中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2013年
9期
560-564
,共5页
刘静%邓文娟%魏素虹%牛瑞兰%许衍甲%权金星%田利民%周淑红%张琦
劉靜%鄧文娟%魏素虹%牛瑞蘭%許衍甲%權金星%田利民%週淑紅%張琦
류정%산문연%위소홍%우서란%허연갑%권금성%전이민%주숙홍%장기
红芪多糖%人脐静脉内皮细胞%高糖%一氧化氮%内皮素-1
紅芪多糖%人臍靜脈內皮細胞%高糖%一氧化氮%內皮素-1
홍기다당%인제정맥내피세포%고당%일양화담%내피소-1
Radix hedysari polysaccharide%Human umbilical vein endothelial cells%High glucose%Nitric oxide%Endothelin-1
目的 研究红芪多糖(HPS)对高糖条件下人脐静脉内皮细胞(HUVEC)合成和释放一氧化氮(NO)和内皮素-1(ET-1)的影响及作用机制.方法 从新鲜脐带中分离HUVEC进行鉴定、培养,细胞分为正常对照组、高糖组(30 mmol/L葡萄糖)和HPS干预组.MTI比色法分析HPS对高糖诱导HUVEC增殖率的影响,流式细胞术Annexin-V/PI双染法检测细胞凋亡,硝酸盐还原酶法检测上清液中NO的水平,分光光度法检测细胞内一氧化氮合酶(NOS)、诱导型一氧化氮合酶(iNOS)的浓度,酶联免疫吸附(ELISA)法检测上清液中ET-1的含量,实时荧光定量聚合酶链反应(RT-PCR)检测细胞内皮型一氧化氮合酶(eNOS)、ET-1和c-Jun氨基末端激酶1(JNK1) mRNA的表达水平.组间差异显著性检验使用单因素方差分析,组间两两比较使用LSD法.结果 高糖组在6 h[(82.4±3.5)％]、24 h[(68.2±1.4)％]、48 h[(63.0 ±2.9)％]的细胞增殖率显著低于对照组(100％,P＜0.05)；HPS干预组细胞增殖率随浓度变化呈现先上升后下降的趋势,其中50 mg/L[(85.3±4.6)％]、100 mg/L[(89.6±1.1)％]、200 mg/L[(88.8±3.6)％]HPS干预组较高糖组增加,差异具有统计学意义(均P ＜0.05)；高糖组NO[(24.84±1.34) μmol/L]、NOS[(0.54 ±0.06) U/ml]含量较正常对照组呈现下降的趋势,iNOS[(0.133±0.015)U/ml]和ET-1[(0.740±0.070) ng/ml]含量则在各时间点均高于对照组,HPS干预组升高不同时间点HUVEC内NO[(23.20 ±0.55) μmol/L]、NOS[(0.46±0.10) U/ml]、以及降低iNOS[(0.08±0.020) U/ml]和ET-1[(0.710 ±0.030) μg/L]的变化,P＜0.05,使其趋向正常水平；HPS可提高高糖所致内皮细胞eNOS mRNA的水平[(0.33 ±0.02)比(0.23 ±0.04)],降低ET-1 mRNA的水平(2.28 ±0.31比2.79±0.29)；高糖组细胞内JNK1 mRNA水平表达(2.95±0.05)较正常对照组(1.00±0.00)显著增加(P＜0.05),而HPS干预组(1.45±0.05)则较高糖组(2.95±0.05)明显减少(P＜0.05).结论 HPS对体外高糖诱导HUVEC损伤具有保护作用,这一作用可能与抑制JNK信号通路有关.
目的 研究紅芪多糖(HPS)對高糖條件下人臍靜脈內皮細胞(HUVEC)閤成和釋放一氧化氮(NO)和內皮素-1(ET-1)的影響及作用機製.方法 從新鮮臍帶中分離HUVEC進行鑒定、培養,細胞分為正常對照組、高糖組(30 mmol/L葡萄糖)和HPS榦預組.MTI比色法分析HPS對高糖誘導HUVEC增殖率的影響,流式細胞術Annexin-V/PI雙染法檢測細胞凋亡,硝痠鹽還原酶法檢測上清液中NO的水平,分光光度法檢測細胞內一氧化氮閤酶(NOS)、誘導型一氧化氮閤酶(iNOS)的濃度,酶聯免疫吸附(ELISA)法檢測上清液中ET-1的含量,實時熒光定量聚閤酶鏈反應(RT-PCR)檢測細胞內皮型一氧化氮閤酶(eNOS)、ET-1和c-Jun氨基末耑激酶1(JNK1) mRNA的錶達水平.組間差異顯著性檢驗使用單因素方差分析,組間兩兩比較使用LSD法.結果 高糖組在6 h[(82.4±3.5)％]、24 h[(68.2±1.4)％]、48 h[(63.0 ±2.9)％]的細胞增殖率顯著低于對照組(100％,P＜0.05)；HPS榦預組細胞增殖率隨濃度變化呈現先上升後下降的趨勢,其中50 mg/L[(85.3±4.6)％]、100 mg/L[(89.6±1.1)％]、200 mg/L[(88.8±3.6)％]HPS榦預組較高糖組增加,差異具有統計學意義(均P ＜0.05)；高糖組NO[(24.84±1.34) μmol/L]、NOS[(0.54 ±0.06) U/ml]含量較正常對照組呈現下降的趨勢,iNOS[(0.133±0.015)U/ml]和ET-1[(0.740±0.070) ng/ml]含量則在各時間點均高于對照組,HPS榦預組升高不同時間點HUVEC內NO[(23.20 ±0.55) μmol/L]、NOS[(0.46±0.10) U/ml]、以及降低iNOS[(0.08±0.020) U/ml]和ET-1[(0.710 ±0.030) μg/L]的變化,P＜0.05,使其趨嚮正常水平；HPS可提高高糖所緻內皮細胞eNOS mRNA的水平[(0.33 ±0.02)比(0.23 ±0.04)],降低ET-1 mRNA的水平(2.28 ±0.31比2.79±0.29)；高糖組細胞內JNK1 mRNA水平錶達(2.95±0.05)較正常對照組(1.00±0.00)顯著增加(P＜0.05),而HPS榦預組(1.45±0.05)則較高糖組(2.95±0.05)明顯減少(P＜0.05).結論 HPS對體外高糖誘導HUVEC損傷具有保護作用,這一作用可能與抑製JNK信號通路有關.
목적 연구홍기다당(HPS)대고당조건하인제정맥내피세포(HUVEC)합성화석방일양화담(NO)화내피소-1(ET-1)적영향급작용궤제.방법 종신선제대중분리HUVEC진행감정、배양,세포분위정상대조조、고당조(30 mmol/L포도당)화HPS간예조.MTI비색법분석HPS대고당유도HUVEC증식솔적영향,류식세포술Annexin-V/PI쌍염법검측세포조망,초산염환원매법검측상청액중NO적수평,분광광도법검측세포내일양화담합매(NOS)、유도형일양화담합매(iNOS)적농도,매련면역흡부(ELISA)법검측상청액중ET-1적함량,실시형광정량취합매련반응(RT-PCR)검측세포내피형일양화담합매(eNOS)、ET-1화c-Jun안기말단격매1(JNK1) mRNA적표체수평.조간차이현저성검험사용단인소방차분석,조간량량비교사용LSD법.결과 고당조재6 h[(82.4±3.5)％]、24 h[(68.2±1.4)％]、48 h[(63.0 ±2.9)％]적세포증식솔현저저우대조조(100％,P＜0.05)；HPS간예조세포증식솔수농도변화정현선상승후하강적추세,기중50 mg/L[(85.3±4.6)％]、100 mg/L[(89.6±1.1)％]、200 mg/L[(88.8±3.6)％]HPS간예조교고당조증가,차이구유통계학의의(균P ＜0.05)；고당조NO[(24.84±1.34) μmol/L]、NOS[(0.54 ±0.06) U/ml]함량교정상대조조정현하강적추세,iNOS[(0.133±0.015)U/ml]화ET-1[(0.740±0.070) ng/ml]함량칙재각시간점균고우대조조,HPS간예조승고불동시간점HUVEC내NO[(23.20 ±0.55) μmol/L]、NOS[(0.46±0.10) U/ml]、이급강저iNOS[(0.08±0.020) U/ml]화ET-1[(0.710 ±0.030) μg/L]적변화,P＜0.05,사기추향정상수평；HPS가제고고당소치내피세포eNOS mRNA적수평[(0.33 ±0.02)비(0.23 ±0.04)],강저ET-1 mRNA적수평(2.28 ±0.31비2.79±0.29)；고당조세포내JNK1 mRNA수평표체(2.95±0.05)교정상대조조(1.00±0.00)현저증가(P＜0.05),이HPS간예조(1.45±0.05)칙교고당조(2.95±0.05)명현감소(P＜0.05).결론 HPS대체외고당유도HUVEC손상구유보호작용,저일작용가능여억제JNK신호통로유관.
Objective To investigate effect of radix hedysari polysaccharide (HPS) on secretion of nitric oxide (NO) and endothelin-1 (ET-1) on human umbilical vein endothelial cells(HUVECs) induced by high glucose and its mechanism.Methods HUVECs were isolated from umbilical vein,and cultured cells were divided into normal control group,high glucose group,HPS group,MTT assay were used to analyze the effect of HPS on HUVECs viability under high glucose.The levels of NO,nitric oxide synthase (NOS) and inducible nitric oxide synthase (iNOS) were measured by colorimetric analysis.The content of ET-1 in clear supernatant were detected by enzyme linked immunosorbent assay (ELISA).The expression of endothelial nitric oxide synthase (eNOS),ET-1 mRNA,c-Jun kinase-1 (JNK1) mRNA were examined by real-time quantitative RT-PCR.Results The cells viability were markedly decreased when HUVECs were treated with 30 mmol/L-glucose for 6 h((82.4 ±3.5)％),24 h((68.2 ± 1.4)％),48 h((63.0 ±2.9) ％),HPS could efficiently prevented cells viability lost(P ＜ 0.05),especially in 50 mg/L((85.3 ±4.6) ％),100 mg/L ((89.6 ± 1.1) ％),200 mg/L ((88.8 ± 3.6) ％),P ＜ 0.05.In high glucose group,the levels of NO ((24.84 ± 1.34) μmol/L) and NOS ((0.54 ± 0.06) U/ml) were increased at early stage and decreased at advanced stage compared with normal control group (P ＜ 0.05).The contents of iNOS ((0.08 ±0.020) U/ml) and ET-1 ((0.710 ± 0.030) ng/ml) were upgraded under high glucose at any time,nevertheless,HPS could balanced the levels of NO((23.20 ±0.55) μmoL/L),NOS((0.46 ±0.10)U/ml),iNOS((0.08 ±0.020) U/ml) and ET-1 ((0.710 ±0.030) μg/L),P ＜0.05.At equal pace,the expressions of eNOS mRNA was up-regulated and ET-1 mRNA was down-regulated in HPS group compared with high glucose group(0.33 ±0.02 vs 0.23 ±0.04,2.28 ±0.31 vs 2.79 ±0.29).The contents of JNK1 mRNA in HUVECs were shown to increased after exposure to high glucose (2.95 ± 0.05),P ＜ 0.05,which were markedly prevented by HPS (1.45 ± 0.05),P ＜ 0.05.Conclusion HPS can protect the injury of HUVECs induced by high glucose in vitro.The mechanism of protection may be associated with blocked JNK signaling pathway.
