J. Biomed. Lab. Sci. 10 (2004) 107 113 Effect of Mori Cortex in the Cardiac Injury Induced by Skin Burn Hye-Jung Moon 1, Hyun Gug Cho 2 and Won-Hark Park 1 1 Department of Biology, Yeungnam University, Kyungbuk 712-749, Korea, 2 Department of Visual Optics, Kyungwoon University, Gumi 730-852, Korea This study was conducted to investigate an effect of Mori Cortex in the cardiac injury following dermal scald burn in rats. Sprague-Dawley rats were induced scald burn (15% of total body surface area). Heart was removed at 5 h postburn and examined with biochemical assay, ultrastructural observations and stereological analysis. The activity of serum aspartate aminotransferase and creatinine was increased at 5 h postburn compared with them of control. Administration of heat extracts of Mori Cortex after scald burn inhibited the production of KC (neutrophil chemoattractant cytokine) and increased the activity of protein kinase C (PKC) in heart tissue. The activity of myeloperoxidase (MPO) in heart tissue was decreased both at 5 h postburn and in case of Mori Cortex administration after scald burn. Ultrastructurally, many contraction bands and separation of intercalated disk induced by scald burn were decreased by administration of heat extracts of Mori Cortex. In stereological analysis, administration of Mori Cortex after scald burn resulted the volume densities of myofibril and mitochondria were increased compared with them of burn control. These data suggest that Mori Cortex may be a useful stuff to the range of available treatments for cardiac injury induced by skin burn. Key Words: Burn, Cardiac injury, Inflammation, Mori Cortex, Protein kinase C 서 화상은범위와깊이에따라전신적인신체반응을유발하는손상으로, 과도한수액손실에따른저혈압, 숔, 급성신장부전등이발생할수있고, 추후상처감염 42) 이나폐렴, 패혈증, 다발성장기기능부전증후군 (multiple organ dysfunction syndrome, MODS) 등으로발전하기도한다 20,34). 피부화상으로인한심근의변화로, 근세포내칼슘의증가와순환계내독성산소대사물의생성증가로인해세포막구조의손상에의한심근수축력저하와 17,22), 근형질세망과사립체의변성 23) 으로인한심부전이발생된다 21). 일부연구에서 TNF-α, IL-1과같은염증성사이토카인이이러한원인인자로밝혀져 39) 염증반응에의한산화적손상기전이전개된다고하였다 13,18). 피부화상에의해손상된심근과염증과의관계에대한연구들을살펴보면 TNF와 IL-1β의합성은세포내칼슘축적을증가시키고, 칼슘과축적은심근세포괴사를야기시키는 * 논문접수 : 2004년 5월 10일수정재접수 : 2004년 6월 8일 별책요청저자 : 조현국, ( 우 ) 730-852 경북구미시산동면인덕리 55, 경운대학교안경광학과 Tel: 054-479-1333, Fax: 054-479-1333 e-mail: hgcho@ikw.ac.kr 론 심근억제물질로작용하며 28,36,43) 호중구로부터유리된독성산소대사물은장기를손상시킨다 7) 고하였다. 호중구는화상후폐, 간, 신장, 장, 근육, 피부에축적되어염증반응을증가시켜산화적손상을야기시키며 8) 호중구로부터유리된유리산소기양의증가는세포막파괴의주요원인이라고하였다 34). 화상과같은열손상에의한세포반응은 protein kinase pathways에의해조절되며, 실험동물을이용한화상모델에서화상후심근부전을일으키는병태생리적기작은 protein kinase C (PKC) 의활성화와수축단백질의변성으로인한수축기능의변화등이관여한다고하여, PKC는화상후심근기능부전을중재하는신호전달에서중요한역할을한다고하였다 19). 활성화된 PKC는화상으로인한여러세포반응들을조절하며호중구를활성화시켜 24,33) 혈관투과성의증대와염증반응을유도한다고하였다 32). PKC는다양한장기에존재하여신호전달경로에관여하는효소로 1), 심장에서는심근수축조절 38), 심근비대 11), 심근보호, 염증과정을조절하는중재자로작용하며 6) 특히심근손상에서염증반응의중요성이대두되어이에대한연구가활발히진행되고있다. 따라서본실험에서는피부화상을유발시킨후, 수분대사를촉진시킴으로써부종을없애는데효능이있다고알려진상백피를열탕추출하고, 흰쥐의구강으로투여하여화상후 - 107 -
손상된심근에효과가있는지검증하고자하였다. 재료및방법 1. 피부화상의유도및처치체중 250 g 내외의외견상건강한 Sprague-Dawley종수컷흰쥐를사용하여 ketamine hydrochloride를복강투여하여마취시킨다음등쪽면의털을깎고 (total body surface area; TBSA, 15%) 37), 100 물로 10초간데인후 5시간후에처치하였다. 동물의처치는효소활성의일중변동을고려하여일정시간에실시하였고, ether 마취하에서복부정중선을따라개복한다음배대동맥으로부터채혈하여실혈사시킨후심장을적출하였다. 적출한심장은생리식염수로씻은후, 혈액을제거한다음무게를측정하였다. 채취한혈액은실온에 30분간방치시켜 3,000 rpm에서 15분간원심분리하고혈청을얻어생화학적활성측정에사용하였다. 2. 검액제조및투여상백피 80 g을열탕기를이용하여 2시간 30분동안열탕시킨후, 진공농축기를이용하여 300 ml가되도록하였다. 농축된약재를화상유발 1시간뒤 5 ml/kg 양으로흰쥐의구강으로직접투여하였다. 3. 혈청내효소활성도측정 1) Aspartate aminotransferase (AST) 활성도측정혈청내 AST의활성측정은 kit 시액 ( 아산제약 ( 주 )) 을사용하였으며, 단위는혈청 ml 당 Karmen unit으로표시하였다. 2) Creatinine 활성도측정혈청내 creatinine의활성도측정은 kit 시액 ( 아산제약 ( 주 )) 을사용하였으며, 단위는 mg/dl로표시하였다. 4. 심근조직내 KC (neutrophil chemoattractant cytokine) 함량측정심근조직내호중구침윤유도물질인 KC의생성정도를측정하기위하여 mouse KC immunoassay kit (R&D, MN, USA) 를사용하였다. 심장을적출하여 4.0 ml의 50 mm potassium phosphate (ph 7.4) 를가하여조직을분쇄한후 (Polytron (Swizland) homogenizer) 3,000 rpm으로 10분간원심분리한다음상등액을채취하였다. 채취한상등액으로 assay procedure 에따라시행하여 microtiter plate reader (BioRad 550, CA, USA) 를이용하여 450 nm에서흡광도를측정하고그함량을계산하였다. 5. 심근조직내 myeloperoxidase (MPO) 활성측정심근조직내호중구의침윤을확인하기위한 MPO 활성측정은 Goldblum 등 15) 의방법에따라시행하였다. 심장을적출하여 4.0 ml의 20 mm potassium phosphate (ph 7.4) 를가하여조직을마쇄하여균질액을만들고 4, 18,000 rpm (Beckman, USA) 에서 30분간원심분리하였다. 그후침전층을 0.5% hexadecyltetramethyl-ammonium bromide (HTAB) 가포함된 4.0 ml의 50 mm potassium phosphate (ph 6.0) 용액에재부유시켜 90초간초음파분쇄 (Vibracell, USA) 하였다. 이후균질액을 60 에서 120분간반응시킨후 ο-dianisidine을사용하여 chromogenic substrate와 hydrogen peroxide가반응을일으킨양을 460 nm에서시간의변화에따른흡광도를측정하여 (UV 1601, Shimadzu) MPO의활성 (U/g of wet heart) 을계산하였다. 6. 심근조직내 protein kinase C (PKC) assay 를위한시료조제심장적출후그무게를측정하고 25 mm Tris-HCl (ph 7.4), 0.5 mm EDTA, 0.5 mm EGTA, 0.05% Triton X-100, 10 nm β-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin으로조제된 extraction buffer 5 ml에담가빙냉하에균질기로마쇄하였다. 마쇄균질액을 14,000 g에서 5분간원심분리시켜상등액을채취한다음 extraction buffer로미리안정화시킨 DEAE cellulose column에통과시켜 200 mm NaCl이포함된 extraction buffer로 PKC-containing fraction을분리하였다. 7. 심근조직내 protein kinase C (PKC) 활성측정 PKC 측정은 SignaTECT protein kinase C assay kit (promega, USA) 를이용해 assay procedure에따라실시하고, scintillation counter (Beckman, USA) 를이용하여 PKC 활성을측정하였다. 8. 심근의미세구조적관찰적출한심장의좌심실부분을세절하여 2.5% glutaraldehyde (0.1 M phosphate buffer, ph 7.4, 4 ) 에 2~4시간전고정시키고, 0.1 M phosphate buffer (ph 7.4) 로충분히세척한후다시 1% osmium tetroxide에 90분간후고정시켰다. 고정이완료된조직은 0.1 M phosphate buffer (ph 7.4) 로세척한다음알코올의농도를순차적으로증가시켜탈수시키고, propylene oxide로치환하여 epoxy resin에침투및포매시킨다음, 37 에서 12시간, 60 에서 48시간동안열중합시켜블럭을제작하였다. 만들어진블럭은초박절편기 (Reichert Supernova, Sweden) 를이용하여 1 µm 두께로박절한다음 1% toluidine blue로염색하여광학현미경하에서관찰부위를선정한다음, 60~70 nm로초박절하여 uranyl acetate와 lead citrate로이중염색한후, 가속전압 75 kv에서투과전자현미 - 108 -
경 (H-600, Hitachi) 으로관찰하였다. 9. 심근의입체해석학적관찰 1) 양평가를위한표본의선택근원섬유와사립체의양적조사를위해미세구조관찰표본을이용하여심근조직의종단면으로잘려진표본을무작위로선택하여 5,000배로촬영하고, 인화과정에서 3배로확대하여 15,000배의사진을얻었다. 2) 체적밀도 (volume density) 근원섬유와사립체의체적밀도측정은 10 d (d=10 mm) 인단일격자 (single lattice) test grid를사용하여 Loud 등 29) 의방법에의해근원섬유의장축에대해 19 의기울기로 grid를배치하여, grid에 intersection되는것을 Park 등 31) 의점계수법 (point counting method) 으로계수한후다음식에적용하였다. V V = Vc V T (Vv = 체적밀도, Vc = 대상물의체적, V T = 전체체적, Pc = intersection 된대상물점의수, P T = 전체점의수 ) 3) 수밀도 (numerical density) 사립체의수밀도계산은 10 d (d=10 mm) 인단일격자 test grid를사용하여계수한후다음식에적용하였다. N V = = Pc P T 1 N A 3/2 1.58 = V V 1/2 Table 1. Changes of serum AST and creatinine activities at 5 h postburn Groups AST (Karmen unit/ml) Creatinine (mg/dl) Control (n=10) 115.0±2.62 0.406±0.0198 Burn 5 h (n=10) 143.0±4.43 0.477±0.0402 Burn + Mo (n=10) 117.3±2.76 0.434±0.0301 Each value represents the mean ± S.E. The numbers of determinations are in the parentheses. Burn + Mo; Mori cortex administration after burn (Nv = 수밀도, N A = 단위면적당대상물의수, Vv = 단위체적 ) 10. 통계처리각실험군비교를위한통계처리는 SPSS WIN 통계프로그램을이용하여유의수준을 0.05로하는일원배치분산분석 (ANOVA) 을실시하였다. 결과 1. 혈청내효소활성변동화상유발후혈청 AST와 creatinine 활성변화를관찰한결과 (Table 1), 화상유발 5시간후혈청 AST의활성은 24.3% 증가되었고, 상백피투여군에서는화상군과비교하여 21.9% 감소되었다. 혈청 creatinine 활성은화상유발 5시간군에서 17.4% 증가되었고, 상백피의투여로화상군보다 9.9% 감소되었다. 2. 화상유발후심근조직내 KC, MPO 및 PKC 활성변동화상유발후심근조직내호중구침윤유도물질의활성을나타내는 KC와호중구침윤정도를나타내는 MPO의활성및 PKC 활성의변화를관찰한결과 (Table 2), 화상유발후 5시간군에서 KC의함량은 6.4% 증가되었고, 상백피의투여로화상군과비교하여 12.3% 감소되었다. 심근조직내 MPO 활성은화상유발후 5시간군이대조군과비교하여볼때약 2.2배감소되었고 (P<0.01), 상백피투여군에서도화상군과같은수준의 MPO 활성이나타났다 (P<0.05). 심근조직내 PKC 활성은대조군과비교하여화상유발후 5시간군에서 76% 증가되었고, 상백피의투여로화상군보다 38.6% 증가되었다. 3. 심근의미세구조적관찰화상으로유도된심근조직의미세구조적관찰결과, 화상유발 5시간후불규칙하게분열된핵이관찰되었고 (Fig. 3b), 수축대의형성과 muscle fraying 현상이나타났다 (Fig. 2). 근 Table 2. Changes of neutrophil chemoattractant cytokine (KC), myeloperoxidase (MPO) and protein kinase C (PKC) activities in heart tissue at 5 h postburn Groups KC (pg/ml) MPO (U/g of weight) PKC (pmol/min/µg) Burn 5 h (n=10) 164.5±0.41 0.022±0.0006 0.025±0.0001 Burn 5 h (n=10) 175.0±1.30 0.010±0.0003 **a) 0.044±0.0004 Burn + Mo (n=10) 155.7±0.32 0.011±0.0004 *a) 0.061±0.0002 Each value represents the mean ± S.E, The numbers of determinations are in the parentheses, Burn + Mo; Mori cortex administration after burn, * ; P<0.05, ** ; P<0.01, a) ; Significant difference compared to control - 109 -
N M M N A B Fig. 1. Electron micrograph of normal cardiac muscle, uranyl acetate and lead citrate stain. Scale bar indicates 2 µm. M: mitochondria, : myofibrils, N: nucleus Fig. 3a. Electron micrograph of cardiac muscle at 5 h postburn, uranyl acetate and lead citrate stain. Enlarged intercalated disk (arrow) and disrupted sarcolemma were observed. Scale bar indicates 2 µm. M: mitochondria, : myofibrils Fig. 3b. Electron micrograph of cardiac muscle at 5 h postburn, uranyl acetate and lead citrate stain. Nucleus (N) was cloven in a few lobes. Scale bar indicates 2 µm. : myofibrils * M * Fig. 2. Electron micrograph of cardiac muscle at 5 h postburn, uranyl acetate and lead citrate stain. Contraction band (*) was shown. Scale bar indicates 2 µm. 세포막의일부가파괴되었고, 사이원반의분리현상도관찰되었다 (Fig. 3a). 상백피투여군에서는화상군에서관찰되었던수축대와 muscle fraying 현상이현저히감소되어나타났으며, 정상적인모습의핵과근세포막이잘보존되어 (Fig. 4) 대조군 (Fig. 1) 과유사한형태로관찰되었다. 4. 심근의입체해석학적관찰피부화상에의한심근조직내에포함된근원섬유, 사립체의양적변화를분석한결과 (Table 3), 근원섬유의체적밀도는화상유발후 5시간군에서대조군과비교하여낮게나타났고, 상백피의투여로현저하게증가되었다 (P<0.05). 화상 Fig. 4. Electron micrograph of cardiac muscle at 5 h postburn administrated with Mori cortex, uranyl acetate and lead citrate stain. ofibrils appeared intact and formed a regular array. Scale bar indicates 2 µm. M: mitochondria, : myofibrils 유발 5시간후사립체의체적밀도는대조군과비교하여볼때 18.7% 감소되었고 (P<0.001), 상백피투여군은화상군보다 9.3% 증가되었다. 1 µm 3 내에포함되는사립체의수를나타내는사립체의수밀도는, 화상유발후 5시간군이대조군과비교하여볼때높게나타났으며상백피투여군에서는사립체수밀도가가장낮게나타났다. - 110 -
Table 3. Results of stereological analysis in heart tissue at 5 h postburn Groups Control (n=10) Burn 5 h (n=10) Burn + Mo (n=10) ofibril 0.450±0.0838 0.448±0.1277 0.484±0.0949 *b) Volume density (µm 3 /µm 3 ) Mitochondria 0.292±0.0937 0.246±0.0795 ***a) 0.269±0.1213 Interstitium 0.256±0.0202 0.304±0.0014 0.246±0.0106 Numerical density (Number/µm 3 ) Mitochondria 11.089±0.4839 12.770±0.8975 9.472±0.6380 Each value represents the mean ± S.E, The numbers of determinations are in the parentheses, Burn + Mo; Mori cortex administration after burn, * ; P<0.05, *** ; P<0.001, a) ; Significant difference compared to control, b) ; Significant difference compared to burn 5 h 고찰응급실에서흔하게볼수있는화상손상은화상부위뿐만아니라 multiple organ failure (MOF) 를유발시키기도한다 4). 그러나이러한 MOF와같은조직손상의병태생리적기작은아직명확히밝혀진바없고몇몇연구들에서국소적인화상손상은조직내산화제들을증가시킴으로인해타장기에손상을유도한다는증거들을보여주고있다 34). 따라서본실험에서는피부열상에의해발생한부종을없애고, 또한열상에의한혈관성부종을포함하는병증에효과가있는상백피 44) 를열탕추출하여피부화상유발후경구투여하고, 화상에의해손상된심근에영향을미치는지생화학적정량법과면역화학적분석법, 미세구조적관찰및입체해석학적분석을통해검토해보고자하였다. 먼저화상에의한심근의손상정도를확인하기위해혈중 AST를측정한결과, 높은활성을나타낸화상군에비해 41) 상백피투여군에서는감소되었고, 심장손상시혈액에서가장먼저활성이증가하는 creatinine은화상군에서높은활성을나타내었지만 19) 상백피의투여로대조군과같은수준으로그활성이감소되어화상으로인한심근손상에효과가있음을확인하였다. 화상은다양한염증반응유발물질들과염증관련효소들의변화를초래하게되고 6), 이러한변화들이심근조직의염증성손상을유도하여 43) 심근기능과형태의변화를일으키는것으로 25) 알려져있다. 화상후일어나는염증반응정도를알아보기위해심근조직내호중구침윤유도물질인 KC와호중구로부터유리되는 MPO의활성을측정하였다. 그결과심근조직내 KC 함량은화상군에서증가되었다가상백피투여군에서감소되는경향으로나타났다. 그러나 MPO 활성은화상군과상백피투여군모두에서정상군과비교하여유의하게감소된활성을보여 KC의함량변화와일치되지않았으나 KC의함량이증가된것은피부화상으로부터심근조직내염증유도물질의상승이일어났음을보여주는것이다 14). 그리고화상군에서 KC가증가되었음에도불구하고 MPO의활성이감소된것은호중구의축적은일시적인현상일뿐대부분의호중구들은화상부로이동하는것으로추측되는데 2,3), 화상손상후신장과회장에서도이와같은결과를보인다고하였다 12). 그리고열손상에대한세포반응은 protein kinase pathways 에의해조절되며그중 PKC는염증반응에관여하는효소로 11), 세포막을통해다양한형태의신호를세포내로전달하여단백질을인산화시켜 phospholipase A 2 (PLA 2 ) 가활성화되면염증반응을유도하기도하고, lipocortin 합성을유도하여세포막인지질로부터 arachidonic acid를생성하게하는 PLA 2 를억제하여염증반응유발물질들의합성을억제함으로써혈관투과성항진, 부종, 백혈구의염증부위로의이동등의일련의염증반응을억제하기도한다 9,26). 이러한 PKC의상반된의견을바탕으로, PKC가염증반응을증가혹은억제시키는지알아보고자 PKC의활성을측정한결과, 화상군에서증가된 PKC 활성은상백피투여로인해더욱높은활성을나타내었다. 즉 PKC의활성증가는 PLA 2 를억제하여 MPO의활성저하를유도하고, 염증유발물질인 KC 생성도저하시켜심근조직내염증반응을감소시키는것으로판단되며, PKC를전신에투여하면화상에대한염증반응을감소시키고심근을보호하는등많은세포반응을변화시킬수있다는보고 19) 와도일치하였다. 염증반응뿐만아니라 PKC는화상에의한심근수축반응과심근보호에도중요한중재자로작용한다고알려져 11) 심근의미세구조적관찰결과와입체해석학적분석을통해확인하였다. 화상에의해심근은심근수축과관련된수축대의형성과 muscle fraying 현상, 불규칙적으로분열된핵등의형태적변형 10,35) 이나타났으나, 상백피의투여로수축대의형성과 muscle fraying 현상이현저히감소되어심근의수축력이향상될수있으며, 전반적으로화상에의한심근의손상정도를어느정도완화시켜줌을확인할수있었다. 이는상백피투여군에서증가된 PKC 활성이수용체의기능과막수송 27) 에관여하는수축을조절하는칼슘의방출을자극하여심근수축력을증가시켰다고판단된다. 심근손상의변화에대한구체적인형태변화를알아보기 - 111 -
위해입체해석학적분석을실시한결과, 화상군에서근원섬유의체적밀도가감소되어심근수축력이저하되었고 40), 상백피의투여로근원섬유의체적밀도는대조군보다증가되어생화학적측정결과에서 PKC 활성이높았음을감안해볼때 PKC 활성이높을수록심근의수축력이증대될것으로예측되었다 5,30). 또한화상군에서감소된사립체의체적밀도는상백피의투여로증가되어상백피는근원섬유와산소부족시세포상해의일차공격대상인사립체 16) 를보호하는효과가있는것으로사료된다. 화상군에서나타난세포간질의증가는심근세포의현저한활동성저하를보여주고있으며, 사립체의체적밀도감소는수밀도증가로이어져사립체각각의활동성저하로인해심근세포의정상적인기능을보상하려는작용으로사립체가분획된것으로판단된다. 이와같은결과들을종합해볼때, 화상으로유도된심근손상은염증반응과관계가있으며상백피는 PKC를활성화시켜염증반응을감소시키고화상으로인해손상된심근의수축력을증가시키는데효능이있는것으로나타났다. 참고문헌 1) Albert CJ and Ford DA (1999): Protein kinase C translocation and PKC-dependent protein phosphorylation during myocardial ischemia. Am J Physiol, 276(45): H642-H650. 2) Arbak S, Ercan F, Hurdag CG, Karabulut O, Gurbuz V, Corak A and Alican I (1999): Acute lung injury following thermal insult to the skin; a light and transmission electron microscopial study. Acta Histoche, 101(3): 255-262. 3) Baskaran H, Yarmush ML and Berthiaume F (2000): Dynamics of tissue neutrophil sequestration after cutaneous burns in rats. J Surg Res, 93: 88-96. 4) Bertin-Maghit M, Goudable J, Dalmas E, Steghens JP, Bouchard C, Gueugniaud PY, Petit P and Delafosse B (2000): Time course of oxidative stress after major burns. Intensive Care Med, 26: 800-803. 5) Buenaventura P, Cao-Danh H, Glynn P, Takeuchi K, Takahashi S, Simplaceany E, McGowan FX and Nido PJ (1995): Protein kinase C activation in the heart: Effects on calcium and contractile proteins. Ann Thorac Surg, 60: S505-S508. 6) Cain BS, Meldrum DR and Harken AH (1999): Protein kinase C in normal and pathologic myocardial states. J Surg Res, 81: 249-259. 7) Cetinkale O, Belce A, Konukoglu D, Senyuva C, Gumustas MK and Tas T (1997): Evaluation of lipid peroxidation and total antioxidant status in plasma of rats following thermal injury. Burns, 23(2): 114-116. 8) Cetinkale O, Konukoglu D, Senel O, Kemerli GD and Yazar S (1999): Modulating the functions of neutrophils and lipid peroxidation by FK506 in a rat model of thermal injury. Burns, 25(2): 105-112. 9) Chang J, Musser JH and McGregor H (1987): Phospholipase A 2 : Function and pharmacological regulation. Biochemical Pharmacology, 36(15): 2429-2436. 10) Chi L, Yang Z and Huan Y (1998): Effects of abnormal distribution of calcium on impairment of myocardial mechanics in the early stage of thermal injury. Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 14(1): 33-36. 11) Clerk A (2001): Death by protein kinase C inhibitor: a stressful event. J Mol Cell Cardiol, 33: 1773-1776. 12) Dries DJ, Lorenz K and Kovacs EJ (2001): Differential neutrophil traffic in gut and lung after scald injury. J Burn Care Rehabil, 22(3): 203-209. 13) Fagan JM, Ganguly M, Stockman H, Ferland LH and Toner M (1999): Posttranslational modifications of cardiac and skeletal muscle proteins by reactive oxygen species after burn injury in the rat. Ann Surg, 229(1): 106-114. 14) Faunce DE, Llanas JN, Patel PJ, Gregory MS, Duffner LA and Kovacs EJ (1999): Neutrophil chemokine production in the skin following scald injury. Burns, 25: 403-410. 15) Goldblum SE, Wu KE and Jay M (1985): Lung myeloperoxidase as a measurement of leukostasis in rabbits. J Appl Physiol, 1978-1985. 16) Hochachka PW (1986): Defense strategies against hypoxia and hypoyhermia. Science, 231: 234. 17) Horton JW (1996): Oxygen free radicals contribute to postburn cardiac cell membrane dysfunction. J Surg Res, 61: 97-102. 18) Horton JW and White DJ (1995): Role of XO and leukocytes in postburn cardiac dysfunction. J Am Coll Surg, 181(2): 129-137. 19) Horton JW, White DJ and Maass D (1998): PKC inhibition improves ventricular function after thermal trauma. J Trauma, 44(2): 254-265. 20) Horton JW, White DJ, Maass D, Sanders B, Thompson M and Giroir B (1999): Calcium antagonists improve cardiac mechanical performance after thermal trauma. J Surg Res, 87: 39-50. 21) Hu S, Sheng Z, Zhou B, Guo Z, Lu J, Xue L, Jin H, Sun X, Sun S, Li J and Lu Y (1998): Study on delay two-phase multiple organ dysfunction syndrome. Chin Med J(Engl), 111(2): 101-108. 22) Jeschke MG, Low JF, Spies M, Vita R, Hawkins HK, Herndon DN and Barrow RE (2001): Cell proliferation, apoptosis, - 112 -
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