持续去甲肾上腺素输注对早期脓毒症大鼠肾脏的保护作用及其机制研究

目的探讨持续去甲肾上腺素输注对早期脓毒症大鼠肾脏是否存在保护作用及其可能机制。方法30只SPF级健康雄性SD大鼠采用随机数字表法随机分为5组（6只／组）。对照组：腹腔注射生理盐水，并开始持续输注生理盐水1ml／h。脂多糖（lipopolysaccharide，LPS）组和低剂量、中剂量和高剂量去甲肾上腺素组：腹腔注射LPS10mg／kg，LPS组持续输注生理盐水1ml／h，低、中、高剂量组分别持续输注0．06、0．30、0．60μg／（kg·min）去甲肾上腺素溶液1ml／h，均持续输注24h处死大鼠。检测2、6h大鼠血清炎症因子肿瘤坏死因子（tumor necrosis factor，TNF）-α、白细胞介素（interleukin，IL）-1β、IL-6及IL-10水平，24h大鼠血清C-反应蛋白（CRP）、肌酐（Cr）和尿素氮（BUN）水平，膜电位和氧化应激相关指标，电镜观察肾脏线粒体肿胀程度，显微镜观察肾脏病理变化。结果与对照组比较，2hLPS组TNF—α浓度为（2203．3±1028．7）pg／ml、IL-1β（2214．5±457．0）pg／ml、IL-6（7784．7±248．2）pg／ml及IL-10（1076．1±368．4）pg／ml，均明显升高（P〈0．05）；24hCRP（0．35±0．24）mg／L，Cr（30．8±11．5）μmol／L，BUN（7．7±1．8）mmol／L，一氧化氮（1057．4±172．9）μmol／gprot，均明显升高（P〈0．01），肾脏线粒体膜电位0．0464±0．0185，明显下降（P〈0．01）。与LPS组比较，2h低剂量组TNF-d浓度为（506．8±301．7）pg／ml、IL-1β（415．6±178．0）pg／ml及IL-α（381．7±171．0）pg／ml，均明显下降（P〈0．05），24hBUN下降为（5．2±1．4）mmol／L（P〈0．05），线粒体膜电位上升为0．3474±0．1526（P〈0．05）；2h中剂量组TNF-α浓度为（323．9±227．9）pg／ml、IL-1β（700．0±246．2）pg／ml，IL-10（282．6±134．4）pg／ml，均明显下降（P〈0．05），24hCRP下降为（0．17±0．08）mg／L（P〈0．05），线粒体膜电位上升为0．3775±0．1437（P〈0．05）；高剂量组2hTNF-α浓度为（378．7±89．8）pg／ml、IL-1β（945．7±264．4）pg／ml，明显下降（P〈0．05），24hCRP（0．19±0．12）mr／L、Cr（23．2±3．4）μmol/L均明显下降（P〈0．05）。电镜观察显示LPS组线粒体包膜模糊及基质空泡化、凝固，显微镜观察显示LPS组肾组织间隙水肿，单核-巨噬细胞浸润，肾小球皱缩，肾小管上皮细胞肿胀、空泡变性等，中剂量组病理损伤明显减轻。结论持续去甲肾上腺素输注对早期脓毒症大鼠肾脏有一定的保护作用，其机制与降低炎症因子风暴的水平、减轻氧化应激损害、改善线粒体功能相关。

Renal protective effect and mechanism of continuous infusion of norepinephrine in rat with early stage sepsis

Objective To investigate the protective effect and possible mechanisms of continuous infusion of norepinephrine in kidney of septic rats in the early stage. Methods Thirty healthy male SD rats of SPF level were randomly divided into five groups. Rats in control group were given intraperitoneal injection of saline and began a continuous infusion of saline （ 1 ml/h）. Rats in LPS group and the intervention group （low-dose,medium-dose and high-dose norepinephrine group） were given intraperitoneal injection of LPS 10 mg/kg. LPS group began a continuous infusion of saline （ 1 ml/h） while low, medium and high dose groups began continuous infusion of different norepinephrine solution （0. 06,0. 3,0. 6μg/（kg·min） ]. Rats were sacrificed after 24 hours infusion. We detected serum inflammatory cytokines E tumor necrosis factor （TNF）-α, intefleukin（IL）-1β, IL-6 and IL-10] in 2 h and 6 h by ELISA. Rat serum CRP, Cr and BUN, swelling and membrane potential of kidney mitochondria and oxidative stress-related indicators were tested in 24 h. We also observed renal pathologic changes by electronic microscopy and biopsy. Results Compared with the control group,serum levels of TNF-α （2 203.3 ± 1 028.7） pg/ml] ,IL-1β （2 214. 5 ±457.0） pg/ml], IL-6 （7784.7 ±248.2） pg/ml] and IL-10 （1 076. 1 ±368.4） pg/ml] were statistically higher in LPS group in 2 h （P 〈0. 05） ;CRP （0. 35 ±0. 24） mg/L] ,Cr （30. 8 ± 11.5） μmol/L] ,BUN （7. 7 ± 1.8 ） mmol/L ], NO （ 1 057.4 ± 172.9 ） μmol/gprot ] were statistically higher （ P 〈 0.01 ）, membrane potential of kidney mitochondria （0. 046 4 ±0. 018 5 ） decreased statistically （ P 〈 0.01 ）. Compared with LPS group, serum levels of TNF-α （ 506. 8 ± 301.7 ） pg/ml ], IL-1β （ 415.6 ± 178. 0 ） pg/ml ], and IL-10 （ 381.7 ± 171.0） pg/ml ] significantly decreased in low-dose group in 2 h （ P 〈 0.05 ）, BUN E （5.2 ± 1.4） mmol/Ll decreased （P 〈0.05） ,mitochondrial membrane potential （0. 347 4 ± 0. 152 6） increased in 24 h （ P 〈 0. 05 ） ; serum levels of TNF-α （ 323. 9 ± 227.9 ） pg/ml ], IL-1β （ 700. 0 ± 246. 2 ） pg/ml ], and IL-10E （282. 6 ±134.4） pg/ml] significantly decreased statistically in medium-dose group in 2 h （P 〈 0.05） ,CRP （0. 17 ±0. 08 ） mg/L ] decreased statistically （ P 〈0. 01 ）, mitochondrial membrane potential （0. 377 5 ± 0. 143 7 ） increased in 24 h （P 〈 0. 05 ） ; serum levels of TNF-α （378.7 ± 89. 8） pg/ml ], IL-1β （945.7 ±264. 4） pg/ml ] significantly decreased in high-dose group in 2 h （P 〈0. 05） ,CRP E （0. 19 ±0. 12） mg/L ] and Cr （ 23.2 ± 3.4 ） vmol/L ] decreased in 24 h （ P 〈 0. 05 ）. Mitochondrial matrix coated fuzzy, vacuoles and coagulation were found in LPS group by electronic microscopy examination. Interstitial edema, monocyte-macrophage infiltration, glomerular shrinkage, tubular epithelial cell swelling, empty bubble degeneration were found in LPS group by microscopy examination. Pathological damage was alleviated in mediumdose group. Conclusion Continuous infusion of norepinephrine plays a protective role on renal function in rats with sepsis in the early stage. The intervention protect rat kidney by reducing levels of inflammatory cytokines, oxidative stress and mitochondrial damage.