丹参酮ⅡA磺酸钠对尿毒症毒素作用下人脐静脉内皮细胞功能的影响

doi:10.13481/j.1671-587X.20240209

摘要/Abstract

摘要：

目的探讨丹参酮ⅡA磺酸钠（STS）对尿毒症毒素作用下人脐静脉内皮细胞（hUVECs）功能的影响，并阐明其作用机制。方法将hUVECs进行传代培养并分为空白对照组、尿毒症毒素刺激组、尿毒症毒素+STS组和尿毒症毒素+STS+细胞外信号调节激酶（ERK）抑制剂组，其中后2组中STS的浓度为10 mg·L^-1；先给予各组剪切力刺激，剪切力大小为12 dyn·cm^-2；采用CCK-8法测定各组细胞增殖活性，Western blotting法检测各组细胞中ERK、核因子κB（NF-κB）和Ⅰ型胶原蛋白表达水平，实时荧光定量PCR （RT-qPCR）法检测细胞中ERK、NF-κB和Ⅰ型胶原mRNA表达情况；原位末端转移酶标记技术（TUNEL）法检测各组细胞凋亡率。结果 CCK-8法检测，在剪切力作用后，尿毒症毒素刺激组和尿毒症毒素+STS+ERK抑制剂组细胞增殖活性低于尿毒症毒素+STS组（P<0.01）。Western blotting法检测，与尿毒症毒素组比较，尿毒症毒素+STS组细胞中ERK、NF-κB和Ⅰ型胶原蛋白表达水平升高（P<0.01）；抑制ERK信号通路后，与空白对照组、尿毒症毒素组和尿毒症毒素+STS组比较，尿毒症毒素+STS+ERK抑制剂组细胞中ERK、NF-κB和Ⅰ型胶原蛋白表达水平明显降低（P<0.01）。RT-qPCR法检测，与尿毒症毒素组比较，尿毒症毒素+STS组细胞中ERK、NF-κB和Ⅰ型胶原mRNA表达水平升高（P<0.01）；抑制ERK通路后，与空白对照组、尿毒症毒素组和尿毒症毒素+STS组比较，尿毒症毒素+STS+ERK抑制剂组细胞中ERK、NF-κB和Ⅰ型胶原mRNA表达水平明显降低（P<0.01）。TUNEL法检测，尿毒症毒素+STS组的细胞凋亡率小于尿毒症毒素刺激组和尿毒症毒素+STS+ERK抑制剂组（P<0.05）。结论一定浓度STS能通过ERK信号通路调节NF-κB和Ⅰ型胶原mRNA及蛋白表达来改善尿毒症毒素作用下的内皮细胞增殖，减少细胞凋亡。

关键词: 血液透析, 动静脉内瘘, 人脐静脉内皮细胞, 尿毒症, 丹参酮ⅡA磺酸钠

Abstract:

Objective To discuss the effect of sodium tanshinone ⅡA sulfonate （STS） on the function of human umbilical vein endothelial cells （hUVECs） after treated with uremic toxin， and to clarify its mechanism. Methods The hUVECs were passaged and divided into blank control group， uremic toxin-stimulation group， uremic toxin + STS group， and uremic toxin + STS + extracellular signal-regulated kinase （ERK） inhibitor group. The concentration of STS used in the last two groups was 10 mg·L^-1. The shear stress stimulation at 12 dyn·cm^-2 was applied to the cells in various groups. The proliferation activities of the cells in various groups were detected by CCK-8 assay； the expression levels of ERK， nuclear factor kappa B （NF-κB）， and type Ⅰ collagen proteins in the cells in various groups were detected by Western blotting method；the expression levels of ERK， NF-κB， and type Ⅰ collagen mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method；the apoptotic rates the cells in various groups were detected by TUNEL method. Results The CCK-8 assay results showed that after treated with shear stress， the probiferation activitres of the cells in uremic toxin-stimulation group and uremic toxin + STS + ERK inhibitor group were lower than that in uremic toxin + STS group （P<0.01）. The Western blotting results showed that compared with uremic toxin group， the expression levels of ERK， NF-κB， and type Ⅰ collagen proteins in the cells in uremic toxin + STS group were increased （P<0.01）. After inhibiting the ERK pathway， compared with blank control group， uremic toxin group， and uremic toxin + STS group， the expression levels of ERK， NF-κB， and type Ⅰ collagen proteins in the cells in uremic toxin + STS + ERK inhibitor group were significantly decreased （P<0.01）. The RT-qPCR results showed that compared with uremic toxin group， the expression levels of ERK， NF-κB， and type Ⅰ collagen mRNA in the cells in uremic toxin + STS group were increased （P<0.01）. After inhibiting the ERK signaling pathway， compared with blank control group， uremic toxin group， and uremic toxin + STS group， the expression levels of ERK， NF-κB， and type Ⅰ collagen mRNA in the cells in uremic toxin + STS + ERK inhibitor group were significantly decreased （P<0.01）.The TUNEL method detection results showed that the apoptotic rate in the cells in uremic toxin + STS group was lower than those in uremic toxin-stimulation group and uremic toxin + STS + ERK inhibitor group （P<0.05）. Conclusion A certain concentration of STS can improve the proliferation of the endothelial cells and reduce the apoptosis of the cells after treated with uremic toxins by modulating the expressions of NF-κB and type Ⅰ collagen mRNA and proteins through the ERK signaling pathway.

Key words: Hemodialysis, Arterial-venous fistula, Human umbilical vein endothelial cell, Uremia, Sodium tanshinone ⅡA sulphonase

中图分类号:

R459.5

王立华,贾岚,陈海燕,杨波,王喆,毕学青. 丹参酮ⅡA磺酸钠对尿毒症毒素作用下人脐静脉内皮细胞功能的影响[J]. 吉林大学学报(医学版), 2024, 50(2): 364-370.

Lihua WANG,Lan JIA,Haiyan CHEN,Bo YANG,Zhe WANG,Xueqing BI. Effect of tanshinone ⅡA on function of human umbilical vein endothelial cells after treated with uremic toxin[J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 364-370.

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参考文献 25

1	DEMBER L M， BECK G J， ALLON M， et al. Effect of clopidogrel on early failure of arteriovenous fistulas for hemodialysis： a randomized controlled trial［J］. JAMA， 2008， 299（18）： 2164-2171.
2	LEE T， ULLAH A， ALLON M， et al. Decreased cumulative access survival in arteriovenous fistulas requiring interventions to promote maturation［J］. Clin J Am Soc Nephrol， 2011， 6（3）： 575-581.
3	林燕婷，吴禹池，杨敏，等. 中医药促进血液透析患者动静脉内瘘成熟的用药规律［J］. 中国中西医结合肾病杂志， 2020， 21（3）： 224-227.
4	SHANG Q H， XU H， HUANG L. Tanshinone ⅡA： a promising natural cardioprotective agent［J］. Evid Based Complement Alternat Med， 2012， 2012： 716459.
5	TIAN X H， WU J H. Tanshinone derivatives： a patent review （January 2006-September 2012）［J］. Expert Opin Ther Pat， 2013， 23（1）： 19-29.
6	DUAN H T， MA L J， LIU H G， et al. Tanshinone ⅡA attenuates epithelial-mesenchymal transition to inhibit the tracheal narrowing［J］. J Surg Res， 2016， 206（1）： 252-262.
7	DURAN C L， KAUNAS R， BAYLESS K J. S1P synergizes with wall shear stress and other angiogenic factors to induce endothelial cell sprouting responses［J］. Methods Mol Biol， 2018， 1697： 99-115.
8	BASHAR K， CONLON P J， KHEIRELSEID E A， et al. Arteriovenous fistula in dialysis patients： factors implicated in early and late AVF maturation failure［J］. Surgeon， 2016， 14（5）： 294-300.
9	GARCÍA-JÉREZ A， LUENGO A， CARRACEDO J， et al. Effect of uraemia on endothelial cell damage is mediated by the integrin linked kinase pathway［J］.J Physiol， 2015， 593（3）： 601-618.
10	MORRIS S T， MCMURRAY J J， RODGER R S， et al. Impaired endothelium-dependent vasodilatation in uraemia［J］. Nephrol Dial Transplant， 2000， 15（8）： 1194-1200.
11	毛俐婵，王文荣，蒋贤辉，等. 中药熏洗联合穴位注射对自体动静脉内瘘成熟影响的临床研究［J］. 浙江中西医结合杂志， 2018， 28（5）： 384-386.
12	CHUNG J， KIM K H， AN S H， et al. Coxsackievirus and adenovirus receptor mediates the responses of endothelial cells to fluid shear stress［J］. Exp Mol Med， 2019， 51（11）： 1-15.
13	YOSHINO D， SAKAMOTO N， SATO M. Fluid shear stress combined with shear stress spatial gradients regulates vascular endothelial morphology［J］. Integr Biol， 2017， 9（7）： 584-594.
14	CHENG C， TEMPEL D， VAN HAPEREN R， et al. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress［J］. Circulation， 2006， 113（23）： 2744-2753.
15	HEO K S， FUJIWARA K， ABE J. Shear stress and atherosclerosis［J］. Mol Cells， 2014， 37（6）： 435-440.
16	CHEN L J， CHUANG L， HUANG Y H， et al. MicroRNA mediation of endothelial inflammatory response to smooth muscle cells and its inhibition by atheroprotective shear stress［J］. Circ Res， 2015， 116（7）： 1157-1169.
17	RUZE A， ZHAO Y W， LI H， et al. Low shear stress upregulates the expression of fractalkine through the activation of mitogen-activated protein kinases in endothelial cells［J］. Blood Coagul Fibrinolysis， 2018， 29（4）： 361-368.
18	AKIMOTO S， MITSUMATA M， SASAGURI T， et al. Laminar shear stress inhibits vascular endothelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21（Sdi1/Cip1/Waf1）［J］. Circ Res， 2000， 86（2）： 185-190.
19	SAKAO S， TARASEVICIENE-STEWART L， LEE J D， et al. Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells［J］. FASEB J， 2005， 19（9）： 1178-1180.
20	BUROTTO M， CHIOU V L， LEE J M， et al. The MAPK pathway across different malignancies： a new perspective［J］. Cancer， 2014， 120（22）： 3446-3456.
21	SAMATAR A A， POULIKAKOS P I. Targeting RAS-ERK signalling in cancer： promises and challenges［J］. Nat Rev Drug Discov， 2014， 13（12）： 928-942.
22	NACKMAN G B， FILLINGER M F， SHAFRITZ R， et al. Flow modulates endothelial regulation of smooth muscle cell proliferation： a new model［J］. Surgery， 1998， 124（2）： 353-360；discussion 360-361.
23	SONG M， FINLEY S D. Mechanistic characterization of endothelial sprouting mediated by pro-angiogenic signaling［J］. Microcirculation， 2022， 29（2）： e12744.
24	KYRIAKIDIS N C， COBO G， DAI L， et al. Role of uremic toxins in early vascular ageing and calcification［J］. Toxins， 2021， 13（1）： 26.
25	JIA L， WANG L H， WEI F， et al. Effects of Caveolin-1-ERK1/2 pathway on endothelial cells and smooth muscle cells under shear stress［J］. Exp Biol Med， 2020， 245（1）： 21-33.

Group	ERK	Type Ⅰ collagen	NF-κB
Blank control	0.040±0.010	0.040±0.010	0.530±0.060
Uremic toxin-stimulation	0.020±0.008^*	0.250±0.018^*	0.220±0.010^*
Uremic toxin+STS	0.030±0.010^△	0.340±0.030^△	0.410±0.030^△
Uremic toxin+STS+ERK inhibitor	0.002±0.010^#	0.140±0.020^#	0.230±0.010^#

Group	ERK	Type Ⅰ collagen	NF-κB
Blank control	1.00±0.01	1.00±0.01	1.00±0.01
Uremic toxin-stimulation	0.53±0.03^*	0.44±0.03^*	0.56±0.18^*
Uremic toxin+STS	0.73±0.05^△	0.81±0.03^△	0.76±0.01^△
Uremic toxin+STS+ERK inhibitor	0.28±0.01^#	0.44±0.02^#	0.33±0.02^#

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