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1 中西医结合学报２００７年７月第５卷第４期 Jo urn al o f C hi nese In t eg r a tiv e M edicin e Ju ly ２００７ V ol ５ N o ４４２７参附注射液对肝缺血再灌注大鼠血浆前列环素和血栓素 A ２及肝组织 A TP 酶的影响彭松林顾玺戴朝六黄勇赵阳２中国医科大学盛京医院肝胆乳腺外科辽宁沈阳０００４２山东省枣庄市市立医院普外科山东枣庄２７７００目的探讨参附注射液对大鼠肝缺血再灌注损伤的保护作用及其机制方法２４只 Wis ta r 大鼠随机分为模型组和参附注射液 S hen f u Injectio n SF 治疗组 S F 治疗组大鼠腹腔注射参附注射液０ m l k g 模型组大鼠给予相同剂量的生理盐水两组均采用 Pri ng le s 法阻断肝门缺血５ min 再灌注 h 和３ h 测定血浆血栓素 B２ t hr om bo xane B２ T XB２和６酮前列腺素 F α ６ ket o pr os ta glandin F α ６ ket o P GF α 以及肝组织匀浆 Na K A T P 酶 Ca A T P 酶的变化并观察肝组织形２２态学改变结果再灌注３ h S F 治疗组血浆 T XB２低于模型组６ ket o P GF α 高于模型组两者比值 T XB２６ ket o P GF α 低于模型组再灌注 h ３ h S F 治疗组 N a K A T P 酶和 Ca ２２组肝实质细胞和线粒体损伤明显减轻 A T P 酶活性高于模型组 SF 治疗结论参附注射液对肝缺血再灌注损伤有保护作用其机制与降低 T XA ２ P G I２比值提高 N a K A T P 酶和 Ca ２２ A T P 酶活性有关关键词缺血再灌注损伤血栓素 A ２前列环素 Na K A T P 酶 Ca A T P 酶２２中图分类号 R６５７３文献标识码 A 文章编号６７２９７７２００７０４０４２７０５ Effects of Shenfu Injection on prostacyclin thromboxane A2 and activities of ATPases in rats exposed to hepatic ischemia reperfusion injury Song lin PENG Xi GU Chao liu DAI Yong HUANG Yong ZHAO ２ Dep ar tment of H epato biliar y and M am ma ry Sur ger y T he Second Hospital Ch ina M edical U niversit y Shen yang Liao ning Pr ovince ０００４ C hina ２ Dep ar tment of G eneral Sur ge ry Zaozhuan g M unicipal H os pital Zaozhua ng Sh andon g P ro vince ２７００ C hina Objective To explore the effects of Shenfu Injection on prostacyclin thromboxane A２ and the activities of ATPases in rats exposed to hepatic ischemia reperfusion injury Methods Twenty four male Wistar rats weighing ２００２５０ g were randomly divided into two groups Shenfu Injection SF treated group rats were treated with Shenfu Injection of ０ ml kg through intraperitoneal injection and untreated group rats were administered wi th normal saline at the same dose and served as a control group Hepatic ischemia was caused by Pringle s maneuver and lasted for fifteen minutes and then one hour or three hour reperfusion was performed Venous blood samples for the measurement of thromboxane B２ TXB２ and ６ keto prostaglandin F α ６ keto PGF α were collected three hours after reperfusion Liver tissue samples were collected one hour or three hours after reperfusion for the measurement of Na K ２２ ATPase and Ca Mg ATPase and for morphological studies Results Plasma TXB２ was lower in the SF treated group than that in the untreated group after three hour reperfusion P ００５ while ６ keto PGF α was higher in the SF treated group than that in the untreated group P ００５ The ratio of TXB２ and ６ keto PGF α was significantly lower in the SF treated group than that in the ２２ untreated group P ００５ The activities of Na K ATPase and Ca Mg ATPase in the SF treated group were improved obviously A three hour reperfusion after fifteen minute ischemia caused important hepatic histological alterations Marked structural abnormali ties were observed in the untreated group such 基金项目辽宁省教育厅重大应用基础研究基金 N o ２００４C０５２ Co rrespondence Prof C hao liu DAI T el ０２４８３９５５２３２ E mail daicl cmu２h com

2 ４２８ Jo ur nal of C h in ese In t eg r a ti ve M edicin e J ul y ２００７ V ol ５ N o ４中西医结合学报２００７年７月第５卷第４期 as massive hepatocyte swelling necrosis mitochondria edema and vacuolar changes In the SF treated group hepatic tissue injury was reduced significantly Conclusion Shenfu Injection protects hepatic tissue from ischemia reperfusion injury and such protective effects are achieved by decreasing the ratio of thromboxane A２ and prostacyclin and increasing the activities ２２ of Na K ATPase and Ca Mg ATPase Keywords ischemia reperfusion injury thromboxane A２ prostacyclin Na K ATPase Ca Mg ATPase ２２ Pe ng S L Gu X Dai C L H uang Y Zhao Y J Ch in Integ r Med Zhong X i Yi J ie H e X ue Bao ２００７５４４２７４３ R eceived Sep tem ber ３２００６ pub lish ed o nlin e July ５２００７ Free fu ll text PD F in available at w w w jcimjournal co m 肝脏缺血再灌注损伤是肝切除严重肝外伤以立即置于液氮中冷冻用锡箔纸包裹置于液氮中保及肝脏移植手术中经常遇到的问题和导致术后肝功存再转至８０冰箱中保存待测 Na K A T P 能衰竭的重要原因研究表明缺血再灌注导致肝脏酶和 Ca 损伤的机制与细胞能量代谢障碍线粒体功能受损３细胞内钙超载氧自由基产生一些细胞因子的合成３与释放等有关参附注射液由人参附子提取物组成其有效成分为人参皂苷和乌头类生物碱本文通过观察参附注射液对血栓素 A ２ t hr o m bo xa ne ２２ A T P 酶然后处死大鼠检测指标与方法血浆 T X A ２和 P G I２的稳定代谢产物血浆血栓素 B２ t hro m bo xane B２ T XB２和６酮前列腺素 F α ６ ket o pr os ta glandi n F α ６ ket o P GF α 测定用放免法进行测定６ keto P GF α 和 T XB２试剂盒 A ２ T X A２和前列环素 pr ostacyclin P G I２ Na 购自北京解放军总医院科技开发中心放免所按照注射液保护肝缺血再灌注损伤的机制３２ 1 盒购自南京建成生物工程研究所底物三磷酸腺苷钠购自中国科学院上海生物化学研究所用电子天 K A T P 酶和 Ca ２２ A T P 酶的影响探讨参附材料与方法说明书进行操作肝组织 A T P 酶水平的测定 A T P 酶测试２４只 Wist ar 大鼠体质量平称取冰冻肝脏组织００ m g 先用比色法按考马斯注射液 Sh enfu Injection S F 治疗组 S F 治疗组量蛋白含量 g L 测定管吸光度标准管吸光０３５０ ml kg 腹腔注射次 d 连续给药６ d 明测定组织匀浆中 A T P 酶的活性 A T P 酶活力动物分组与给药２００２５０ g 随机分为肝缺血再灌注模型组和参附给予参附注射液雅安三九药业有限公司批号亮蓝试剂盒说明书操作测定肝组织匀浆的蛋白含度标准管浓度 g L 同样用比色法按测试盒说第６天于手术前３０ min 给药模型组大鼠同样方 μ mol Pi m g pr ot h 测定管吸光度对术给药期间常规自由喂养给水 μm ol m l 反应体系中样本稀释倍数６组法给予相同剂量的生理盐水两组均于第６天手２动物模型及取材术前２ h 禁食自由饮水０水合氯醛３ ml k g 腹腔注射麻醉后剃除腹部体毛局部消毒取腹正中切口开腹开腹后离断肝周韧带包括镰状韧带肝胃韧带冠状韧带消除肝照管吸光度标准管吸光度标准管浓度织中蛋白含量 m g pro t ml ３３组织细胞学改变分别采用光学显微镜和 T JE M ２００EX 透射电子显微镜观察４统计学方法肝组织 AT P 酶水平和血浆 T XB２６ keto PGF α 测定结果以 x ± s 表示应用 SPSS 脏侧枝循环用 Pri ng le s 法无创伤微血管夹夹闭门静脉肝动脉胆总管使肝缺血持续５ min 后０统计软件进行统计分析采用成组 t 检验松开血管夹恢复肝脏血供然后关腹分别于再灌 2 注 h 和３ h 再次开腹先取一次性５ m l 无菌注射２结果血浆 T XB２６ keto P GF α 浓度肝脏缺血器吸取消炎痛 ED T A N a２０２ ml 自下腔静脉肝下５ min 再灌注３ h 后 S F 治疗组血浆 T XB２浓度较３５００ r min 上海手术器械厂８０２型离心沉淀器 P GF α 比值明显降低 P ００５见表段快速抽取静脉血３ ml 即刻在空针内颠倒混匀模型组低而６ ket o P GF a 浓度升高 T XB２６ ket o ４离心５ min 分离血浆２０保存用取肝２２组织 PBS 液冲洗后分别置于０福尔马林溶液中和 Ca 组织镊子快速夹取大小约 cm 和０５ c m 两块肝３３肝组织中 A T P 酶水平肝缺血５ min 再灌注 h 和３ h S F 治疗组肝组织中 Na K A T P 酶２２ A T P 酶水平均明显高于模型组 P 固定和２５戊二醛溶液中固定用于光学显微镜００５见表２和电镜检查快速切取剩余肝组织 PBS 液冲洗后２３组织细胞学改变光镜下模型组肝细胞浊肿

中西医结合学报 2007 年 7 月第 5 卷第 4 期 Journ al o f C hinese Int egra tiv e M edicin e, July 2007 ; V ol.5, N o.

3 h 血浆 TXB 2 6 keto PGF 1 α 浓度及其比值 Table 1 Plasma concentrations of TXB2 and 6 keto PGF1 α and their ratio after three hour reperfusion ( x ± s ) Group n T XB 2 ( ng / L) 6 ke t o PG F 1a (n g/ L) T

表 2 再灌注 1 h 和 3 h 肝组织中 ATP 酶水平 Table 2 Levels of hepatic tissue ATPases after one or three hour reperfusion [ x ± s, μmol Pi/(mg prot h)] 1 h 3 h G roup n N a + K + A T Pase Ca 2 + M g 2 + A T Pase N

3 中西医结合学报 2007 年 7 月第 5 卷第 4 期 Journ al o f C hinese Int egra tiv e M edicin e, July 2007 ; V ol.5, N o 大片坏死, 肝窦和微血管内明显淤血 SF 治疗组肝窦内淤血和组织损伤明显减轻, 可见小灶状坏死电镜下模型组肝细胞膜缺损不连续, 甚至溶解 ; 细胞器中线粒体损伤尤为明显, 大量线粒体空泡变性外膜溶解 ; 核异染色质边集凝集成块, 核膜不规则 ; SF 治疗组肝细胞损伤较轻, 胞膜较完整, 线粒体和内质网较丰富, 核内充盈, 但亦有染色质边集见图 1 ~ 4 表 1 再灌注 3 h 血浆 TXB 2 6 keto PGF 1 α 浓度及其比值 Table 1 Plasma concentrations of TXB2 and 6 keto PGF1 α and their ratio after three hour reperfusion ( x ± s ) Group n T XB 2 ( ng / L) 6 ke t o PG F 1a (n g/ L) T XB 2 /6 k et o PG F 1 a Untrea ted ± ± ± 0.24 S F trea ted ± ± ± 0.03 P < 0.05, vs untreat ed group. 表 2 再灌注 1 h 和 3 h 肝组织中 ATP 酶水平 Table 2 Levels of hepatic tissue ATPases after one or three hour reperfusion [ x ± s, μmol Pi/(mg prot h)] 1 h 3 h G roup n N a + K + A T Pase Ca 2 + M g 2 + A T Pase N a + K + A T Pase Ca 2 + M g 2 + A T Pase Unt rea t ed ± ± ± ± 0.65 S F trea te d ± ± ± ± 2.93 P < 0.05, vs untreat ed group. 图 1 模型组肝细胞浊肿窦内淤血大片坏死 (HE 染色, 100) Figure 1 Massive hepatocyte swelling, necro sis, sinusoidal and microvascular congestion in untreated group (HE staining, 100) 图 3 模型组肝细胞及其线粒体严重损伤 ( 透射电镜, 8 000) Figure 3 Severe injuries of hepatocytes and mitochondria in untreated group (transmission electron microscope, 8 000) 图 2 SF 治疗组肝窦内淤血和组织损伤明显减轻, 可见小片状坏死 (HE 染色, 100) Figure 2 Sinusoidal congestion and tissue injury were reduced significantly in SF treated group (HE staining, 100) 图 4 SF 治疗组肝细胞和线粒体损伤减轻 ( 透射电镜, 8 000) Figure 4 Injuries of hepatocytes and mitochondria were reduced in SF treated group (transmission electron microscope, 8 000)

4 430 中西医结合学报 2007 年 7 月第 5 卷第 4 期 Jo ur nal of C hin ese Int egra ti ve M edicin e, J ul y 2007 ; V ol.5, N o.4 3 讨论肝脏缺血再灌注损伤首先表现的是微循环障碍, 具有血管活性的细胞因子导致的微循环收缩是导致肝缺血再灌注损伤的一个重要因素 K ondo [ 1 ] 等报告在肝脏缺血期, 常温下缺血 20 min 就导致肝血窦直径和窦后小静脉直径在缺血末分别缩小 25 % 和 20 %, 而且缺血后的肝功能障碍与这种微循环的收缩有关 T X A2 和 PGI2 是花生四烯酸代谢的两个产物 PGI2 具有很强的扩血管作用, 同时通过升高 ca M P 加强渗透屏障内源性 PG I2 的反应性血管舒张作用维持比较短暂, 更为重要的是 PGI2 可能激活依赖 Ca 2 + 激活的 K + 通道和依赖 A T P 激活的 K + [ 2, 3 ] 通道, 这些离子通道的开放可使细胞膜超极化, 减少细胞内钙超载和起到持久舒张血管的作 [ 4 ~ 6 ] 用而 T X A 2 是一种很强的缩血管物质, 能增加微血管的通透性研究表明缺血再灌注后 PGI2 [ 7 ] 的释放下降或 T X A2 的释放增加, 在再灌注损伤 [ 8 ] 中起着重要作用肝缺血再灌注时 T X A2 明显升高, 早期更趋明显, 晚期下降 ;PGI2 的含量亦增高, 但增高的梯度远低于 T X A2 ;T X A 2 /PGI2 比值在肝缺血再灌注早期增高明显, 再灌注 24 h 接近正 [ 9 ] 常用 T X A2 合成酶抑制剂抑制 TX A 2 的释放或 [ 1 0 ] 用受体拮抗剂阻断 TXA 2 效应 [ 7 ] 或通过提高内源性 PGI2 水平 [ 8 ], 用 PGI2 拟似剂, 都可起到防护缺血 [ 2 ] 再灌注损伤的作用 Xiao 等报告当血栓素受体缺乏时也并不减轻心脏的缺血再灌注损伤, 而当 PGI2 受体缺乏时却明显加重心脏缺血再灌注损伤 ; 内源性 PGI2 水平下降比 T XA2 水平升高更易导致再灌注后的组织损伤因此, 改善 PGI2 与 TX A 2 二者比例失衡是防护缺血再灌注损伤的一个重要机制 Na + K + A T P 酶和 Ca 2 + M g 2 + A T P 酶是反映细胞能量代谢和建立跨膜的离子梯度维持细胞膜电位与细胞内外离子平衡的两个重要的蛋白酶, 在生理条件下又称依赖 A T P 的膜结合蛋白酶肝脏缺血再灌注后二者的活性降低, 热缺血时降低更 [ 1 1 ] 为严重, 且与肝细胞死亡的方式相关谷天祥 [ 1 2 ] 等已证实二者泵功能抑制是心肌缺血再灌注后发生在亚细胞水平 Ca 2 + 超载的直接原因肝缺血再灌注后 Na + K + A T P 酶和 Ca 2 + Mg 2 + A T P 酶活性降低, 导致亚细胞水平 Na + Ca 2 + M g 2 + 分布, 从而引起细胞内 N a + Ca 2 + 超载和 M g 2 + 重新低 Na + K + A T P 酶活性下降引起细胞内 Na + 超载又激活细胞膜上 Na + Ca 2 + Ca 2 + 降交换蛋白, 使更多进入细胞内 ; 同时还引起线粒体中 Ca 2 + 释放, 最终导致胞内 Ca 2 + 超载细胞内 M g 2 + 减少, 一方面削弱它对 Ca 2 + 增多的生理拮抗作用, 另一方面, 低 M g 2 + 本身还可降低细胞内 30 多种酶的活力, 抑制能量合成, 增加细胞膜通透性和减少蛋白质合成, 结果细胞内晶体渗透压增高水增加细胞肿胀, 最终导致细胞死亡提高二者的活性有助于减轻组织 [ 1 3 ] 的缺血再灌注损伤本实验大鼠肝脏在缺血 15 min 再灌注 3 h 后出现明显的组织损伤, 肝细胞肿胀坏死 ; 电镜下大量线粒体水肿和空泡变性在再灌注 3 h 参附注射液通过降低 TXA 2 和提高 PGI2 改善 PGI2 与 T X A2 二者的比例失衡 ; 在再灌注 1 h 3 h 参附注射液明显提高 Na + K + A T P 酶和 Ca 2 + M g 2 + A T P 酶的活性 ; 同时在再灌注 3 h 组织损伤明显减轻因此参附注射液通过改善缺血再灌注后 PGI2 与 T X A2 的比例失衡, 和提高 N a + K + A T P 酶与 Ca 2 + M g 2 + A T P 酶的活性保护大鼠肝脏缺血再灌注损伤 REFERENCES 1 Ko ndo T, T odoroki T, Hirano T, et al. Impact o f ischemia reperfusion injury on dimensio nal changes o f hepa tic microvessels. Res E xp Med (Berl) ; 198 (2) : Xiao CY, Hara A, Yuhki K,et al.roles o f prostaglan din I2 and thromboxane A2 in cardiac ischemia reperfu sion injury : a study using mice lacking their respective rece pto rs. Circulatio n ;104(18) : Schubert R,Serebrya ko v V N, Me wes H,et al.iloprost dilates rat small arteries : role of K (A T P) and K(Ca) channel activation by c AM P depende nt protein kinase. Am J Physiol.1997 ; 272 (3 pt 2) : H1147 H No de K, Kitakaze M, Kosaka H,et al.brady kinin me dia tio n of Ca 2 + activated K + channels reg ulates co ro nary blood flo w in ischemic myocardium. Circulation ; 95(6) : Gross GJ, Auchampach JA. Blocka de of A T P sensitive po tassiu m channels prevents m yocardial preconditioning in dogs.circ Res.1992 ;70(2) : de Wit C, Bolz SS, Pohl U. Interaction of endo thelial autacoids in microvascular control. Z Kardiol.2000 ;89 ( suppl 9) : Ⅸ / M yers SI, Horto n JW, He rnandez R, et al.pento xif yl line protects splanchnic prostacyclin synthesis during mesenteric ischemia/reperfusion. Prosta glandins.1994 ; 47(2) : Jiang XW, Kam bara K, Go toh N, et al.ef fects of lo w dose Beraprost sodium, a stable prostaglandin I 2

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TGF-β AngⅡ B SD ~ 220g SPF. SCXK No SYXK ~ 25 40% ~ 70% OR37G-C

TGF-β AngⅡ B SD ~ 220g SPF. SCXK No SYXK ~ 25 40% ~ 70% OR37G-C 2017 7 43 4 Journal of Southwest Minzu University Natural Science Edition Jul. 2017 Vol. 43 No. 4 doi10. 11920 /xnmdzk. 2017. 04. 008 1 1 1 2 1 1 3 1. 610212 2. 6100413. 610031 AngⅡ TGF-β. SD 60 180 ~