中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2014年
10期
753-757
,共5页
胡雪梅%皇甫健%刘洁%李彩萍%邓浩华%吴玉文%徐焱成
鬍雪梅%皇甫健%劉潔%李綵萍%鄧浩華%吳玉文%徐焱成
호설매%황보건%류길%리채평%산호화%오옥문%서염성
冬凌草甲素%血管内皮细胞%TAp63%沉默信息调节因子1%炎症
鼕凌草甲素%血管內皮細胞%TAp63%沉默信息調節因子1%炎癥
동릉초갑소%혈관내피세포%TAp63%침묵신식조절인자1%염증
Oridonin%V ascular endothelial cells%TAp63%Silent information regulator 1%Inflammation
目的 研究冬凌草甲素(oridonin)对高糖高脂诱导的人脐静脉内皮细胞(HUVECs)损伤的保护作用机制.方法 体外培养HUVECs,噻唑蓝法(MTT)确定oridonin作用的最佳浓度;将细胞分为空白对照组、高糖高脂+oridonin预处理组(以下简称为oridonin预处理组,oridonin预处理细胞24 h)、高糖高脂组(高糖高脂的浓度为33.3 mmol/L葡萄糖+500 μmol/L棕榈酸,处理48 h),利用流式细胞仪检测细胞凋亡率,观察各组细胞凋亡情况;酶联免疫吸附法(ELISA)检测上清液中肿瘤坏死因子仅(TNF-α)、白介素6(IL-6)的浓度,以观察细胞的炎症情况;实时定量聚合酶链反应(RT-PCR)分析HUVECs内TAp63、沉默信息调节因子1(Sirt1)mRNA的表达水平;Western blotting分析TAp63、Sirt1及凋亡相关基因磷酸化细胞外调节蛋白激酶(p-ERK)、B淋巴细胞瘤2(Bcl-2)的蛋白表达水平.两组间比较采用￡检验,多组间采用单因素方差分析.结果 Oridonin作用于HUVECs细胞24 h的最佳浓度为5 μmol/L.高糖高脂组HUVECs细胞内TAp63(mRNA及蛋白)、Sirt1 (mRNA及蛋白)、Bcl-2蛋白水平明显低于空白对照组(t=-22.50、-6.02、-16.22、-6.72、-7.71,均P＜0.05),HUVECs凋亡率、细胞内p-ERK蛋白表达及上清中炎症因子TNF-α、IL-6浓度明显高于空白对照组(t=14.34、12.59、17.37、17.90,均P＜0.05).与高糖高脂组相比,oridonin预处理组HUVECs内TAp63 mRNA和蛋白、Sirt1mRNA和蛋白、Bcl-2蛋白表达明显升高(t=5.85、5.06、4.55、3.04、5.81,均P＜0.05),HUVECs凋亡率、细胞内p-ERK蛋白表达及上清中炎症因子TNF-α及IL-6的浓度明显下降(t=-7.11、-7.81、-10.51、-10.36,均P＜0.05).结论 冬凌草甲素能够抑制高糖高脂诱导的HUVECs的凋亡,可能与其抑制高糖高脂诱导的炎症反应和(或)增加TAp63、Sirt1、Bcl-2的表达,且降低p-ERK的表达有关.
目的 研究鼕凌草甲素(oridonin)對高糖高脂誘導的人臍靜脈內皮細胞(HUVECs)損傷的保護作用機製.方法 體外培養HUVECs,噻唑藍法(MTT)確定oridonin作用的最佳濃度;將細胞分為空白對照組、高糖高脂+oridonin預處理組(以下簡稱為oridonin預處理組,oridonin預處理細胞24 h)、高糖高脂組(高糖高脂的濃度為33.3 mmol/L葡萄糖+500 μmol/L棕櫚痠,處理48 h),利用流式細胞儀檢測細胞凋亡率,觀察各組細胞凋亡情況;酶聯免疫吸附法(ELISA)檢測上清液中腫瘤壞死因子僅(TNF-α)、白介素6(IL-6)的濃度,以觀察細胞的炎癥情況;實時定量聚閤酶鏈反應(RT-PCR)分析HUVECs內TAp63、沉默信息調節因子1(Sirt1)mRNA的錶達水平;Western blotting分析TAp63、Sirt1及凋亡相關基因燐痠化細胞外調節蛋白激酶(p-ERK)、B淋巴細胞瘤2(Bcl-2)的蛋白錶達水平.兩組間比較採用￡檢驗,多組間採用單因素方差分析.結果 Oridonin作用于HUVECs細胞24 h的最佳濃度為5 μmol/L.高糖高脂組HUVECs細胞內TAp63(mRNA及蛋白)、Sirt1 (mRNA及蛋白)、Bcl-2蛋白水平明顯低于空白對照組(t=-22.50、-6.02、-16.22、-6.72、-7.71,均P＜0.05),HUVECs凋亡率、細胞內p-ERK蛋白錶達及上清中炎癥因子TNF-α、IL-6濃度明顯高于空白對照組(t=14.34、12.59、17.37、17.90,均P＜0.05).與高糖高脂組相比,oridonin預處理組HUVECs內TAp63 mRNA和蛋白、Sirt1mRNA和蛋白、Bcl-2蛋白錶達明顯升高(t=5.85、5.06、4.55、3.04、5.81,均P＜0.05),HUVECs凋亡率、細胞內p-ERK蛋白錶達及上清中炎癥因子TNF-α及IL-6的濃度明顯下降(t=-7.11、-7.81、-10.51、-10.36,均P＜0.05).結論 鼕凌草甲素能夠抑製高糖高脂誘導的HUVECs的凋亡,可能與其抑製高糖高脂誘導的炎癥反應和(或)增加TAp63、Sirt1、Bcl-2的錶達,且降低p-ERK的錶達有關.
목적 연구동릉초갑소(oridonin)대고당고지유도적인제정맥내피세포(HUVECs)손상적보호작용궤제.방법 체외배양HUVECs,새서람법(MTT)학정oridonin작용적최가농도;장세포분위공백대조조、고당고지+oridonin예처리조(이하간칭위oridonin예처리조,oridonin예처리세포24 h)、고당고지조(고당고지적농도위33.3 mmol/L포도당+500 μmol/L종려산,처리48 h),이용류식세포의검측세포조망솔,관찰각조세포조망정황;매련면역흡부법(ELISA)검측상청액중종류배사인자부(TNF-α)、백개소6(IL-6)적농도,이관찰세포적염증정황;실시정량취합매련반응(RT-PCR)분석HUVECs내TAp63、침묵신식조절인자1(Sirt1)mRNA적표체수평;Western blotting분석TAp63、Sirt1급조망상관기인린산화세포외조절단백격매(p-ERK)、B림파세포류2(Bcl-2)적단백표체수평.량조간비교채용￡검험,다조간채용단인소방차분석.결과 Oridonin작용우HUVECs세포24 h적최가농도위5 μmol/L.고당고지조HUVECs세포내TAp63(mRNA급단백)、Sirt1 (mRNA급단백)、Bcl-2단백수평명현저우공백대조조(t=-22.50、-6.02、-16.22、-6.72、-7.71,균P＜0.05),HUVECs조망솔、세포내p-ERK단백표체급상청중염증인자TNF-α、IL-6농도명현고우공백대조조(t=14.34、12.59、17.37、17.90,균P＜0.05).여고당고지조상비,oridonin예처리조HUVECs내TAp63 mRNA화단백、Sirt1mRNA화단백、Bcl-2단백표체명현승고(t=5.85、5.06、4.55、3.04、5.81,균P＜0.05),HUVECs조망솔、세포내p-ERK단백표체급상청중염증인자TNF-α급IL-6적농도명현하강(t=-7.11、-7.81、-10.51、-10.36,균P＜0.05).결론 동릉초갑소능구억제고당고지유도적HUVECs적조망,가능여기억제고당고지유도적염증반응화(혹)증가TAp63、Sirt1、Bcl-2적표체,차강저p-ERK적표체유관.
Objective To investigate the protective role of oridonin in the vascular endothelial cells dysfunction induced by high glucose and lipid.Methods The optimum concentration of oridonin on human umbilical vein endothelial cells(HUVECs)were determined by MTT assay.The model of HUVECs was established and divided into 3 groups:control group,oridonin group(oridonin treated for 24 h,with high glucose and high palmitic),high glucose and palmitic acid group 33.3 mmol/L glucose.HUVECs were pretreated with oridonin under the condition of 33.3 mmol/L glucose and 500 μmol/L palmitic acid,while other groups received 33.3 mmol/L glucose and 500 μmol/L palmitic acid and dimethyl sulfoxide(DMSO).After 48 h treatment,the concentration of tumor necrosis factor-α (TNF-α)and interleukin-6 (IL-6) in the supernatant was detected by the enzyme-linked immunosorbent assay (ELISA) to observethe inflammation state while flow cytometry was used to detect the cell apoptosis of each group,the mRNA expression of TAp63 and Silent information regulator 1 (Sirt1) in HUVECs was detected by the real time polymerase chain reaction(real time PCR).And the protein expression of TAp63,Sirt1,phosphorylated extracellular regulated protein kinases (p-ERK) and B-cell lymphoma 2(Bcl-2) were determined by Western-blotting.Result The optimum concentration of oridonin treating on HUVECs was 5 μmol/L when effect for 24 h.Compared with the control group,the apoptosis rate,the protein expression of phosphated ERK,and the concentration of inflammation factors(TNF-α and IL-6) in the supernatant were significantly higher (t=14.34,12.59,17.37,17.90,P＜0.05 all above) while the expression of TAp63 mRNA and protein,Sirt1 mRNA and protein,Bcl-2 protein was significantly lower in the HUVECs in the high glucose and palmitic acid group (t=-22.50,-6.02,-16.22,-6.72,-7.71,all P＜0.05).But in the oridonin group which were treated with 33.3 mmol/L glucose and 500 μmol/L palmitic acid,the pretreatment with oridonin significantly induced the expression of TAp63 mRNA and protein(t=5.85,5.06,all P＜0.05),Sirt 1 mRNA and protein level(t=4.55,3.04,all P＜0.05)and Bcl-2 protein (t=5.81,P＜0.05).Furthermore,the apoptosis rate,the protein expression of phosphated ERK,and the concentration of TNF-α and IL-6 in the supernatant were significantly reduced(t=-7.11,-7.81,-10.51,-10.36,all P＜0.05).Conclusion Oridonin could protect vein endothelial cells from the damage induced by high glucose and palmitic acid,which may be related to the inhibition inflammation induced by high glucose and palmitic acid,or to increasing the expression of TAp63,Sirt1,Bcl-2 and decreasing the expression of pERK.
