中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2013年
6期
441-446
,共6页
马碧蔓%刘朝晖%梁志科%关丽君%汪新龙
馬碧蔓%劉朝暉%樑誌科%關麗君%汪新龍
마벽만%류조휘%량지과%관려군%왕신룡
肺疾病,慢性阻塞性%呼吸肌%蛋白酶体抑制剂
肺疾病,慢性阻塞性%呼吸肌%蛋白酶體抑製劑
폐질병,만성조새성%호흡기%단백매체억제제
Pulmonary disease,chronic obstructive%Respiratory muscles%Proteasome inhibitors
目的 研究蛋白酶体抑制剂MG-132抑制烟草烟雾所致慢性阻塞性肺疾病(简称慢阻肺)大鼠模型骨骼肌萎缩的可能机制.方法 采用反复烟草烟雾暴露联合气管内注入脂多糖法成功制备成年雄性SD大鼠慢阻肺模型,分为非干预组、高剂量组(MG-132 0.1 mg·kg-1·d-1)和低剂量组(MG-132 0.05 mg·kg-1·d-1),每组12只,另设对照组.各组分别于实验1周和4周处死6只大鼠,分离大鼠膈肌并称重,采用酶联免疫吸附试验法测定大鼠血清和膈肌中肿瘤坏死因子-α(TNF-α)的表达,逆转录-PCR法和Western blot法检测大鼠膈肌内的肌萎缩F-box蛋白(MAFbx)、核因子-κB p65和抑制性因子κB-α(IκB-α)mRNA和蛋白表达.多组间比较采用方差分析,两组间比较采用t检验,相关性检验采用Pearson直线相关性分析.结果 实验1周和4周非干预组[(0.83±0.09)mg和(1.01±0.06) mg]、高剂量组[(0.85±0.02) mg和(1.11 ±0.06) mg]及低剂量组[(0.83±0.03) mg和(1.04 ±0.02) mg]大鼠的膈肌重量均低于对照组[(0.99 ±0.06) mg和(1.20±0.04) mg],高剂量组和低剂量组大鼠膈肌重量下降程度均小于非干预组,高剂量组干预4周时更明显；实验4周时在高剂量组和低剂量组膈肌中TNF-α含量[(106±8)ng/L和(122±7)ng/L]显著低于非干预组[(143 ±24) ng/L],高剂量组膈肌中核因子-κBp65和MAFbx蛋白表达水平(1.13±0.04和1.27±0.05)及mRNA含量(2.17±0.42和1.74±0.14)显著低于非干预组(1.32±0.04和1.44±0.07及2.81 ±0.31和4.87±0.34),IκB-α基因和蛋白表达水平(0.96±0.08和0.83 ±0.06)显著高于非干预组(0.25±0.02和0.58±0.06),差异均有统计学意义(t值为1.57～24.9,P＜0.05).各组大鼠血清和膈肌中TNF-α表达水平与膈肌中核因子-κBp65蛋白和mRNA表达水平呈显著正相关(r值为0.672 ～0.875,均P＜0.01),与IκB-α蛋白和mRNA表达水平呈显著负相关(r值为-0.656 ～-0.927,均P＜0.01).结论 蛋白酶体抑制剂MG-132可能通过抑制IκB-α的泛素化降解,从而稳定核因子-κB活性,抑制泛素蛋白酶体途径介导的慢阻肺大鼠膈肌萎缩.
目的 研究蛋白酶體抑製劑MG-132抑製煙草煙霧所緻慢性阻塞性肺疾病(簡稱慢阻肺)大鼠模型骨骼肌萎縮的可能機製.方法 採用反複煙草煙霧暴露聯閤氣管內註入脂多糖法成功製備成年雄性SD大鼠慢阻肺模型,分為非榦預組、高劑量組(MG-132 0.1 mg·kg-1·d-1)和低劑量組(MG-132 0.05 mg·kg-1·d-1),每組12隻,另設對照組.各組分彆于實驗1週和4週處死6隻大鼠,分離大鼠膈肌併稱重,採用酶聯免疫吸附試驗法測定大鼠血清和膈肌中腫瘤壞死因子-α(TNF-α)的錶達,逆轉錄-PCR法和Western blot法檢測大鼠膈肌內的肌萎縮F-box蛋白(MAFbx)、覈因子-κB p65和抑製性因子κB-α(IκB-α)mRNA和蛋白錶達.多組間比較採用方差分析,兩組間比較採用t檢驗,相關性檢驗採用Pearson直線相關性分析.結果 實驗1週和4週非榦預組[(0.83±0.09)mg和(1.01±0.06) mg]、高劑量組[(0.85±0.02) mg和(1.11 ±0.06) mg]及低劑量組[(0.83±0.03) mg和(1.04 ±0.02) mg]大鼠的膈肌重量均低于對照組[(0.99 ±0.06) mg和(1.20±0.04) mg],高劑量組和低劑量組大鼠膈肌重量下降程度均小于非榦預組,高劑量組榦預4週時更明顯；實驗4週時在高劑量組和低劑量組膈肌中TNF-α含量[(106±8)ng/L和(122±7)ng/L]顯著低于非榦預組[(143 ±24) ng/L],高劑量組膈肌中覈因子-κBp65和MAFbx蛋白錶達水平(1.13±0.04和1.27±0.05)及mRNA含量(2.17±0.42和1.74±0.14)顯著低于非榦預組(1.32±0.04和1.44±0.07及2.81 ±0.31和4.87±0.34),IκB-α基因和蛋白錶達水平(0.96±0.08和0.83 ±0.06)顯著高于非榦預組(0.25±0.02和0.58±0.06),差異均有統計學意義(t值為1.57～24.9,P＜0.05).各組大鼠血清和膈肌中TNF-α錶達水平與膈肌中覈因子-κBp65蛋白和mRNA錶達水平呈顯著正相關(r值為0.672 ～0.875,均P＜0.01),與IκB-α蛋白和mRNA錶達水平呈顯著負相關(r值為-0.656 ～-0.927,均P＜0.01).結論 蛋白酶體抑製劑MG-132可能通過抑製IκB-α的汎素化降解,從而穩定覈因子-κB活性,抑製汎素蛋白酶體途徑介導的慢阻肺大鼠膈肌萎縮.
목적 연구단백매체억제제MG-132억제연초연무소치만성조새성폐질병(간칭만조폐)대서모형골격기위축적가능궤제.방법 채용반복연초연무폭로연합기관내주입지다당법성공제비성년웅성SD대서만조폐모형,분위비간예조、고제량조(MG-132 0.1 mg·kg-1·d-1)화저제량조(MG-132 0.05 mg·kg-1·d-1),매조12지,령설대조조.각조분별우실험1주화4주처사6지대서,분리대서격기병칭중,채용매련면역흡부시험법측정대서혈청화격기중종류배사인자-α(TNF-α)적표체,역전록-PCR법화Western blot법검측대서격기내적기위축F-box단백(MAFbx)、핵인자-κB p65화억제성인자κB-α(IκB-α)mRNA화단백표체.다조간비교채용방차분석,량조간비교채용t검험,상관성검험채용Pearson직선상관성분석.결과 실험1주화4주비간예조[(0.83±0.09)mg화(1.01±0.06) mg]、고제량조[(0.85±0.02) mg화(1.11 ±0.06) mg]급저제량조[(0.83±0.03) mg화(1.04 ±0.02) mg]대서적격기중량균저우대조조[(0.99 ±0.06) mg화(1.20±0.04) mg],고제량조화저제량조대서격기중량하강정도균소우비간예조,고제량조간예4주시경명현；실험4주시재고제량조화저제량조격기중TNF-α함량[(106±8)ng/L화(122±7)ng/L]현저저우비간예조[(143 ±24) ng/L],고제량조격기중핵인자-κBp65화MAFbx단백표체수평(1.13±0.04화1.27±0.05)급mRNA함량(2.17±0.42화1.74±0.14)현저저우비간예조(1.32±0.04화1.44±0.07급2.81 ±0.31화4.87±0.34),IκB-α기인화단백표체수평(0.96±0.08화0.83 ±0.06)현저고우비간예조(0.25±0.02화0.58±0.06),차이균유통계학의의(t치위1.57～24.9,P＜0.05).각조대서혈청화격기중TNF-α표체수평여격기중핵인자-κBp65단백화mRNA표체수평정현저정상관(r치위0.672 ～0.875,균P＜0.01),여IκB-α단백화mRNA표체수평정현저부상관(r치위-0.656 ～-0.927,균P＜0.01).결론 단백매체억제제MG-132가능통과억제IκB-α적범소화강해,종이은정핵인자-κB활성,억제범소단백매체도경개도적만조폐대서격기위축.
Objective To investigate the effect of the proteasome inhibitor MG-132 on skeletal muscle atrophy in a rat model of chronic obstructive pulmonary disease (COPD) and its potential mechanisms.Methods The COPD rat model was established by instillation of LPS and exposure to the cigarette smoke.Then the COPD rats were randomly divided into 3 groups (each group n =12):COPD model control group,MG-132 high dose group (MG-132 0.1 mg · kg-1 · d-1) and low dose group (MG-132 0.05 mg· kg-1 · d-1),and normal control group.After 1 week and 4 week,6 rats of each group were sacrificed,and then the following parameters were determined:the weight of the diaphragm muscle,the concentration of TNF-α in the serum and diaphragm via enzyme-linked immunosorbent assay (ELISA).Muscle atrophy F-box protein (MAFbx),NF-κBp65,and IκB-α mRNA levels were determined by RT-PCR.The protein levels of MAFbx,NF-κBp65 and IκB-α in diaphragm were measured by Western blot.The single factor analysis of variance was used for statistical analysis among the groups,while t test was used for comparison between 2 groups,and Pearson linear correlation analysis was also performed.Results The weight of diaphragm muscle from 1 week and 4 week normal control group [(0.99 ± 0.06) mg and (1.20 ±0.04) mg] were reduced as compared to those of COPD model control group [(0.83 ±0.09) mg and (1.01 ±0.06) mg],high dose group [(0.85 ±0.02) mg and (1.11 ±0.06) mg],and low dose group [(0.83 ± 0.03) mg and (1.04 ± 0.02) mg].The reduction of diaphragm muscle weight in the high dose group and the low dose group was significantly less than that in the COPD model control group,with a more marked difference as compared with the 4 week high dose group.The TNF-α levels in diaphragm from 4 week high dose group [(106 ± 8) ng/L] and low dose group [(122 ± 7) ng/L] were decreased as compared to that of the COPD model control group [(143 ± 24) ng/L].The levels of NF-κBp65 and MAFbx mRNA from the 4 week high dose group (2.17 ±0.42) and low dose group (1.74 ±0.14) and the protein expression (1.13 ± 0.04 and 1.27 ± 0.05) were also decreased as compared to those of the COPD model control group (mRNA 2.81 ± 0.31 and 4.87 ± 0.34,protein expression 1.32 ± 0.04 and 1.44 ±0.07).The levels of IκB-α mRNA and protein expression (0.96 ± 0.08 and 0.83 ± 0.06) were higher than those of the COPD model control group (0.25 ± 0.02 and 0.58 ± 0.06),(t =1.57-24.9,P ＜ 0.05).The levels of the TNF-α levels in serum and diaphragm were correlated positively with the levels of MAFbx and NF-κBp65 mRNA and protein expression (r =0.672-0.875,P ＜ 0.01),but negatively with the levels of IκB-α mRNA and protein expression (r =-0.656--0.927,P ＜ 0.01).Conclusions The proteasome inhibitor MG-132 significantly inhibited IκB-α degradation thus preventing NF-κB activation.This effect resulted in preventing skeletal muscle atrophy in the COPD rats.
