摘要目的:探讨西维来司他钠（SV）对脓毒症急性肾损伤（AKI）大鼠的保护作用及机制。方法:按照随机数字表法将64只雄性Wistar大鼠分为假手术组（Sham组）、盲肠结扎穿孔术致脓毒症组（CLP组）、脓毒症+SV低剂量组（SL组，术后12 h、24 h尾静脉注射SV 50 mg/kg）、脓毒症+SV高剂量组（SH组，术后12 h、24 h尾静脉注射SV 100 mg/kg），每组16只，记录大鼠48 h存活情况，并于制模后48 h处死大鼠并取材。采用酶联免疫吸附试验（ELISA）检测血清中肾损伤分子-1（KIM-1）、白细胞介素（IL-1β、IL-6）、肿瘤坏死因子-α（TNF-α）和中性粒细胞弹性蛋白酶（NE）水平；苏木素-伊红（HE）染色后，光镜下观察肾脏组织病理学改变并进行肾小管损伤评分；Masson染色检测肾脏组织胶原容积分数（CVF）；蛋白质免疫印迹试验（Western blotting）检测肾脏组织中磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（AKT）、磷酸化AKT（p-AKT）、核转录因子-κB p65（NF-κB p65）和NE的蛋白表达；免疫组化法检测肾脏组织中p-PI3K、p-AKT、NF-κB p65的阳性面积。结果:与Sham组相比，CLP组大鼠术后48 h存活率明显下降；组织病理学结果显示大片肾小管上皮细胞和刷状缘脱落，肾小管内管型形成，部分肾小管轻度萎缩，肾小球充血，肾间质炎症细胞浸润，肾小管损伤评分升高；肾间质纤维化病变明显，CVF增加；血清中KIM-1、IL-1β、IL-6、TNF-α及NE水平明显升高；肾脏组织中炎症通路相关蛋白p-PI3K/PI3K、p-AKT/AKT、NF-κB p65及NE表达均明显增加，提示CLP组脓毒症大鼠肾损伤，PI3K/AKT炎症通路活化。与CLP组相比，给予低剂量或高剂量SV干预后大鼠48 h存活率虽无明显增加（68.75%、75.00%比56.25%，均 P>0.05），但肾脏损伤有所缓解，具体表现为：肾小管损伤评分下降〔分：2（1，2）、1（1，1）比2（2，3），均 P<0.05〕，CVF显著降低〔（22.36±0.86）%、（18.74±1.05）%比（58.38±0.79）%，均 P<0.05〕，血清KIM-1、IL-1β、IL-6、TNF-α及NE水平明显降低〔KIM-1（ng/L）：145.03±8.88、117.58±7.02比158.22±12.00，IL-1β（ng/L）：108.32±9.00、92.98±8.06比133.78±8.48，IL-6（ng/L）：124.33±10.11、115.42±8.17比165.19±5.70，TNF-α（ng/L）：321.56±19.29、289.68±21.57比424.88±22.76，NE（mol/L）：93.84±9.14、75.01±10.56比113.45±6.39，均 P<0.05〕，肾脏组织炎症通路相关蛋白p-PI3K/PI3K、p-AKT/AKT、NF-κB p65及NE的蛋白表达明显下降（p-PI3K/PI3K：0.93±0.06、0.67±0.04比1.27±0.08，p-AKT/AKT：0.78±0.09、0.47±0.05比0.96±0.12，NF-κB p65/GAPDH：1.43±0.13、0.85±0.08比1.88±0.17，NE/GAPDH：1.45±0.06、0.91±0.04比1.71±0.08，均 P<0.05），肾脏组织p-PI3K、p-AKT、NF-κB p65表达明显降低〔p-PI3K阳性面积：（13.36±1.84）%、（8.03±1.12）%比（21.56±1.20）%，p-AKT阳性面积：（21.57±0.91）%、（15.21±2.76）%比（30.81±2.12）%，NF-κB p65阳性面积：（25.17±1.38）%、（17.07±2.11）%比（37.85±2.50）%，均 P<0.05〕。SH组血清炎症因子水平和肾脏组织PI3K/AKT通路相关蛋白、NF-κB p65、NE蛋白表达水平及p-PI3K、p-AKT、NF-κB p65阳性面积等指标均较SL组进一步降低（均 P<0.05）。 结论:SV对脓毒症合并AKI模型大鼠能起到一定的保护作用，其作用机制可能与抑制PI3K/AKT通路相关，且高剂量SV疗效更佳。

abstractsObjective:To explore the protective effect of sivelestat (SV) against sepsis-induced acute kidney injury (AKI) and its molecular mechanism.Methods:According to the random number table method, 64 male Wistar rats were divided into sham operation group (Sham group), sepsis due to cecal ligation and puncture group (CLP group), low dose of SV treatment group (SL group, 50 mg/kg SV was injected into the tail vein at 12 hours and 24 hours after CLP), and high dose of SV treatment group (SH group, 100 mg/kg SV was injected into the tail vein at 12 hours and 24 hours after CLP), with 16 rats in each group. 48 hours after CLP, the 48-hour survival of rats were recorded, all rats were sacrificed and samples were harvested. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of kidney injury molecule-1 (KIM-1), interleukins (IL-1β, IL-6), tumor necrosis factor-α (TNF-α) and neutrophil elastase (NE). Hematoxylin-eosin (HE) staining was used to observe histopathological changes and assess renal tubule injury score. Masson staining was used to detect the collagen volume fraction (CVF) of kidney tissue. Western blotting was used to detect the protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphorylation PI3K (p-PI3K), protein kinase B (AKT), phosphorylation AKT (p-AKT), nuclear factor-κB p65 (NF-κB p65) and NE. The protein expressions of p-PI3K, p-AKT, NF-κB p65 were detected by immunohistochemistry.Results:Compared with Sham group, the 48-hour survival rate of CLP group was significantly reduced. Histopathological results showed that large tubular epithelial cells and brush margins were shed, tubular casts were formed, some tubular atrophy, glomerular hyperemia, renal interstitial inflammatory cell infiltration and increased renal tubular injury score. Renal interstitial fibrosis was obvious and CVF increased. The levels of KIM-1, IL-1β, IL-6, TNF-α and NE in serum were significantly elevated in the CLP group. The proteins expression of inflammatory pathway-related p-PI3K/PI3K, p-AKT/AKT, NF-κB p65 and NE were significantly increased in kidney tissue. It suggested that septic rats had renal injury and the PI3K/AKT inflammatory pathway was activated. Compared with CLP group, there was no significant difference in 48-hour survival in SL group and SH group (68.75%, 75.00% vs. 56.25%, both P > 0.05), but kidney injury was significantly relieved. Specifically: renal tubular injury score and CVF significantly decreased [tubular injury score: 2 (1, 2), 1 (1, 1) vs. 2 (2, 3); CVF: (22.36±0.86)%, (18.74±1.05)% vs. (58.38±0.79)%, all P < 0.05]; the serum levels of KIM-1, IL-1β, IL-6, TNF-α and NE also decreased significantly [KIM-1 (ng/L): 145.03±8.88, 117.58±7.02 vs. 158.22±12.00; IL-1β (ng/L): 108.32±9.00, 92.98±8.06 vs. 133.78±8.48; IL-6 (ng/L): 124.33±10.11, 115.42±8.17 vs. 165.19±5.70; TNF-α (ng/L): 321.56±19.29, 289.68±21.57 vs. 424.88±22.76, NE (mol/L): 93.84±9.14, 75.01±10.56 vs. 113.45±6.39, all P < 0.05]; the proteins expression of inflammatory pathway-related p-PI3K/PI3K, p-AKT/AKT, NF-κB p65 and NE were significantly decreased (p-PI3K/PI3K: 0.93±0.06, 0.67±0.04 vs. 1.27±0.08; p-AKT/AKT: 0.78±0.09, 0.47±0.05 vs. 0.96±0.12; NF-κB p65/GAPDH: 1.43±0.13, 0.85±0.08 vs. 1.88±0.17; NE/GAPDH: 1.45±0.06, 0.91±0.04 vs. 1.71±0.08, all P < 0.05), the positive expressions of p-PI3K, p-AKT and NF-κB p65 in kidney tissue were decreased [p-PI3K positive expression area: (13.36±1.84)%, (8.03±1.12)% vs. (21.56±1.20)%; p-AKT positive expression area: (21.57±0.91)%, (15.21±2.76)% vs. (30.81±2.12)%; NF-κB p65 positive expression area: (25.17±1.38)%, (17.07±2.11)% vs. (37.85±2.50)%, all P < 0.05]. Serum inflammatory factor level, and PI3K/AKT pathway related protein, NF-κB p65, NE protein expression level and p-PI3K, p-AKT, NF-κB p65 positive area and other indicators in renal tissue in SH group were further lower than those in SL group (all P < 0.05). Conclusions:SV can ameliorate sepsis-induced AKI. The mechanism may be related to the inhibition of PI3K/AKT pathway, and high dose of SV has better efficacy.