齐墩果酸通过PPARγ调控人脐静脉内皮细胞抗氧化损伤作用

doi:10.3877/cma.j.issn.2095-1221.2017.06.003

目的

探讨齐墩果酸（OA）对ox-LDL诱导的内皮细胞氧化损伤的作用及其机制。

方法

实验分组：对照组，ox-LDL模型组，ox-LDL+OA（10μmol/L，20μmol/L，40?μmol/L）组，ox-LDL+OA（10μmol/L，20μmol/L，40μmol/L）+GW9662，GW9662单独处理组。采用MTT法检测HUVECs细胞活力；酶联免疫吸附试验法检测HUVECs细胞SOD活力、GSH活力以及MDA含量；活性氧检测试剂盒检测HUVECs细胞ROS水平；Western blot检测HUVECs细胞PPARγ蛋白表达水平，所有指标的检测都进行生物学重复。采用方差分析和两样本t检验进行统计学分析。

结果

MTT结果显示，ox-LDL组的细胞存活率为（49.17±0.62）﹪，OA（10μmol/L、20μmol/L、40μmol/L）预处理后存活率分别为（68.51±1.16）﹪、（82.64±0.73）﹪、（92.37±0.13）﹪，可减弱ox-LDL对HUVECs细胞存活率的降低且呈剂量依赖性关系，差异具有统计学意义（t = 24.35，26.18，35.17；P = 0.034，0.027，0.008）。本研究还发现，OA对ox-LDL诱导的HUVECs细胞SOD、GSH活性的降低和MDA、ROS水平的增加具有抑制作用且呈剂量依赖关系。ox-LDL组的细胞SOD、GSH、ROS和MDA水平分别为（16.12±0.06）μmol/g、（132.16?±?2.11）μmol/g、（2.63±0.02）kU/g、（158.12±0.39）﹪，ox-LDL+OA（10μmol/L）组的细胞SOD、GSH、ROS和MDA水平分别为（10.60±0.14）?μmol/g、（108.36±2.05）?μmol/g、（2.41?±0.21）kU/g、（136.18±1.24）﹪，ox-LDL+OA（20μmol/L）组的细胞SOD、GSH、ROS和MDA水平分别为（13.28±0.09）μmol/g、（129.58±0.09）μmol/g、（2.26±0.15）kU/g、（126.43±1.51）﹪，ox-LDL+OA（40μmol/L）组的细胞SOD、GSH、ROS和MDA水平分别为（14.86±0.16）μmol/g、（131.47±0.76）μmol/g、（2.14±0.08）kU/g、（112.39±1.07）﹪（F = 26.38，31.27，56.82，41.16；P?=?0.005，0.004，0.002，0.003）。Western blot结果显示，OA有效促进HUVECs细胞PPARγ蛋白水平提高。与ox-LDL+OA（20μmol/L）组（65.37±0.15）﹪比较，ox-LDL+OA（20?μmol/L）+ GW9662组的细胞活力为（52.89±0.16）﹪，差异有统计学意义（t =16.47，P = 0.035）。进一步发现ox-LDL+OA（10μmol/L）组SOD、GSH、MDA、ROS水平为（10.58?±?0.13）μmol/g、（102.46?±?0.06）μmol/g、（2.42?±?0.08）kU/g、（144.38?±2.02）﹪，ox-LDL+OA（10μmol/L）+GW9662组的SOD、GSH、MDA、ROS水平分别为（8.42±0.05）μmol/g、（88.38±0.48）μmol/g、（2.83?±?0.01）kU/g、（154.41?±?1.04）﹪，两组比较差异有统计学意义（t = 38.47，39.25，43.69，41.27；P = 0.008，0.008，0.006，0.006）；ox-LDL+OA（20μmol/L）组SOD、GSH、MDA、ROS水平（13.25?±?0.05）μmol/g、（122.59?±?0.33）μmol/g、（2.23±0.16）kU/g、（123.94±?0.15）﹪，ox-LDL+ OA（20?μmol/L）+ GW9662组的SOD、GSH、MDA、RO水平分别为（10.59±?0.12）μmol/g、（106.42?±?0.15）μmol/g、（2.61?±?0.07）kU/g、（138.12?±?1.15）﹪，两组比较差异有统计学意义（t = 46.08，38.11，49.35，35.59；P = 0.005，0.008，0.004，0.009）；ox-LDL+OA（40μmol/L）组SOD、GSH、MDA、ROS水平分别为（15.88±0.14）μmol/g、（140.26±1.05）μmol/g、（2.02± 0.13）kU/g、（187.52?±?0.68）﹪，ox-LDL+OA（40μmol/L）+GW9662组的SOD、GSH、MDA、RO水平（13.65?±?0.03）μmol/g、（124.61?±?1.27）μmol/g、（2.49±0.04）kU/g、（126.51±?0.73）﹪，两组比较差异有统计学意义（t = 48.04，38.62，45.14，50.13；P = 0.004，0.008，0.005，0.002），此外，本研究还发现，抑制PPARγ后，OA抑制ox-LDL诱导的HUVECs细胞的氧化损伤仍存在剂量效应。

结论

OA可以通过PPARγ抑制x-LDL诱导HUVECs细胞氧化损伤。

关键词: 齐墩果酸, ox-LDL, 氧化损伤, PPARγ, 人脐静脉内皮细胞

Objective

To investigate the effects of oleanolic acid (OA) on oxidative damage in human vascular endothelial cells (HUVECs) and its underlying mechanism.

Methods

The groups are as followings: the control group, ox-LDL alone group, ox-LDL+OA (10μmol/L,20μmol/L, 40μmol/L) group, ox-LDL+OA (10μmol/L, 20μmol/L, 40μmol/L) +GW9662 group, and GW9662 alone group. The cell viability was measured using MTT assay. The levels of SOD, GSH as well as MDA were detected by enzyme linked immunosorbent assay (Elisa) . The level of ROS was detected using reactive oxygen species detection kit. The expression level of PPARγwas measured by Western blot. All indexed are performed biology-repeatedly. The statistical analysis was used with Analysis of Variance and Kruskal-WallisHtest.

Results

OA (10μmol/L, 20μmol/L,40μmol/L) significantly attenuated the cell viability of HUVECs inhibited by ox-LDL (100μg/ml) in a dose-dependent manner and each cell viability is (68.51±1.16) ﹪, (82.64±0.73) ﹪, (92.37?±?0.13) ﹪ (t = 24.35, 26.18, 35.17; P = 0.034, 0.027, 0.008) . OA reversed oxidative damage was induced by ox-LDL including decreases in the levels of SOD and GSH, and increases in the level of MDA and ROS in HUVECs. The levels of SOD, GSH, ROS and MDA in ox-LDL group are (16.12±0.06) μmol/g, (132.16±2.11) μmol/g, (2.63±0.02) kU/g, (158.12?±0.39) ﹪; the levels of SOD, GSH, ROS and MDA in ox-LDL and OA (10μmol/L) group are (13.28?±0.09) μmol/g, (129.58±0.09) μmol/g, (2.26±0.15) kU/g, (126.43±1.51) ﹪respectively; the levels of SOD, GSH, ROS and MDA in ox-LDL and OA (20μmol/L) group is (13.28±0.09) μmol/g, (129.58?±?0.09) μmol/g, (2.26±0.15) kU/g, (126.43±1.51) ﹪respectively; the levels of SOD, GSH, ROS and MDA in ox-LDL and OA (40μmol/L) group are (14.86±0.16) μmol/g, (131.47±0.76) μmol/g, (2.14?±0.08) kU/g, (112.39±1.07) ﹪respectively (F = 26.38, 31.27, 56.82, 41.16; P = 0.005, 0.004, 0.002, 0.003) . Western blot result showed that OA attenuated downregulation of PPARγprotein induced by ox-LDL in HUVECs. Our result also showed that PPARγinhibitor GW9662 reversed the attenuation of OA on ox-LDL-caused inhibition of cell viability: the cell viability in ox-LDL+OA (20μmol/L) +GW9662 group (t = 16.47,P = 0.035) . In addition, we found that PPARγinhibitor GW9662 reversed the improvement of OA on ox-LDL-caused oxidative damage: the levels of SOD, GSH, MDA and ROS in ox-LDL+OA (10μmol/L) +GW9662 group are (8.42±0.05) μmol/g, (88.38±0.48) μmol/g, (2.83±0.01) kU/g, (154.41±1.04) ﹪ (t = 38.47, 39.25, 43.69, 41.27; P = 0.008, 0.008, 0.006, 0.006) respectively; the levels of SOD, GSH, MDA and ROS in ox-LDL+OA (20μmol/L) +GW9662 group are (10.59±0.12) μmol/g, (106.42±0.1) μmol/g, (2.61?±?0.07) kU/g, (138.12±1.15) ﹪ (t?= 46.08, 38.11, 49.35, 35.59,P =0.005, 0.008, 0.004, 0.009) respectively; the levels of SOD, GSH, MDA and ROS in ox-LDL+OA (40μmol/L) +GW9662 group was (13.65.±0.03) μmol/g, (124.61±1.27) ?μmol/g, (2.49±0.04) ?kU/g, (126.51±0.73) ﹪ (t = 48.04, 38.62, 45.14, 50.13;P = 0.004, 0.008, 0.005, 0.002) respectively. Furthermore, we also found that after inhibiting PPAR gamma, OA still dose dependently alleviated ox-LDL-caused oxidative damage of HUVECs cells.

Conclusion

OA inhibits oxidative damage induced by ox-LDL and PPARγplays a regulatory role in the protection of OA against ox-LDL-induced oxidative damage of HUVECs.

Key words: Oleanolic acid, Ox-LDL, Oxidative damage, PPARγ, Human umbilical vein endothelial cells

图1 OA对ox-LDL抑制HUVECs细胞活力的影响

表1 OA对ox-LDL抑制HUVECs细胞氧化损伤的影响(±s)

分组	样本数	SOD（μmol/g）	GSH（μmol/g）	MDA（kU/g）	ROS相对荧光强度（﹪）
对照组	3	16.12±0.06	132.16±2.11	2.03±0.04	100.04±0.45
ox-LDL	3	5.43±0.08^a	49.72±1.28^a	2.63±0.02^a	158.12±0.39^a
ox-LDL+OA（10 μmol/L）	3	10.60±0.14^b	108.36±2.05^b	2.41±0.21^b	136.18±1.24^b
ox-LDL+OA（20 μmol/L）	3	13.28±0.09^b	129.58±0.09^b	2.26±0.15^b	126.43±1.51^b
ox-LDL+OA（40 μmol/L）	3	14.86±0.16^b	131.47±0.76^b	2.14±0.08^b	112.39±1.07^b
F值	?	26.38	31.27	56.82	41.16
P值	?	0.005	0.004	0.002	0.003

表1 OA对ox-LDL抑制HUVECs细胞氧化损伤的影响(±s)

分组	样本数	SOD（μmol/g）	GSH（μmol/g）	MDA（kU/g）	ROS相对荧光强度（﹪）
对照组	3	16.12±0.06	132.16±2.11	2.03±0.04	100.04±0.45
ox-LDL	3	5.43±0.08^a	49.72±1.28^a	2.63±0.02^a	158.12±0.39^a
ox-LDL+OA（10 μmol/L）	3	10.60±0.14^b	108.36±2.05^b	2.41±0.21^b	136.18±1.24^b
ox-LDL+OA（20 μmol/L）	3	13.28±0.09^b	129.58±0.09^b	2.26±0.15^b	126.43±1.51^b
ox-LDL+OA（40 μmol/L）	3	14.86±0.16^b	131.47±0.76^b	2.14±0.08^b	112.39±1.07^b
F值	?	26.38	31.27	56.82	41.16
P值	?	0.005	0.004	0.002	0.003

图2 OA对ox-LDL抑制HUVECs细胞PPARγ蛋白表达水平的影响

图3 PPARγ抑制剂对OA抗ox-LDL抑制HUVECs细胞活力的影响

表2 PPARγ抑制剂对OA抗ox-LDL抑制HUVECs细胞氧化损伤的影响(±s)

分组	样本数	SOD（μmol/g）	GSH（μmol/g）	MDA（kU/g）	ROS相对荧光强度（﹪）
对照组	3	17.05±0.12	135.28±2.14	2.16±0.15	100.53±1.08
ox-LDL	3	6.41±0.04^a	50.38±0.13^a	2.71±0.12^a	160.05±0.46^a
ox-LDL+OA（10 μmol/L）	3	10.58±0.13^b	102.46±0.06^b	2.42±0.08^b	144.38±2.02^b
ox-LDL+OA（20 μmol/L）	3	13.25±0.05^b	122.59±0.33^b	2.23±0.16^b	123.94±0.15^b
ox-LDL+OA（40 μmol/L）	3	15.88±0.14^b	140.26±1.05^b	2.02±0.13^b	187.52±0.68^b
oxLDL+OA(10 μmol/L)+GW9662	3	8.42.±0.05^c	88.38±0.48^c	2.83±0.01^c	154.41±1.04^c
oxLDL+OA(20 μmol/L)+GW9662	3	10.59.±0.12^c	106.42±0.15^c	2.61±0.07^c	138.12±1.15^c
oxLDL+OA(40 μmol/L)+GW9662	3	13.65.±0.03^c	124.61±1.27^c	2.49±0.04^c	126.51±0.73^c
GW9662	3	16.02±0.13	130.14±2.04	1.98±0.29	103.15±0.29
F值	?	35.08	42.31	26.84	33.73
P值	?	0.003	0.001	0.004	0.003

表2 PPARγ抑制剂对OA抗ox-LDL抑制HUVECs细胞氧化损伤的影响(±s)

分组	样本数	SOD（μmol/g）	GSH（μmol/g）	MDA（kU/g）	ROS相对荧光强度（﹪）
对照组	3	17.05±0.12	135.28±2.14	2.16±0.15	100.53±1.08
ox-LDL	3	6.41±0.04^a	50.38±0.13^a	2.71±0.12^a	160.05±0.46^a
ox-LDL+OA（10 μmol/L）	3	10.58±0.13^b	102.46±0.06^b	2.42±0.08^b	144.38±2.02^b
ox-LDL+OA（20 μmol/L）	3	13.25±0.05^b	122.59±0.33^b	2.23±0.16^b	123.94±0.15^b
ox-LDL+OA（40 μmol/L）	3	15.88±0.14^b	140.26±1.05^b	2.02±0.13^b	187.52±0.68^b
oxLDL+OA(10 μmol/L)+GW9662	3	8.42.±0.05^c	88.38±0.48^c	2.83±0.01^c	154.41±1.04^c
oxLDL+OA(20 μmol/L)+GW9662	3	10.59.±0.12^c	106.42±0.15^c	2.61±0.07^c	138.12±1.15^c
oxLDL+OA(40 μmol/L)+GW9662	3	13.65.±0.03^c	124.61±1.27^c	2.49±0.04^c	126.51±0.73^c
GW9662	3	16.02±0.13	130.14±2.04	1.98±0.29	103.15±0.29
F值	?	35.08	42.31	26.84	33.73
P值	?	0.003	0.001	0.004	0.003

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Oleanolic acid inhibits oxidative damage of HUVECs via PPARγ

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Oleanolic acid inhibits oxidative damage of HUVECs via PPARγ