된결과를나타내었다. Lee 등의연구에서는신장피질전체를이용한반면본연구에서는사구체만분리하여실험에이용하였는데, Western 분석결과 TGF-β1 단백발현이대조군에비해당뇨군에서의미있게증가되었으며, 이러한증가가 LAB 투여로유의하게억제되어 LAB가신장피질의주요구조인신세뇨관뿐만아니라사구체내 TGF-β1 단백의발현증가도억제시킴을알수있었다. 본연구에서는 TGF-β1과 fibronectin 단백의발현증가가 mrna 수준에서의변화에기인한것인지를증명하기위해 RT-PCR을시행하였는데, 대조군에비해당뇨군에서 TGF-β1과 fibronectin mrna 발현이증가되었으며, 이러한발현증가가 LAB 투여로유의하게억제된결과를보여 LAB의효과가 mrna 수준에서나타남을처음으로증명하였다. LAB가당뇨사구체내 TGF-β1과 fibronectin의발현증가를억제시키는기전에대해서는아직확실하게밝혀져있지는않으나, 메산지움세포에서 LAB가활성산소족생성과 PKC 활성을억제한 30

37 것으로미루어보아생체내에서도이와관련이있을것으로추정이되나향후이에대한연구가추가적으로필요할것으로사료된다. 이상으로본연구에서는 STZ로당뇨가유발된백서에서사구체내 TGF-β1과 fibronectin의발현이증가되었으며, 이러한발현증가가 LAB에의해억제됨을확인하였다. 따라서 LAB가당뇨병성신병증의발생및진행예방에유용한약제로사용될수있을것으로생각된다. 31

38 V. 결론 본연구에서는 LAB가당뇨병성신병증의중요매개인자인 TGFβ1과세포외기질의주요성분인 fibronectin의사구체내 mrna 및단백발현에미치는영향을알아보고자 STZ로당뇨가유발된백서에서 LAB를 12주간투여한후사구체내 TGF-β1과 fibronectin의발현을관찰하여다음과같은결과를얻었다. 1. 체중당신장무게는당뇨군에비해 LAB 투여당뇨군에서유의하게적었으며 (1.79 ± 0.11% vs ± 0.06%, p<0.01), 24시간요단백배설량도 LAB 투여로의의있게감소되었다 (90.6 ± 11.3 mg/day vs ± 5.1 mg/day, p<0.01). 2. 사구체내 TGF-β1 mrna와단백발현은대조군에비해당뇨군에서각각 190% 와 140% 증가되었으며 (p<0.05), LAB 투여로당뇨사구체내 TGF-β1 mrna와단백발현증가가각각 89% 와 73% 억제되었다 (p<0.05). 3. Fibronectin mrna 발현은대조군에비해당뇨군에서 180% 증가되었으며 (p<0.05), LAB 투여당뇨군에서는당뇨군에비해사구체내 fibronectin mrna의발현증가가 61% 억제되었다. 32

39 4. 면역조직화학염색을시행한결과, 당뇨군에서대조군에비해사구체내 TGF-β1과 fibronectin 단백의발현이유의하게증가되어있었으며 (2.1 ± 0.3 vs. 0.7 ± 0.1, 2.3 ± 0.2 vs. 0.8 ± 0.1, p<0.05), LAB 투여당뇨군에서는당뇨군에비해의의있게 TGF-β1과 fibronectin 단백발현증가가감소된소견을보였다. 이상의결과로, STZ로당뇨가유발된백서에서관찰되는사구체내 TGF-β1과 fibronectin mrna와단백발현의증가가 LAB 투여로억제됨을확인하였다. 따라서 LAB가당뇨병성신병증의발생및진행예방에유용한약제로사용될수있을것으로사료된다. 33

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51 The effect of lithospermate B (LAB) on the expression of TGF-β1 and fibronectin in diabetic glomeruli Hoon Young Choi Department of Medicine The Graduate School, Yonsei University (Directed by Professor Dae-Suk Han) Background: Lithospermate B (LAB) is an active component isolated from Salvia miltiorrhizae, a Chinese herbal medicine traditionally used for the treatment of diabetic complications. Previous study reported that LAB suppressed TGF-β1 and fibronectin protein expression in diabetic kidney. This study was performed to see whether LAB inhibits TGF-β1 and fibronectin mrna as well as protein expression in diabetic glomeruli. Methods: Eighteen male Sprague-Dawley rats weighing g were used. Six were injected with diluent (C) and 12 with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-induced diabetic rats were left untreated (DM, n=6) or were treated with 10mg/kg/day LAB by gavage for 12 weeks (DM+LAB, n=6). 45

52 Glomeruli were isolated by sieving technique. RT-PCR, Western blot, and immunohistochemistry were performed. Results: The ratio of kidney weight to body weight in DM (1.79 ± 0.11%) was significantly higher than C (0.58 ± 0.03%) and DM+LAB (1.14 ± 0.06%). Compared to C, 24-hour urinary protein excretion was significantly higher in the DM (19.8 ± 3.2 mg/day vs ± 11.3 mg/day, p<0.05), and LAB treatment inhibited the increase in proteinuria in DM (31.6 ± 5.1 mg/day, p<0.05). TGF-β1 mrna and protein expressions in 12-week DM glomeruli were increased significantly (140%, 190% compared to C, respectively, p<0.05). LAB treatment inhibited the increase in TGF-β1 mrna expression by 89%, and TGF-β1 protein expression by 73% (p<0.05). Fibronectin mrna expression in DM+LAB glomeruli were also significantly reduced by 61% compared with DM glomeruli (180% compared to C, p<0.05). LAB treatment significantly ameliorated increased glomerular TGF-β1 and fibronectin protein expression in DM as assessed by immunohistochemistry (p<0.05). 46

53 Conclusion: TGF-β1 and fibronectin mrna and protein expressions were increased in DM glomeruli and this increment was effectively ameliorated by LAB treatment. Our data suggest that LAB may be used as a new therapeutic agent for the treatment of diabetic nephropathy. Key Words: diabetic nephropathy, lithospermate B, TGF-β1, fibronectin 47