本文關鍵詞：咪唑克生對膿毒癥小鼠的器官保護及抗氧化應激分子機制研究　出處：《重慶醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 咪唑克生 膿毒癥 氧化應激 核因子E2相關因子2

【摘要】：目的:探討咪唑克生(IDA)對脂多糖誘導膿毒癥小鼠的器官保護作用及抗氧化應激分子機制。方法:(1)將60只成年C57BL/6小鼠隨機分為對照組(12只,腹腔注射磷酸鹽緩沖液)、LPS組(12只,腹腔注射LPS 10mg/kg)、低劑量組(12只,腹腔注射LPS 10mg/kg和IDA 1mg/kg)、中劑量組(12只,腹腔注射LPS 10mg/kg和IDA 2mg/kg)和高劑量組(12只,腹腔注射LPS10mg/kg和IDA 4mg/kg),于制模后24h處死所有小鼠,采集血和肝、肺、腎臟器組織標本。全自動生化分析儀檢測血清丙氨酸氨基轉(zhuǎn)移酶(ALT)和天冬氨酸氨基轉(zhuǎn)移酶(AST)的水平;化學比色法檢測肝組織勻漿中丙二醛(MDA)含量以及總超氧化物歧化酶(SOD)、過氧化氫酶(CAT)、總谷胱甘肽過氧化物酶(GPx)的活性;蘇木精-伊紅染色觀察肝、肺、腎組織病理學改變;Western Blot法檢測肝組織勻漿中HO-1、NQO-1、Nrf2蛋白表達;免疫組化法檢測肝、腎組織Nrf2的表達。(2)體外培養(yǎng)小鼠巨噬細胞系RAW264.7細胞,隨機分為四組:對照組、LPS組、治療組及IDA組,對照組僅用完全培養(yǎng)基培養(yǎng),LPS組給予10μg/ml LPS刺激,LPS+IDA組同時給予10μg/ml LPS+100μM IDA作用,IDA組給予100μM IDA處理。以2,7-二氫二氯熒光素二乙酸酯(DCFH-DA)為熒光探針,運用流式細胞儀及熒光顯微鏡檢測細胞內(nèi)活性氧(ROS)水平;Western Blot法檢測細胞中HO-1、NQO-1、Nrf2蛋白表達,細胞免疫熒光檢測細胞內(nèi)HO-1的表達。結(jié)果:(1)LPS刺激24h,LPS組小鼠血清中ALT、AST水平均顯著高于對照組[ALT(U/L):88.10±12.05比35.93±3.02;AST(U/L):311.97±75.04比136.77±10.80;均P0.01]。IDA能有效降低LPS所致的小鼠血清轉(zhuǎn)氨酶的異常升高[ALT(U/L):低劑量組68.40±5.17比88.10±12.05(P=0.06),中劑量組51.10±5.66比88.10±12.05(P0.01),高劑量組44.27±9.10比88.10±12.05(P㩳0.01);AST(U/L):低劑量組257.73±51.97比311.97±75.04(P=0.36),中劑量組194.37±20.84比311.97±75.04(P=0.06),高劑量組161.13±27.91比311.97±75.04(P㩳0.05)]。肝組織勻漿中,LPS組的MDA水平顯著高于對照組[(6.37±1.45)nmol/mg比(1.13±0.25)nmol/mg,P0.01],抗氧化蛋白酶(SOD、CAT及GPx)的活性顯著降低[SOD(U/mg):0.97±0.35比3.83±0.71;CAT(U/mg):30.67±7.02比70.33±8.50;GPx(U/mg):8.33±2.52比30.33±5.51,;均P0.01]。然而,咪唑克生治療則可呈劑量依賴性地減少丙二醛(MDA)水平[低劑量組(4.83±0.80)nmol/mg比(6.37±1.45)nmol/mg(P=0.18),中劑量組(3.20±0.62)nmol/mg比(6.37±1.45)nmol/mg(P0.05),高劑量組(2.30±0.46)nmol/mg比(6.37±1.45)nmol/mg(P㩳0.01)]及改善抗氧化蛋白酶(SOD、CAT及GPx)活性的抑制[SOD(U/mg):低劑量組1.47±0.31比0.97±0.35(P=0.14),中劑量組2.36±0.47比0.97±0.35(P0.05),高劑量組3.27±0.70比0.97±0.35(P㩳0.01);CAT(U/mg):低劑量組50.33±13.50比30.67±7.02(P=0.09),中劑量組67.67±13.50比30.67±7.02(P㩳0.05),高劑量組80.67±13.01比30.67±7.02(P㩳0.01);GPx(U/mg):低劑量組15.67±4.04比8.33±2.52(P=0.06),中劑量組21.33±4.51比8.33±2.52(P㩳0.05),高劑量組35.33±9.07比8.33±2.52(P㩳0.01)]。IDA還可明顯改善小鼠肝、肺、腎的病理改變,并且可增加肝組織中HO-1、NQO-1及Nrf2及腎組織中Nrf2的表達。(2)體外實驗中,與對照組相比,RAW264.7細胞受到LPS刺激后,細胞內(nèi)ROS明顯增加(P㩳0.01),而IDA能顯著降低LPS誘導的ROS異常升高(P㩳0.05)。同時,與LPS組相比,LPS+IDA組中HO-1、NQO-1及Nrf2的蛋白表達明顯增加。結(jié)論:IDA通過激活巨噬細胞Nrf2信號通路發(fā)揮抗氧化應激作用,從而減輕小鼠膿毒癥時的多器官損害。
[Abstract]:Objective: To investigate the effects of idazoxan (IDA) on lipopolysaccharide induced septic mouse organ protective effect and antioxidative molecular mechanism. Methods: (1) a total of 60 adult C57BL/6 mice were randomly divided into control group (12 rats, intraperitoneal injection of phosphate buffer), LPS group (12 rats, intraperitoneal injection of LPS 10mg/kg). The low dose group (12 rats, intraperitoneal injection of LPS 10mg/kg and IDA 1mg/kg), middle dose group (12 rats, intraperitoneal injection of LPS 10mg/kg and IDA 2mg/kg) and high dose group (12 rats, intraperitoneal injection of LPS10mg/kg and IDA 4mg/kg), in the system of 24h after all the mice were sacrificed, blood and liver, lung and kidney for tissue samples. The serum alanine amino transferase automatic biochemical analyzer (ALT) and aspartate aminotransferase (AST) levels were measured; liver tissue homogenate colorimetric method (MDA) content and superoxide dismutase (SOD), catalase (CAT), total glutathione. Oxidation Peroxidase (GPx) activity; hematoxylin eosin staining of liver, lung, renal pathological change; HO-1, detection of Western in liver tissue homogenate in Blot NQO-1, Nrf2 protein expression; immunohistochemistry of liver, expression of Nrf2 in renal tissue. (2) mouse macrophage RAW264.7 cells cultured in vitro. Were randomly divided into four groups: control group, LPS group, IDA group and treatment group, the control group only with complete medium, LPS group was given 10 g/ml LPS stimulation, while group LPS+IDA was treated with 10 g/ml LPS+100 M IDA, IDA group was given 100 M IDA to two 2,7-. Two hydrogen chloride two fluorescein acetate (DCFH-DA) as a fluorescence probe by flow cytometry and fluorescence microscopy to detect intracellular reactive oxygen species (ROS); HO-1, Western Blot in NQO-1 cells, the expression of Nrf2 protein expression, cell immunofluorescence detection of HO-1 in cells. Results: (1) LPS stimulated 24h, serum LPS group mice in ALT, AST 姘村鉤鍧囨樉钁楅珮浜庡鐓х粍[ALT(U/L):88.10鹵12.05姣，

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