肠淋巴途径在失血-脂多糖二次打击大鼠心肌损伤中的作用

Role of intestinal lymphatic in myocardial injury in rats receiving hemorrhage and lipopolysaccharide challenge

目的探讨肠系膜淋巴管结扎干预失血-脂多糖（LPS）致大鼠心肌损伤的作用机制。方法将雄性Wistar大鼠按随机数字表法分为假手术组、未结扎组、结扎组；以失血-LPS复制二次打击动物模型，结扎组于失血后行肠系膜淋巴管结扎术以阻断肠淋巴液回流。创伤后24h处死各组大鼠制备心肌组织匀浆，检测髓过氧化物酶（MPO）、ATP酶活性以及肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）含量；制备心肌病理切片，用原位末端缺刻标记法（TUNEL）检测细胞凋亡率，免疫组化法检测bcl-2和hax蛋白表达。结果未结扎组大鼠心肌MPO[（0．23±0．08）U／g]、TNF-α[（9．99±2．74）μg／g]、IL-6[（31．57±12．71）μg／g]均显著高于假手术组[MPO：（O．12±0．03）U／g、TNF-α：（4．17±1．35）μg/g、IL-6：（17．86±5．17）μg／g，均P〈0．01]，ATP酶活性显著低于假手术组；结扎组大鼠心肌MPO[（0．13±0．03）U／g]、TNF-α[（5．57±1．65）μg／g]、IL-6[（23．24±5．95）弘g／g]均显著低于未结扎组（P〈0．05或P〈0．01），ATP酶活性显著高于未结扎组。未结扎组心肌细胞凋亡率[（22．7±6．9）％]、心肌细胞bax蛋白表达（104．5±11．4）显著高于假手术组[凋亡率：（3．8±1．2）％，bax蛋白：142．1±10．9]和结扎组[凋亡率：（8．4±2．8）％，bax蛋白：128．4±9．6]，bcl-2蛋白表达（196．4±19．3）显著低于假手术组（132．2±12．3）和结扎组（165．1±11．6，均P〈0．01）。结论肠系膜淋巴管结扎通过降低炎症介质TNF-α、IL-6水平，提高bcl-2蛋白表达和心肌细胞膜ATP酶活性，从而干预失血-LPS致大鼠心肌损伤。

Objective To explore the effect of mesenteric lymph duct ligation in protecting myocardial injury in rats after hemorrhage and lipopolysaccharide （LPS） challenge. Methods Wistar rats were randomly divided into the sham group, non-ligation group and ligation group, and the ＂two-hit＂ injury model was reproduced by hemorrhage and LPS administration. Mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group after hemorrhage. After 24 hours of the assault, myocardial tissue was harvested and homogenized. The activity of myeloperoxidase （MPO） and ATPase and the contents of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined in myocardial homogenate. At the same time, myocardium was pathologically studied. Apoptosis cell rate of myocardium was determined by method of terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL）, the expression of bcl-2 and bax protein was determined by immunohistochemical method. Results After hemorrhage and LPS challenge, the level of MPO （（0. 23 ±0.08） U/g], TNF-α （（9. 99 ±2.74） μg/g] and IL-6 [（31.57 ±12.71）μg/g] in myocardial homogenate of non-ligation group were significantly higher than those of sham group （MPO.. （0. 12±0, 03） U/g, TNF-α （4. 17±1.35） μg/g, IL-6： （17.86±5.17）μg/g, respectively, all P 〈 0. 013, and the activity of ATPase was significantly lower. MPO （（0.13 ±0.03） U/g ], TNF-α [（5.57±1.65）μg/g] and IL-6 [（23.24± 5.95） μg/g3 in myocardial homogenate of ligation group were significantly lower （P〈0. 05 or P〈0.01）, and the ATPase activity was significantly higher compared with non-ligation group. The apoptosis rate （（22.7± 6.9）%] and expression of bax protein （104. 5± 11.4） of myocardial cells in non-ligation group were significantly higher compared with sham group （apoptosis rate： （3.8±1.2）%, bax protein： 142.1±10.93 and ligation group （apoptosis rate： （8.4±2.8）%, bax protein： 128.4±9.6], and expression of bcl-2 protein of non-ligation group （196.4±19.3） was significantly lower than that of sham group （132. 2±12. 3） and ligation group （165.1±11.6, all P〈0. 01）. Conclusion The results demonstrate that the mesenteric lymph duct ligation ameliorate the myocardial injury in rats subjected to hemorrhage and LPS by reducing the inflammatory mediators of TNF-α and IL-6 levels, upregulating the bcl-2 protein expression and improving membrane ATPase activity of myocardium.