橘皮苷对大鼠重症急性胰腺炎的治疗作用及机制

Therapeutic effect of hesperidin on severe acute pancreatitis in rats and its mechanism

目的 探讨橘皮苷对大鼠重症急性胰腺炎（SAP）治疗作用的机制.方法 将60只雄性SD大鼠按随机数字表法分为假手术组、SAP模型组、地塞米松组（5 mg/kg）、低剂量橘皮苷组（10 mg/kg）和高剂量橘皮苷组（20 mg/kg）,每组12只.经胰管逆行注射3.5％牛磺胆酸钠建立SAP大鼠模型,假手术组注射生理盐水;术后5 min经股静脉注射相应药物.术后24 h观察动物存活情况,测定血清淀粉酶水平、腹水量和胰腺相对比重;苏木素-伊红（HE）染色后镜下观察胰腺组织病理学改变;酶联免疫吸附试验（ELISA）检测血清和胰腺组织白细胞介素（IL-1β、IL-6）和肿瘤坏死因子-α（TNF-α）水平;蛋白质免疫印迹试验（Western Blot）检测Toll样受体4（TLR4）表达以及白细胞介素-1受体相关激酶（IRAK1）、核转录因子-κB（NF-κB）磷酸化水平.结果 与SAP模型组比较,低、高剂量橘皮苷治疗后24 h动物存活率提高（83.3％、100％比58.3％）,腹水量减少（mL：7.36±0.91、6.10±1.02比13.82±2.06）,血清淀粉酶水平降低（U/L：1081.48±78.23、1048.58±49.97比1990.37±127.27）,胰腺相对比重降低〔（7.52±1.02）％、（5.59±0.96）％比（11.22±0.96）％〕,胰腺组织病理损伤减轻;高剂量橘皮苷组血清和胰腺组织炎性因子水平明显降低〔血清IL-1β（ng/L）：68.08±10.49比130.30±23.35,IL-6（ng/L）：63.88±10.47比158.41±21.38,TNF-α（ng/L）：10.42±1.49比18.16±2.01;胰腺IL-1β（pg/μg）：13.87±1.84比20.08±1.66,IL-6（pg/μg）：21.90±3.12比38.13±3.57,TNF-α（pg/μg）：1.88±0.20比4.26±0.58〕;低、高剂量橘皮苷均可抑制TLR4表达及IRAK1、NF-κB磷酸化（以假手术组为100,TLR4/β-actin：91.9±15.6、83.7±11.2比168.5±9.0,p-IRAK1/IRAK1：117.4±7.6、104.7±11.5比173.5±15.8,p-NF-κB p65/NF-κB p65：119.9±9.3、105.8±12.6比174.1±13.0）,差异均有统计学意义（均P〈0.05）.地塞米松治疗SAP的作用与高剂量橘皮苷相当.结论 橘皮苷可降低SAP大鼠24 h死亡率,治疗SAP的机制可能与抑制胰腺TLR4/IRAK1/NF-κB信号通路、减少炎性因子释放有关,高剂量橘皮苷（20 mg/kg）作用更为显著.

Objective To investigate the protective effect of hesperidin on severe acute pancreatitis （SAP） in rats and its related mechanism.Methods Sixty male Sprague-Dawley （SD） rats were randomly divided into five groups （n = 12 in each group）： sham group, SAP model group, dexamethasone group （5 mg/kg）, low and high dose of hesperidin groups （10 mg/kg and 20 mg/kg）. SAP rats were administered a retrograde infusion of 3.5% sodium taurocholate solution into the biliopancreatic duct after laparotomy. Sham rats were administered with equivalent saline. The treatment was intravenously injected 5 minutes after operation through femoral vein. After 24 hours, the survival of animals was observed, the level of serum amylase, the volume of ascites and the relative specific gravity of the pancreas were measured; the pathological changes of pancreatic tissue were observed by Hematoxylin-eosin （HE） staining; the levels of serum and pancreatic tissue interleukin （IL-1β, IL-6） and tumor necrosis factor-α （TNF-α） were detected by enzyme linked immunosorbent assay （ELISA）; the expression of Toll-like receptor 4 （TLR4）, the phosphorylation of IL-1 receptor associated kinase （IRAK1） and nuclear factor-κB （NF-κB） were detected by Western Blot.Results Compared with SAP model group, the 24-hour survival rate were increased in low and high dose of hesperidin groups （83.3%, 100% vs. 58.3%）, the volume of ascites were reduced （mL： 7.36±0.91, 6.10±1.02 vs. 13.82±2.06）, the levels of serum amylase were reduced （U/L： 1081.48±78.23, 1048.58±49.97 vs. 1990.37±127.27）, the relative specific gravity of the pancreas were reduced [（7.52±1.02）%, （5.59±0.96）% vs. （11.22±0.96）%], and the pathological damage of pancreatic tissue were reduced; the levels of serum and pancreatic tissue inflammatory factors were reduced in high dose hesperidin group [serum IL-1β （ng/L）： 68.08±10.49 vs. 130.30±23.35, IL-6 （ng/L）： 63.88±10.47 vs. 158.41±21.38, TNF-α（ng/L）： 10.42±1.49 vs. 18.16±2.01; pancreas IL-1β （pg/μg）： 13.87±1.84 vs. 20.08±1.66, IL-6 （pg/μg）： 21.90±3.12vs. 38.13±3.57, TNF-α （pg/μg）： 1.88±0.20 vs. 4.26±0.58]; the expression of TLR4, and the phosphorylation levels of IRAK1 and NF-κB were decreased in low and high dose of hesperidin groups （the sham operation group was 100, TLR4/β-actin： 91.9±15.6, 83.7±11.2 vs. 168.5±9.0, p-IRAK1/IRAK1： 117.4±7.6, 104.7±11.5 vs. 173.5±15.8, p-NF-κB p65/NF-κB p65： 119.9±9.3, 105.8±12.6 vs. 174.1±13.0）, with statistically significant differences （allP 〈 0.05）. The effects of dexamethasone were similar to that of high dose of hesperidin.Conclusions Hesperidin could significantly protect SAP rats, and this protection was related to the inhibition of TLR4/IRAK1/NF-κB signaling pathway, and to the reduction of pro-inflammatory cytokine expressions. The effect of high dose hesperidin （20 mg/kg） was more significant.