姜黄素对脓毒症大鼠肝细胞的剂量保护效应

The dose-dependent protective effect of curcumin on hepatocyte of rats with sepsis

目的：观察不同剂量姜黄素对脓毒症大鼠肝细胞的保护作用。方法按随机数字表法将100只雄性SD大鼠分为假手术（Sham）组、脓毒症组及低、中、高剂量姜黄素干预组（L-cur、M-cur、H-cur组），每组20只。采用盲肠结扎穿孔术（CLP）制备脓毒症动物模型；Sham组仅开腹取出盲肠后还纳关腹。L-cur、M-cur、H-cur组于术后即刻腹腔注射姜黄素50、100、150mg/kg，Sham组和脓毒症组给予等量生理盐水。分别于术后2、6、12、24h处死5只大鼠，取肝组织和血液标本，镜下观察肝组织病理改变；用原位末端缺刻标记试验（TUNEL）检测肝细胞凋亡情况并计算凋亡指数（AI），用酶联免疫吸附试验（ELISA）检测血清降钙素原（PCT）、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）水平。结果光镜下观察显示，脓毒症组肝组织损伤严重，凋亡细胞增多，且随时间延长损伤程度和凋亡细胞进一步增加；但姜黄素各剂量组损伤程度明显轻于脓毒症组，以中剂量尤甚。Sham组术后各时间点肝细胞AI及血清PCT、TNF-α和IL-1β水平均无明显变化。脓毒症组术后2h起肝细胞AI及血清PCT、TNF-α和IL-1β水平即较Sham组明显升高〔AI：（23.59±2.00）%比（2.02±0.13）%，PCT（μg/L）：2.41±0.21比0.81±0.01，TNF-α（ng/L）：217.28±14.24比80.02±2.26，IL-1β（ng/L）：61.84±3.21比25.78±1.29，均P＜0.05〕，且随时间延长呈逐渐升高趋势，AI于术后24h达峰值〔（52.05±1.31）%〕，PCT、TNF-α和IL-1β均于术后12h达峰值〔分别为（8.68±0.58）μg/L、（314.13±14.39）ng/L、（132.24±2.58）ng/L〕。姜黄素干预后可明显降低脓毒症大鼠肝细胞AI及血清PCT、TNF-α和IL-1β水平，以M-cur组降低尤为显著〔AI：2h为（11.56±0.96）%比（23.59±2.00）%，24h为（30.35±1.20）%比（52.05±1.31）%；PCT（μg/L）：2h为1.13±0.19比2.41±0.21，12h为5.09±0.42比8.68±0.58；TNF-α（ng/L）：2h为124.73±7.47比217.28±14.24，12h为168.68±6.95比314.13±14.39；IL-1β（ng/L）：2h为35.05±1.00比61.84±3.21，12h为84.06±3.42比132.24±2.58，均P＜0.05〕。结论姜黄素能抑制脓毒症大鼠肝细胞炎症反应，阻止肝细胞凋亡，从而对肝脏起到保护作用，以100mg/kg中剂量姜黄素作用最强。

Objective To observe the protective effect of different doses of curcumin on hepatocytes of rats with sepsis. Methods 100 healthy male Sprague-Dawley （SD） rats were randomly divided into sham operation group, sepsis group, and low, medium, high dose curcumin intervention groups （L-cur, M-cur, H-cur groups）, with 20 rats in each group. The animal model of sepsis was reproduced by cecal ligation and puncture （CLP） method, and in the sham operation group the cecum was just taken out and returned. In the L-cur, M-cur, H-cur groups curcumin was immediately injected after CLP with a dose of 50, 100, 150 mg/kg, respectively, and the rats in sham operation group and sepsis group were given the same amount of normal saline. Five rats in each group were sacrificed at 2, 6, 12, 24 hours after operation, and the hepatic tissues and blood samples were obtained. The pathological changes in hepatic tissues were observed under a microscope, and hepatocytes apoptosis and apoptosis index （AI） of hepatocytes were determined with transferase-mediated deoxyuridine triphosphate-biotin nick end labeling （TUNEL） method, and the levels of serum procalcitonin （PCT）, tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） were determined with enzyme linked immunosorbent assay （ELISA） method. Results Microscopic examination showed that the damage degree of hepatic tissues was significantly increased in sepsis group; the number of apoptotic cells and damage degree of hepatic tissues were increased gradually over time. The damage degree of hepatic tissues in curcumin groups was lessened as compared with sepsis group, especially in M-cur group. There were no significant changes in AI and serum PCT, TNF-α, and IL-1β levels at any of the time points tested in the sham operation group. The AI, serum PCT, TNF-α, and IL-1β levels in the sepsis group were significantly higher than those in the sham operation group from 2 hours after operation on [AI： （23.59±2.00）% vs. （2.02±0.13）%, PCT （μg/L）： 2.41±0.21 vs. 0.81±0.01, TNF-α （ng/L）： 217.28±14.24 vs. 80.02±2.26, IL-1β （ng/L）： 61.84±3.21 vs. 25.78±1.29, all P 〈 0.05], and they showed a gradually increasing tendency. AI reached peak value at 24 hours after operation [（52.05±1.31）%]; PCT, TNF-α and IL-1β reached the peak values at 12 hours after operation [（8.68±0.58） μg/L, （314.13±14.39） ng/L, （132.24±2.58） ng/L, respectively]. Curcumin intervention significantly reduced the levels of AI, TNF-α, PCT and IL-1β in hepatocytes of septic rats, especially in M-cur group [AI： （11.56±0.96）% vs. （23.59±2.00）% at 2 hours, （30.35±1.20）% vs. （52.05±1.31）% at 24 hours; PCT （μg/L）： 1.13±0.19 vs. 2.41±0.21 at 2 hours, 5.09±0.42 vs. 8.68±0.58 at 12 hours; TNF-α （ng/L）： 124.73±7.47 vs. 217.28±14.24 at 2 hours, 168.68±6.95 vs. 314.13±14.39 at 12 hours; IL-1β （ng/L）： 35.05±1.00 vs. 61.84±3.21 at 2 hours, 84.06±3.42 vs. 132.24±2.58 at 12 hours; all P 〈 0.05]. Conclusions Curcumin can inhibit the inflammatory reaction of hepatocytes of rats, prevent apoptosis, and protect the hepatocytes of rats with sepsis. The concentration of curcumin with the most significant effect is 100 mg/kg, which is the medium dosage.