PQ401对脂多糖诱导的急性腹膜炎小鼠的保护作用及其机制研究

发布时间：2018-07-23 10:18

【摘要】：目的：PQ401是一种选择性的胰岛素样生长因子1受体(IGF-1R)抑制剂。本研究旨在观察PQ401对脂多糖(LPS)诱导的急性腹膜炎的保护效应,并探讨其作用机制,以期为腹膜炎的临床治疗提供新策略。方法：采用LPS诱导建立C57BL/6小鼠急性腹膜炎模型。将小鼠随机分配为对照组、模型组、PQ401组(25mg/kg、50mg/kg、100mg/kg)。给药12h后将小鼠全部处死,采集小鼠血和肝组织,分别提取血清和蛋白后冻存。观察小鼠的身体状态,评价PQ401对小鼠精神状态和活动的影响。通过酶联免疫吸附试验(ELISA)检测血清和肝组织匀浆中的肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-1β、IL-6)的浓度,评价PQ401对急性腹膜炎小鼠炎症因子的影响。全自动生化分析仪检测血清丙氨酸转氨酶(ALT)活性、天冬氨酸转氨酶(AST)活性及总胆红素(TBIL)浓度,评价PQ401对腹膜炎小鼠肝脏的影响。通过蛋白印迹实验(Western blot)检测小鼠肝细胞中核转录因子-κB (NF-κB)的表达,探究PQ401抗炎作用的分子机制。在体外实验中,通过LPS诱导RAW264.7细胞建立体外炎症模型。给予不同浓度PQ401治疗后,不同时间点收集细胞上清液和细胞蛋白。ELISA法检测细胞上清的炎症因子TNF-α、IL-1β、IL-6的浓度,评价PQ401对巨噬细胞的抗炎作用。通过Western blot检测IGF-1R磷酸化水平,评价PQ401与IGF-1R磷酸化的关系。通过加入IGF-1和IGF-IRa/p shRNA慢病毒转染两种方式,评价IGF-1R信号通路与PQ401抗炎效应的关系。通过Western blot检测巨噬细胞的NF-κB信号通路,评价PQ401抗炎作用对NF-κB信号通路的影响。结果：在LPS诱导的小鼠急性腹膜炎模型中,模型组小鼠精神萎靡、活动减少,PQ401 (25mg/kg,50mg/kg、100mg/kg)组小鼠表现明显好于模型组。PQ401能有效降低LPS诱导的急性腹膜炎小鼠血清中TNF-α、IL-1β、IL-6的浓度,降低肝组织TNF-α、IL-1β的浓度。PQ401对小鼠血清ALT、AST的活性及TBIL的浓度无明显影响。PQ401对肝细胞中NF-K B的表达无明显影响。在体外实验中,PQ401 (0.1μM、1μM、10μM)分别作用6h、12 h、24 h,能有效降低LPS诱导的巨噬细胞释放炎症因子TNF-α、IL-1β、IL-6的浓度。PQ401能抑制炎性细胞中IGF-1R磷酸化。加入IGF-1活化IGF-1R或者IGF-IRα/β shRNA慢病毒转染巨噬细胞低表达IGF-1R,均不能影响PQ401的抗炎效应。PQ401对LPS诱导的NF-K B信号通路无影响。结论：PQ401在体内外均具有抗炎作用。但是PQ401的抗炎效应与抑制IGF-1R磷酸化无相关性,说明IGF-1R不是PQ401发挥抗炎效应的有效靶点。PQ401对LPS诱导的NF-K B信号通路无影响,说明PQ401可能通过其他信号通路发挥抗炎作用。
[Abstract]:Objective: PQ 401 is a selective insulin-like growth factor 1 receptor (IGF 1 R) inhibitor. The aim of this study was to observe the protective effect of PQ401 on lipopolysaccharide (LPS) -induced acute peritonitis and to explore its mechanism in order to provide a new strategy for the clinical treatment of peritonitis. Methods: acute peritonitis was induced by LPS in C57BL / 6 mice. Mice were randomly assigned to control group and model group PQ401 (25 mg / kg 50 mg 路kg ~ (-1) 路kg ~ (-1) 100 mg 路kg ~ (-1) 路kg ~ (-1). After 12 hours of administration, all mice were killed, blood and liver tissues were collected, serum and protein were extracted and frozen. To observe the physical state of mice and evaluate the effect of PQ 401 on the mental state and activity of mice. The levels of tumor necrosis factor- 伪 (TNF- 伪) and interleukin-6 (IL-1 尾 -IL-6) in serum and liver homogenate were determined by Elisa to evaluate the effect of PQ401 on inflammatory factors in mice with acute peritonitis. The activity of alanine aminotransferase (alt), aspartate aminotransferase (AST) and total bilirubin (TBIL) were measured by automatic biochemical analyzer to evaluate the effect of PQ401 on the liver of peritonitis mice. The expression of nuclear transcription factor-魏 B (NF- 魏 B) in mouse hepatocytes was detected by Western blot assay (blot) to explore the molecular mechanism of anti-inflammatory effect of PQ401. In vitro inflammatory model of RAW264.7 cells induced by LPS was established. After treatment with different concentrations of PQ401, the supernatant and the level of TNF- 伪 IL-1 尾 IL-6 in the supernatant were collected at different time points to evaluate the anti-inflammatory effect of PQ401 on macrophages. The phosphorylation level of IGF-1R was detected by Western blot, and the relationship between PQ401 and IGF-1R phosphorylation was evaluated. The relationship between IGF-1R signaling pathway and anti-inflammatory effect of PQ401 was evaluated by adding IGF-1 and IGF-IRap-shRNA lentivirus transfection. The NF- 魏 B signaling pathway of macrophages was detected by Western blot to evaluate the effect of PQ401 on NF- 魏 B signaling pathway. Results: in the model of acute peritonitis induced by LPS, the mice in the model group were depressed in spirit and decreased in activity (25 mg / kg, 50 mg / kg, 100 mg / kg), which was better than that in the model group. PQ401 could effectively reduce the concentration of TNF- 伪 and IL-1 尾 IL-6 in the serum of mice with acute peritonitis induced by LPS. PQ401 had no effect on the activity of serum alt and TBIL. PQ401 had no effect on the expression of NF-K B in hepatocytes. In vitro, PQ401 (0.1 渭 M 1 渭 M 10 渭 M) could effectively reduce the concentration of inflammatory factor TNF- 伪, IL-1 尾 and IL-6 in LPS induced macrophages. PQ401 could inhibit the phosphorylation of IGF-1R in inflammatory cells. Addition of IGF-1 activated IGF-1R or IGF-IR 伪 / 尾 shRNA lentivirus transfected macrophages with low expression of IGF-1R could not affect the anti-inflammatory effect of PQ401. PQ401 had no effect on LPS-induced NF-K B signaling pathway. Conclusion in vivo and in vitro, WPQ401 has anti-inflammatory effect. However, the anti-inflammatory effect of PQ401 was not related to the inhibition of IGF-1R phosphorylation, indicating that IGF-1R was not an effective target for PQ401 to play an anti-inflammatory effect. PQ401 had no effect on LPS-induced NF-K B signaling pathway, suggesting that PQ401 might play an anti-inflammatory role through other signaling pathways.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R572.2

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