摘要
目的 探讨预防性使用非瑟酮(Fisetin)对急性前列腺损伤大鼠的保护作用及机制.方法 4～6周龄成年雄性Wistar大鼠通过对大鼠前列腺左、右腹侧叶各注射2针3％角叉菜胶生理盐水溶液,每针50 μl,共注射200 μl的方法,构建急性前列腺损伤大鼠模型.将36只大鼠采用随机数字表法分为6组(每组6只),假手术(Sham)组、模型组、Fisetin低剂量组(25 mg/kg)、中剂量组(50 mg/kg)、高剂量组(100 mg/kg)、阳性药物组(普乐安片1.08 g/kg),Fisetin治疗组及阳性药物组分别在造模前3 d每天1次,造模当天及造模后3 d每天1次(共7次)进行药物灌胃治疗.Sham组和模型组给予相同剂量的生理盐水,在注射角叉菜胶后3 d后处死大鼠,收集前列腺组织,通过苏木精-伊红(HE)染色分析每组大鼠前列腺组织病理学变化;酶联免疫吸附试验(ELISA)、实时荧光定量(RT-qPCR)、免疫组织化学法(IHC)分析炎性因子表达,蛋白质印迹法(Western blot)检测炎性因子及Toll样受体4(TLR4)、核因子-κB(NF-κB)与磷酸化核因子-κB(p-NF-KB)的蛋白表达水平.多组间比较采用单因素方差分析,两组间比较采用t检验.结果 模型组大鼠前列腺组织中IL-6、IL-1β、TNF-α的蛋白表达水平明显高于Sham组、Fisetin高剂量组和阳性药物组[IL-6:(245.10± 7.73)pg/ml 比(77.65±6.28)、(191.10±10.20)、(197.80±9.31)pg/ml,t=6.632、4.237、3.924,P＜0.05;0.90±0.05 比 0.61±0.05、0.36±0.08、0.31±0.07,t=4.218、5.918、6.684,P＜0.05;IL-1β:(55.59±4.06)pg/ml 比(19.32±2.68)、(40.32±2.75)、(40.22±2.06)pg/ml,t=3.453、3.126、3.385,P＜0.05;1.07±0.08 比 0.66±0.02、0.46±0.05、0.37±0.06,t=5.253、6.951、7.018,P＜0.05;TNF-α:(188.40±14.36)pg/ml 比(45.52±7.54)、(125.50±5.92)、(118.60± 14.89)pg/ml,t=7.158、4.135、3.372,P＜0.05;0.84±0.06 比 0.53±0.05、0.44±0.04、0.40± 0.02,t=4.824、5.827、6.785,P＜0.05],差异有统计学意义.模型组大鼠前列腺组织中TLR-4、NF-κB/p-NF-κB水平明显高于Sham组、Fisetin高剂量组和阳性药物组(TLR-4:0.87±0.04比0.12±0.01、0.30±0.03、0.44±0.04,t=7.453、11.236、7.953,P＜0.05;NF-κB/p-NF-κB:1.38± 0.04 比 1.01±0.04、0.50±0.04、0.42±0.05,t=8.582、16.639、16.328,P＜0.05),差异有统计学意义.结论 Fisetin可能通过抑制TLR4/NF-κB信号通路减轻前列腺损伤大鼠的炎症水平,进而改善前列腺病变.
Abstract
Objective To investigate the protective effect and mechanism of prophylactic use of Fisetin on acute prostate injury in rats.Methods A rat model of acute prostate injury was established by injecting 2 needles of 3％sterile carrageenan saline solution into the left and right ventral lobes of the pros-tate of adult male Wistar rats(purchased from Wuhan Wanqian Jiaxing Biological Technology Co.,Ltd.),each needle of 50 μl,a total of 200 μl.The rats were randomly divided into 6 groups(6 cases each):sham operation group,model group,Fisetin low dose group(25 mg/kg),Fisetin middle dose group(50 mg/kg),Fisetin high dose group(100 mg/kg),positive drug group(1.08 g/kg).Fisetin treatment group and positive drug group were treated with intragastric administration once a day for 3 days before modeling,once a day on the day of modeling and 3 days after modeling(7 times in total).The sham opera-tion group and the model group were given the same dose of normal saline.The rats were sacrificed 3 days after injection of carrageenan.The prostate tissue was collected and the pathological changes of prostate tis-sue in each group were analyzed by hematoxylin-eosin(HE)staining.The expression of inflammatory fac-tors was analyzed by enzyme-linked immunosorbent assay(ELISA),real-time fluorescence quantitative PCR(RT-qPCR)and immunohistochemistry.The protein expression levels of inflammatory factors,toll-like receptor 4(TLR4),nuclear factor kappa B(NF-κB)and phosphorylated nuclear factor kappa B(p-NF-KB)were detected by Western blotting.One-way analysis of variance was used for comparison be-tween multiple groups,and t test was used for comparison between two groups.Results In the prostate tissues of rats,IL-6,IL-1β and TNF-α protein expression levels were significantly higher in the model group than in the Sham group,the Fisetin high-dose group,and the positive drug group[IL-6:(245.10± 7.73)vs.(77.65±6.28),(191.10±10.20),(197.80±9.31)pg/ml,t=6.632,4.237,3.924,P＜0.05;0.90±0.05 vs.0.61±0.05,0.36±0.08,0.31±0.07,t=4.218,5.918,6.684,P＜0.05;IL-1 β:(55.59±4.06)vs.(19.32±2.68),(40.32±2.75),(40.22±2.06)pg/ml,t=3.453,3.126,3.385,P＜0.05;1.07±0.08 vs.0.66±0.02,0.46±0.05,0.37±0.06,t=5.253,6.951,7.018,P＜0.05;TNF-α:(188.40±14.36)vs.(45.52±7.54),(125.50±5.92),(118.60± 14.89)pg/ml,t=7.158,4.135,3.372,P＜0.05;0.84±0.06 vs.0.53±0.05,0.44±0.04,0.40±0.02,t=4.824,5.827,6.785,P＜0.05].In the model group,the levels of TLR-4 and NF-κB/p-NF-κB in the prostate tissues were significantly higher than those in the Sham group,the Fisetin high-dose group,and the positive drug group(TLR-4:0.87±0.04 vs.0.12±0.01,0.30±0.03,0.44± 0.04,t=7.453,11.236,7.953,P＜0.05;NF-κB/p-NF-κB:1.38±0.04 vs.1.01±0.04,0.50± 0.04,0.42±0.05,t=8.582,16.639,16.328,P＜0.05).Conclusion Fisetin may reduce the level of inflammation in rats with prostate injury by inhibiting TLR4/NF-κB signaling pathway,thereby impro-ving prostate lesions.
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