中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2015年
2期
110-114
,共5页
许世清%刘虹麟%娄晋宁%王在%彭亮%房青%游嘉%邓婷婷%郭彬%张文健
許世清%劉虹麟%婁晉寧%王在%彭亮%房青%遊嘉%鄧婷婷%郭彬%張文健
허세청%류홍린%루진저%왕재%팽량%방청%유가%산정정%곽빈%장문건
C肽%系膜细胞%氧化应激%蛋白激酶A%晚期糖基化终末产物
C肽%繫膜細胞%氧化應激%蛋白激酶A%晚期糖基化終末產物
C태%계막세포%양화응격%단백격매A%만기당기화종말산물
C-peptide%Mesangial cells%Oxidative stress%Protein kinase A%Advanced glycation end products
目的:研究C肽对晚期糖基化终末产物(AGEs)诱导的大鼠肾小球系膜细胞氧化应激的影响及其机制。方法将大鼠肾小球系膜细胞按照下列分组培养:对照组:正常培养基培养;AGEs组:培养基中含有200 mg/L AGEs;AGEs+C肽组:培养基中含有200 mg/L AGEs和5μmol/L C肽;AGEs+C肽+H89组:预先用含有H89(终浓度10μmol/L)的培养基培养30 min后加入AGEs(终浓度200 mg/L)和C肽(终浓度5μmol/L)。采用荧光法检测细胞内活性氧的水平,应用格里斯反应检测细胞一氧化氮(NO)的生成水平,采用实时荧光定量聚合酶链反应(RT-PCR)或Western blotting检测晚期糖基化终末产物受体(RAGE)、蛋白激酶A(PKA)、尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)和诱导型一氧化氮合酶(iNOS)的表达水平。采用非参数检验(Kruskal-Wallis H检验)进行组间比较,采用Mann-Whitney U检验进行两两比较。结果 AGEs组系膜细胞生成ROS和NO较对照组增多(分别为193.7±6.4比136.1±4.9和27.2±4.7比15.5±0.7,均U=0,P<0.05),C肽可以抑制ROS和NO的产生, AGEs+C肽组的ROS和NO水平与AGEs组差异有统计学意义(分别为136.9±14.3比193.7±6.4;16.0±2.1比27.2±4.7;均U=0,P<0.05)。与对照组相比,AGEs上调RAGE的表达,下调PKA的表达,并且AGEs可上调NOX4和iNOS的表达(分别为0.565±0.027比0.148±0.006,0.085±0.035比0.518±0.019,0.912±0.055比0.105±0.012,0.279±0.003比0.126±0.004,均U=0,P<0.05);C肽处理组较AGEs组RAGE的表达下调,PKA的表达上调,NOX4和iNOS的表达下调(分别为0.159±0.003比0.565±0.027,0.594±0.079比0.085±0.035,0.085±0.005比0.912±0.055,0.071±0.016比0.279±0.003,均U=0,P<0.05)。与C肽组比较,H89组阻断PKA激活后,ROS和NO生成增多(分别为195.7±9.3比149.7±11.7,22.2±1.1比16.4±2.1,均U=0,P<0.05),并且H89逆转C肽下调NOX4和iNOS表达的作用(H89组比C肽组分别为0.455±0.018比0.085±0.005,0.296±0.013比0.071±0.016,均U=0,P<0.05)。结论 C肽通过影响PKA信号通路抑制肾小球系膜细胞氧化应激。
目的:研究C肽對晚期糖基化終末產物(AGEs)誘導的大鼠腎小毬繫膜細胞氧化應激的影響及其機製。方法將大鼠腎小毬繫膜細胞按照下列分組培養:對照組:正常培養基培養;AGEs組:培養基中含有200 mg/L AGEs;AGEs+C肽組:培養基中含有200 mg/L AGEs和5μmol/L C肽;AGEs+C肽+H89組:預先用含有H89(終濃度10μmol/L)的培養基培養30 min後加入AGEs(終濃度200 mg/L)和C肽(終濃度5μmol/L)。採用熒光法檢測細胞內活性氧的水平,應用格裏斯反應檢測細胞一氧化氮(NO)的生成水平,採用實時熒光定量聚閤酶鏈反應(RT-PCR)或Western blotting檢測晚期糖基化終末產物受體(RAGE)、蛋白激酶A(PKA)、尼剋酰胺腺嘌呤二覈苷痠燐痠氧化酶4(NOX4)和誘導型一氧化氮閤酶(iNOS)的錶達水平。採用非參數檢驗(Kruskal-Wallis H檢驗)進行組間比較,採用Mann-Whitney U檢驗進行兩兩比較。結果 AGEs組繫膜細胞生成ROS和NO較對照組增多(分彆為193.7±6.4比136.1±4.9和27.2±4.7比15.5±0.7,均U=0,P<0.05),C肽可以抑製ROS和NO的產生, AGEs+C肽組的ROS和NO水平與AGEs組差異有統計學意義(分彆為136.9±14.3比193.7±6.4;16.0±2.1比27.2±4.7;均U=0,P<0.05)。與對照組相比,AGEs上調RAGE的錶達,下調PKA的錶達,併且AGEs可上調NOX4和iNOS的錶達(分彆為0.565±0.027比0.148±0.006,0.085±0.035比0.518±0.019,0.912±0.055比0.105±0.012,0.279±0.003比0.126±0.004,均U=0,P<0.05);C肽處理組較AGEs組RAGE的錶達下調,PKA的錶達上調,NOX4和iNOS的錶達下調(分彆為0.159±0.003比0.565±0.027,0.594±0.079比0.085±0.035,0.085±0.005比0.912±0.055,0.071±0.016比0.279±0.003,均U=0,P<0.05)。與C肽組比較,H89組阻斷PKA激活後,ROS和NO生成增多(分彆為195.7±9.3比149.7±11.7,22.2±1.1比16.4±2.1,均U=0,P<0.05),併且H89逆轉C肽下調NOX4和iNOS錶達的作用(H89組比C肽組分彆為0.455±0.018比0.085±0.005,0.296±0.013比0.071±0.016,均U=0,P<0.05)。結論 C肽通過影響PKA信號通路抑製腎小毬繫膜細胞氧化應激。
목적:연구C태대만기당기화종말산물(AGEs)유도적대서신소구계막세포양화응격적영향급기궤제。방법장대서신소구계막세포안조하렬분조배양:대조조:정상배양기배양;AGEs조:배양기중함유200 mg/L AGEs;AGEs+C태조:배양기중함유200 mg/L AGEs화5μmol/L C태;AGEs+C태+H89조:예선용함유H89(종농도10μmol/L)적배양기배양30 min후가입AGEs(종농도200 mg/L)화C태(종농도5μmol/L)。채용형광법검측세포내활성양적수평,응용격리사반응검측세포일양화담(NO)적생성수평,채용실시형광정량취합매련반응(RT-PCR)혹Western blotting검측만기당기화종말산물수체(RAGE)、단백격매A(PKA)、니극선알선표령이핵감산린산양화매4(NOX4)화유도형일양화담합매(iNOS)적표체수평。채용비삼수검험(Kruskal-Wallis H검험)진행조간비교,채용Mann-Whitney U검험진행량량비교。결과 AGEs조계막세포생성ROS화NO교대조조증다(분별위193.7±6.4비136.1±4.9화27.2±4.7비15.5±0.7,균U=0,P<0.05),C태가이억제ROS화NO적산생, AGEs+C태조적ROS화NO수평여AGEs조차이유통계학의의(분별위136.9±14.3비193.7±6.4;16.0±2.1비27.2±4.7;균U=0,P<0.05)。여대조조상비,AGEs상조RAGE적표체,하조PKA적표체,병차AGEs가상조NOX4화iNOS적표체(분별위0.565±0.027비0.148±0.006,0.085±0.035비0.518±0.019,0.912±0.055비0.105±0.012,0.279±0.003비0.126±0.004,균U=0,P<0.05);C태처리조교AGEs조RAGE적표체하조,PKA적표체상조,NOX4화iNOS적표체하조(분별위0.159±0.003비0.565±0.027,0.594±0.079비0.085±0.035,0.085±0.005비0.912±0.055,0.071±0.016비0.279±0.003,균U=0,P<0.05)。여C태조비교,H89조조단PKA격활후,ROS화NO생성증다(분별위195.7±9.3비149.7±11.7,22.2±1.1비16.4±2.1,균U=0,P<0.05),병차H89역전C태하조NOX4화iNOS표체적작용(H89조비C태조분별위0.455±0.018비0.085±0.005,0.296±0.013비0.071±0.016,균U=0,P<0.05)。결론 C태통과영향PKA신호통로억제신소구계막세포양화응격。
Objective To investigate the effect of C-peptide on advanced glycation end products (AGE)-induced oxidative stress in rat mesangial cells and its mechanism. Methods Rat mesangial cells were cultured in the normal medium(Control group),or medium with 200 mg/L AGEs(AGEs group),or medium with 200 mg/L AGEs and 5μmol/L C-peptide(AGEs+C-peptide group)or medium with 10μmol/L H89(added in advance,and incubated for 30 min)and 200 mg/L AGEs and 5μmol/L C-peptide(AGEs+C-peptide+H89 group). The intracellular reactive oxygen species(ROS)was detected with fluorescence method,and the supernatant nitric oxide(NO)level was detected by Griess reaction. Real-time PCR and Western blotting were used to detect the expression of the receptor for advanced glycation endproducts (RAGE),protein kinase A(PKA),nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)and inducible nitric oxide synthase(iNOS). Non-parametric Kruskal-Wallis H test and Mann-Whitney U test were used to compare data between groups,and two groups,respectively. Results Compared with control group,the level of ROS and NO increased in AGEs group(193.7±6.4 vs 136.1±4.9;and 27.2±4.7 vs 15.5± 0.7,respectively,all U=0,P<0.05). C-peptide could suppress the production of ROS and NO. In AGEs+C-peptide group,ROS and NO reduced than AGEs group(136.9±14.3 vs 193.7±6.4 and 16.0±2.1 vs 27.2±4.7 respectively,all U=0,P<0.05). Compared with control group,AGEs could up-regulate the expression of RAGE(0.565±0.027 vs 0.148±0.006,U=0,P<0.05)but down-regulate PKA(0.085±0.035 vs 0.518±0.019, U=0,P<0.05). And AGEs increased the expression of NOX4 and iNOS(0.912±0.055 vs 0.105±0.012,and 0.279±0.003 vs 0.126±0.004 respectively,all U=0,P<0.05). Compared with AGEs group,C-peptide down-regulated RAGE(0.159 ± 0.003 vs 0.565 ± 0.027,U=0,P<0.05),up-regulated PKA(0.594 ± 0.079 vs 0.085 ± 0.035,U=0,P<0.05),and down-regulated NOX4 and iNOS(0.085±0.005 vs 0.912±0.055,and 0.071±0.016 vs 0.279 ± 0.003 respectively,all U=0,P<0.05)Compared with C-peptide group,H89 groupinhibited the activation of PKA,increased the production of ROS and NO(195.7 ± 9.3 vs 149.7 ± 11.7 and 22.2 ± 1.1 vs 16.4 ± 2.1,all U=0,P<0.05). Moreover,compared with C-peptide group,the expression of NOX4 and iNOS in H89 group was increased(0.455±0.018 vs 0.085±0.005 and 0.296±0.013 vs 0.071±0.016 respectively,all U=0,P<0.05). Conclusion C-peptide inhibited oxidative stress in mesangial cells through PKA signaling pathway.