【摘要】：目的：PQ401是一種選擇性的胰島素樣生長(zhǎng)因子1受體(IGF-1R)抑制劑。本研究旨在觀察PQ401對(duì)脂多糖(LPS)誘導(dǎo)的急性腹膜炎的保護(hù)效應(yīng),并探討其作用機(jī)制,以期為腹膜炎的臨床治療提供新策略。方法：采用LPS誘導(dǎo)建立C57BL/6小鼠急性腹膜炎模型。將小鼠隨機(jī)分配為對(duì)照組、模型組、PQ401組(25mg/kg、50mg/kg、100mg/kg)。給藥12h后將小鼠全部處死,采集小鼠血和肝組織,分別提取血清和蛋白后凍存。觀察小鼠的身體狀態(tài),評(píng)價(jià)PQ401對(duì)小鼠精神狀態(tài)和活動(dòng)的影響。通過(guò)酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)血清和肝組織勻漿中的腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素(IL-1β、IL-6)的濃度,評(píng)價(jià)PQ401對(duì)急性腹膜炎小鼠炎癥因子的影響。全自動(dòng)生化分析儀檢測(cè)血清丙氨酸轉(zhuǎn)氨酶(ALT)活性、天冬氨酸轉(zhuǎn)氨酶(AST)活性及總膽紅素(TBIL)濃度,評(píng)價(jià)PQ401對(duì)腹膜炎小鼠肝臟的影響。通過(guò)蛋白印跡實(shí)驗(yàn)(Western blot)檢測(cè)小鼠肝細(xì)胞中核轉(zhuǎn)錄因子-κB (NF-κB)的表達(dá),探究PQ401抗炎作用的分子機(jī)制。在體外實(shí)驗(yàn)中,通過(guò)LPS誘導(dǎo)RAW264.7細(xì)胞建立體外炎癥模型。給予不同濃度PQ401治療后,不同時(shí)間點(diǎn)收集細(xì)胞上清液和細(xì)胞蛋白。ELISA法檢測(cè)細(xì)胞上清的炎癥因子TNF-α、IL-1β、IL-6的濃度,評(píng)價(jià)PQ401對(duì)巨噬細(xì)胞的抗炎作用。通過(guò)Western blot檢測(cè)IGF-1R磷酸化水平,評(píng)價(jià)PQ401與IGF-1R磷酸化的關(guān)系。通過(guò)加入IGF-1和IGF-IRa/p shRNA慢病毒轉(zhuǎn)染兩種方式,評(píng)價(jià)IGF-1R信號(hào)通路與PQ401抗炎效應(yīng)的關(guān)系。通過(guò)Western blot檢測(cè)巨噬細(xì)胞的NF-κB信號(hào)通路,評(píng)價(jià)PQ401抗炎作用對(duì)NF-κB信號(hào)通路的影響。結(jié)果：在LPS誘導(dǎo)的小鼠急性腹膜炎模型中,模型組小鼠精神萎靡、活動(dòng)減少,PQ401 (25mg/kg,50mg/kg、100mg/kg)組小鼠表現(xiàn)明顯好于模型組。PQ401能有效降低LPS誘導(dǎo)的急性腹膜炎小鼠血清中TNF-α、IL-1β、IL-6的濃度,降低肝組織TNF-α、IL-1β的濃度。PQ401對(duì)小鼠血清ALT、AST的活性及TBIL的濃度無(wú)明顯影響。PQ401對(duì)肝細(xì)胞中NF-K B的表達(dá)無(wú)明顯影響。在體外實(shí)驗(yàn)中,PQ401 (0.1μM、1μM、10μM)分別作用6h、12 h、24 h,能有效降低LPS誘導(dǎo)的巨噬細(xì)胞釋放炎癥因子TNF-α、IL-1β、IL-6的濃度。PQ401能抑制炎性細(xì)胞中IGF-1R磷酸化。加入IGF-1活化IGF-1R或者IGF-IRα/β shRNA慢病毒轉(zhuǎn)染巨噬細(xì)胞低表達(dá)IGF-1R,均不能影響PQ401的抗炎效應(yīng)。PQ401對(duì)LPS誘導(dǎo)的NF-K B信號(hào)通路無(wú)影響。結(jié)論：PQ401在體內(nèi)外均具有抗炎作用。但是PQ401的抗炎效應(yīng)與抑制IGF-1R磷酸化無(wú)相關(guān)性,說(shuō)明IGF-1R不是PQ401發(fā)揮抗炎效應(yīng)的有效靶點(diǎn)。PQ401對(duì)LPS誘導(dǎo)的NF-K B信號(hào)通路無(wú)影響,說(shuō)明PQ401可能通過(guò)其他信號(hào)通路發(fā)揮抗炎作用。
[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- 偽 (TNF- 偽) 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- 偽 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- 偽 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- 偽, 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 偽 / 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R572.2

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