中国医药导报
中國醫藥導報
중국의약도보
CHINA MEDICAL HERALD
2014年
29期
19-23
,共5页
田飒%彭学军%何宇明%吴蓉
田颯%彭學軍%何宇明%吳蓉
전삽%팽학군%하우명%오용
硫化氢%高糖%氧化应激%沉默信息调节子1
硫化氫%高糖%氧化應激%沉默信息調節子1
류화경%고당%양화응격%침묵신식조절자1
Hydrogen sulfide%High glucose%Oxidative stress%Silent information regulator 1
目的：观察外源性硫化氢(H2S)对高糖诱导人脐静脉内皮细胞(HUVECs)氧化应激损伤的保护作用及其可能机制。方法用25 mmol/L D-葡萄糖(高糖)、外源性H2S供体硫化氢钠(NaHS)(5×10-5、1×10-4、5×10-4、1×10-3和5×10-3 mol/L)及沉默信息调节子1(SIRT1)特异性抑制剂尼克酰胺(40 mmol/L)处理HUVECs细胞株24 h。实验分为对照组、高糖组、甘露醇组、NaHS(5×10-5、1×10-4、5×10-4、1×10-3、5×10-3 mol/L)组、高糖+NaHS(5×10-5、1×10-4、5×10-4、1×10-3和5×10-3 mol/L)组和高糖+NaHS(10-3 mol/L)+尼克酰胺组。采用MTT比色法检测细胞活力，流式细胞术检测细胞内的内活性氧(ROS)水平，Western bolt法检测SIRT1的表达。结果与对照组比较，高糖组细胞活力显著降低[OD值分别为(0.76±0.09)和(0.18±0.01)，t=5.34，P<0.05]，细胞内ROS水平显著增加[ROS水平分别为(356.18±42.96)au和(1183.63±84.31)au，t=6.72，P<0.05]。与高糖组比较，高糖+NaHS(5×10-4、1×10-3和5×10-3 mol/L)组细胞活力显著增加[OD值分别为(0.18±0.01)、(0.39±0.05)、(0.68±0.04)和(0.51±0.08)，t1=3.16，t2=3.95，t3=3.86，均P<0.05]，细胞内 ROS水平显著降低[ROS水平分别为(1183.63±84.31)、(874.32±85.36)、(628.65±54.27)和(439.56±53.64)au，t1=3.46，t2=3.97，t3=5.13，均P<0.05]。与对照组比较，高糖组细胞中SIRT1蛋白表达显著下调[蛋白相对表达量分别为(0.48±0.04)和(0.17±0.03)，t=3.94，P<0.05]。与高糖组比较，高糖+NaHS(10-3 mol/L)组细胞中SIRT1表达显著上调[蛋白相对表达量分别为(0.17±0.03)和(0.59±0.08)，t=4.36，P<0.05]。 SIRT1抑制剂尼克酰胺取消了NaHS抑制高糖诱导的HUVECs细胞活力的降低[高糖+NaHS组和高糖+NaHS+尼克酰胺组OD值分别为(0.52±0.04)和(0.23±0.03)，t =2.98，P <0.05]和细胞内ROS水平的增加[高糖+NaHS组和高糖+NaHS+尼克酰胺组ROS水平分别为(628.65±54.27)au和(1052.84±113.42)au，t=3.76，P<0.05]。结论外源性H2S抑制了高糖诱导的HUVECs氧化应激损伤，其机制可能与H2S上调SIRT1的表达有关。
目的：觀察外源性硫化氫(H2S)對高糖誘導人臍靜脈內皮細胞(HUVECs)氧化應激損傷的保護作用及其可能機製。方法用25 mmol/L D-葡萄糖(高糖)、外源性H2S供體硫化氫鈉(NaHS)(5×10-5、1×10-4、5×10-4、1×10-3和5×10-3 mol/L)及沉默信息調節子1(SIRT1)特異性抑製劑尼剋酰胺(40 mmol/L)處理HUVECs細胞株24 h。實驗分為對照組、高糖組、甘露醇組、NaHS(5×10-5、1×10-4、5×10-4、1×10-3、5×10-3 mol/L)組、高糖+NaHS(5×10-5、1×10-4、5×10-4、1×10-3和5×10-3 mol/L)組和高糖+NaHS(10-3 mol/L)+尼剋酰胺組。採用MTT比色法檢測細胞活力，流式細胞術檢測細胞內的內活性氧(ROS)水平，Western bolt法檢測SIRT1的錶達。結果與對照組比較，高糖組細胞活力顯著降低[OD值分彆為(0.76±0.09)和(0.18±0.01)，t=5.34，P<0.05]，細胞內ROS水平顯著增加[ROS水平分彆為(356.18±42.96)au和(1183.63±84.31)au，t=6.72，P<0.05]。與高糖組比較，高糖+NaHS(5×10-4、1×10-3和5×10-3 mol/L)組細胞活力顯著增加[OD值分彆為(0.18±0.01)、(0.39±0.05)、(0.68±0.04)和(0.51±0.08)，t1=3.16，t2=3.95，t3=3.86，均P<0.05]，細胞內 ROS水平顯著降低[ROS水平分彆為(1183.63±84.31)、(874.32±85.36)、(628.65±54.27)和(439.56±53.64)au，t1=3.46，t2=3.97，t3=5.13，均P<0.05]。與對照組比較，高糖組細胞中SIRT1蛋白錶達顯著下調[蛋白相對錶達量分彆為(0.48±0.04)和(0.17±0.03)，t=3.94，P<0.05]。與高糖組比較，高糖+NaHS(10-3 mol/L)組細胞中SIRT1錶達顯著上調[蛋白相對錶達量分彆為(0.17±0.03)和(0.59±0.08)，t=4.36，P<0.05]。 SIRT1抑製劑尼剋酰胺取消瞭NaHS抑製高糖誘導的HUVECs細胞活力的降低[高糖+NaHS組和高糖+NaHS+尼剋酰胺組OD值分彆為(0.52±0.04)和(0.23±0.03)，t =2.98，P <0.05]和細胞內ROS水平的增加[高糖+NaHS組和高糖+NaHS+尼剋酰胺組ROS水平分彆為(628.65±54.27)au和(1052.84±113.42)au，t=3.76，P<0.05]。結論外源性H2S抑製瞭高糖誘導的HUVECs氧化應激損傷，其機製可能與H2S上調SIRT1的錶達有關。
목적：관찰외원성류화경(H2S)대고당유도인제정맥내피세포(HUVECs)양화응격손상적보호작용급기가능궤제。방법용25 mmol/L D-포도당(고당)、외원성H2S공체류화경납(NaHS)(5×10-5、1×10-4、5×10-4、1×10-3화5×10-3 mol/L)급침묵신식조절자1(SIRT1)특이성억제제니극선알(40 mmol/L)처리HUVECs세포주24 h。실험분위대조조、고당조、감로순조、NaHS(5×10-5、1×10-4、5×10-4、1×10-3、5×10-3 mol/L)조、고당+NaHS(5×10-5、1×10-4、5×10-4、1×10-3화5×10-3 mol/L)조화고당+NaHS(10-3 mol/L)+니극선알조。채용MTT비색법검측세포활력，류식세포술검측세포내적내활성양(ROS)수평，Western bolt법검측SIRT1적표체。결과여대조조비교，고당조세포활력현저강저[OD치분별위(0.76±0.09)화(0.18±0.01)，t=5.34，P<0.05]，세포내ROS수평현저증가[ROS수평분별위(356.18±42.96)au화(1183.63±84.31)au，t=6.72，P<0.05]。여고당조비교，고당+NaHS(5×10-4、1×10-3화5×10-3 mol/L)조세포활력현저증가[OD치분별위(0.18±0.01)、(0.39±0.05)、(0.68±0.04)화(0.51±0.08)，t1=3.16，t2=3.95，t3=3.86，균P<0.05]，세포내 ROS수평현저강저[ROS수평분별위(1183.63±84.31)、(874.32±85.36)、(628.65±54.27)화(439.56±53.64)au，t1=3.46，t2=3.97，t3=5.13，균P<0.05]。여대조조비교，고당조세포중SIRT1단백표체현저하조[단백상대표체량분별위(0.48±0.04)화(0.17±0.03)，t=3.94，P<0.05]。여고당조비교，고당+NaHS(10-3 mol/L)조세포중SIRT1표체현저상조[단백상대표체량분별위(0.17±0.03)화(0.59±0.08)，t=4.36，P<0.05]。 SIRT1억제제니극선알취소료NaHS억제고당유도적HUVECs세포활력적강저[고당+NaHS조화고당+NaHS+니극선알조OD치분별위(0.52±0.04)화(0.23±0.03)，t =2.98，P <0.05]화세포내ROS수평적증가[고당+NaHS조화고당+NaHS+니극선알조ROS수평분별위(628.65±54.27)au화(1052.84±113.42)au，t=3.76，P<0.05]。결론외원성H2S억제료고당유도적HUVECs양화응격손상，기궤제가능여H2S상조SIRT1적표체유관。
Objective To investigate the protective effect of extrogenous hydrogen sulfide (H2S) on the injury of oxida-tive stress induced by high glucose and explore the possible mechanism in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were incubated by 25 mmol/L D-glucose (high glucose) to induce injury and cells were treated with H2S donor sodium bisulfide (NaHS) (5í10-5, 1í10-4, 5í10-4, 1í10-3 and 5í10-3 mol/L) for 24 h. The in-hibitor of silent information regulator 1 (SIRT1) niacinamide was used in the study. The cells were divided into the control group, high glucose group, mannitol group, NaHS (5í10-5, 1í10-4, 5í10-4, 1í10-3, 5í10-3 mol/L)groups, high glucose +NaHS (5í10-5, 1í10-4, 5í10-4, 1í10-3 and 5í10-3 mol/L) groups and high glucose + NaHS (10-3 mol/L) + niacinamide group. MTT assay was used to detect the cell viability. The level of reactive oxygen species (ROS) was measured by flow cytometry. The expression of SIRT1 was tested by Western blot. Results Compared with the control group, the cell viability was significantly decreased [OD were (0.76±0.09) and (0.18±0.01) respectively, t = 5.34, P < 0.05], and the level of ROS was significantly increased [the levels of ROS were (356.18±42.96) au and (1183.63±84.31) au respective-ly, t = 6.72, P< 0.05] in high glucose group. Compared with high glucose group, the cells viability were signifi-cantly increased [OD were (0.18±0.01), (0.39±0.05), (0.68±0.04) and (0.51±0.08) respectively, t1= 3.16, t2= 3.95, t3=3.86, P< 0.05], and the levels of ROS were significantly decreased [the levels of ROS were (1183.63±84.31), (874.32±85.36), (628.65±54.27) and (439.56±53.64) au respectively, t1=3.46, t2=3.97, t3= 5.13, all P< 0.05] in high glucose+NaHS (5í10-4, 10-3 and 5í10-3 mol/L) groups. Compared with the control, the expression of SIRT1 was significantly down-regulated in high glucose group [relative expression levels were (0.48±0.04) and (0.17±0.03) respectively, t =3.94, P < 0.05]. Compared with high glucose group, the expression of SIRT1 was significantly up-regulated in high glucose+NaHS (10-3 mol/L) group [relative expression levels were (0.17±0.03) and (0.59±0.08) respectively, t= 4.36, P<0.05]. Compared with the high glucose+NaHS (10-3 mol/L) group, the cell viability was significantly decreased [OD were (0.52±0.04) and (0.23±0.03) respectively, t=2.98, P<0.05) and the level of ROS was significantly increased [the level of ROS was (628.65±54.27) au and (1052.84±113.42) au, t = 3.76, P< 0.05) in high glucose+NaHS (10-3 mol/L)+niaci-namide group. Conclusion Extrogenous H2S prevents the injury of oxidative stress induced by high glucose in HUVECs, the mechanisms may be related to the up-regulation of SIRT1 induced by H2S.
