Calycosin-7-O-β-D-glucoside promotes oxidative stress-induced cytoskeleton reorganization through integrin-linked kinase signaling pathway in vascular endothelial cells

BMC Complementary and Alternative Medicine

Jing-Xin Xu¹  Hui-Hui Jiang¹  Le Zhou¹  Hong-Zhen Hu¹  Wei Li¹  Wei Sun²  Yue-Hua Jiang¹ 

[1] Affiliated Hospital of Shandong University of Traditional Chinese Medicine, West Wenhua road #42, Jinan 250011, Shandong, PR China;Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, China

关键词: AKT pathway;    Rho/ROCK pathway;    HUVECs;    Calycosin-7-O-β-D-glucoside;   

Others  :  1224048
DOI  :  10.1186/s12906-015-0839-5

 received in 2015-04-25, accepted in 2015-08-28,  发布年份 2015

【 摘 要 】

Background

Dysfunction of vascular endothelium is implicated in many pathological situations. Cytoskeleton plays an importance role in vascular endothelial permeability barrier and inflammatory response. Many Chinese herbs have the endothelial protective effect, of which, “Astragalus membranaceus” is a highly valued herb for treatment of cardiovascular and renal diseases in traditional Chinese medicine, In this study, we tested whether calycosin-7-O-β-D-glucoside (Calycosin), a main effective monomer component of “Astragalus membranaceus”, could protect endothelial cells from bacterial endotoxin (LPS)-induced cell injury.

Methods

Endothelial cell injury was induced by exposing human umbilical vein endothelial cells (HUVECs) to LPS. The effects of calycosin on LPS-induced changes in cell viability, apoptosis rate, cell migration, nitric oxide synthase (NOS), generationof intracellular reactive oxygen species (ROS) and cytoskeleton organization were determined. Microarray assay was employed to screen the possible gene expression change. Based on the results of microarray assay, the expression profile of genes involved in Rho/ROCK pathway and AKT pathway were further evaluated with quantitative real-time RT-PCR or western blot methods.

Results

Calycosin improved cell viability, suppressed apoptosis and protected the cells from LPS-induced reduction in cell migration and generation of ROS, protein level of NOS at a comparable magnitude to that of Y27632 and valsartan. Similar to Y27632 and valsartan, Calycosin, also neutralized LPS-induced actomyosin contraction and vinculin protein aggregation. Microarray assay, real-time PCR and western blot results revealed that LPS induced expression of FN, ITG A5, RhoA, PI3K (or PIP2 in western blotting), FAK, VEGF and VEGF R2, and inhibited expression of MLCP. We believed multiple pathways involved in the regulation of calycosin on HUVECs. Calycosin are considered to be able to activate MLCP through promoting the generation of NO, decreasing PMLC, suppressing the cytoskeleton remodeling caused by activation of Rho/ROCK pathway and inhibiting AKT pathway by decreasing VEGF, VEGF R2 and PI3K level.

Conclusion

Calycosin protected HUVEC from LPS-induced endothelial injury, possibly through suppression of Rho/ROCK pathway and regulation of AKT pathway.

【 授权许可】

   
2015 Jiang et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

【 参考文献 】

• [1]Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest. 2003; 111(5):649-58.
• [2]Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction. A marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol. 2003; 23(2):168-75.
• [3]Masumoto A, Mohri M, Shimokawa H, Urakami L, Usui M, Takeshita A. Suppression of coronary artery spasm by the Rho-kinase inhibitor fasudil in patients with vasospastic angina. Circulation. 2002; 105(13):1545-7.
• [4]Zeuke S, Ulmer AJ, Kusumoto S, Katus HA, Heine H. T LR4-mediated inflammatory activation of human coronary artery endothelial cells by LPS. Cardiovasc Res. 2002; 56(1):126-34.
• [5]Shaw LH, Chen WM, Tsai TH. Identification of multiple ingredients for a Traditional Chinese Medicine preparation (bu-yang-huan-wu-tang) by liquid chromatography coupled with tandem mass spectrometry. Molecules. 2013; 18(9):11281-98.
• [6]Ma X, Zhang K, Li H, Han S, Ma Z, Tu P. Extracts from Astragalus membranaceus limit myocardial cell death and improve cardiac function in a rat model of myocardial ischemia. J Ethnopharmacol. 2013; 149(3):720-8.
• [7]Wang X, Li Y, Yang X, Yao J. Astragalus polysaccharide reduces inflammatory response by decreasing permeability of LPS-infected Caco2 cells. Int J Biol Macromol. 2013; 61:347-52.
• [8]Kim JI, Jang HS, Park KM. Endotoxin-induced renal tolerance against ischemia and reperfusion injury is removed by iNOS, but not eNOS, gene-deletion. BMB Report. 2010; 43(9):629-34.
• [9]Polli-Lopes AC, Estofolete CF, Oliani SM, Zucoloto S, Cunha FQ, Gil CD. Myenteric denervation in gastric carcinogenesis: differential modulation of nitric oxide and annexin-A1. Int J Clin Exp Pathol. 2013; 6(1):13-23.
• [10]De Luisi A, Ferrucci A, Coluccia AM, Ria R, Moschetta M, de Luca E, Pieroni L, Maffia M, Urbani A, Di Pietro G, Guarini A, Ranieri G, Ditonno P, Berardi S, Caivano A, Basile A, Cascavilla N, Capalbo S, Quarta G, Dammacco F, Ribatti D, Vacca A. Lenalidomide restrains motility and overangiogenic potential of bone marrow endothelial cells in patients with active multiple myeloma. Clin Cancer Res. 2011; 17(7):1935-46.
• [11]Takase H, Matsumoto K, Yamadera R, Kubota Y, Otsu A, Suzuki R, Ishitobi H, Mochizuki H, Kojima T, Takano S, Uchida K, Takahashi S, Ema M. Genome-wide identification of endothelial cell–enriched genes in the mouse embryo. Blood. 2012; 120(4):914-23.
• [12]Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature. 2001; 410(6824):37-40.
• [13]Yamamoto K, Hamada H, Shinkai H, Kohno Y, Koseki H, Aoe T. The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades. J Biol Chem. 2003; 278(36):34525-32.
• [14]Enomoto A, Murakami H, Asai N, Morone N, Watanabe T, Kawai K, Murakumo Y, Usukura J, Kaibuchi K, Takahashi M. Akt/pkb regulates actin organization and cell motility via girdin/ape. Dev Cell. 2005; 9(3):389-402.
• [15]Kitamura T, Asai N, Enomoto A, Maeda K, Kato T, Ishida M, Jiang P, Watanabe T, Usukura J, Kondo T, Costantini F, Murohara T, Takahashi M. Regulation of vegf-mediated angiogenesis by the akt/pkb substrate girdin. Nat Cell Biol. 2008; 10(3):329-37.
• [16]Kimura K, Fukata Y, Matsuoka Y, Bennett V, Matsuura Y, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of the association of adducin with actin filaments by rho-associated kinase (rho-kinase) and myosin phosphatase. J Biol Chem. 1998; 273(10):5542-8.
• [17]Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev. 2006; 86(1):279-367.
• [18]Gundersen GG, Cook TA. Microtubules and signal transduction. Curr Opin Cell Biol. 1999; 11(1):81-94.
• [19]Kim SB, Kang OH, Keum JH, Mun SH, Seo YS, Choi JG, Kim MR, Rho JR, Shin DW, Kil KJ, Kwon DY. Anti-inflammatory effects of Danggui Liuhuang Decoction in RAW 264.7 cells. Chin J Integr Med. 2012; 12(3):1-7.
• [20]Madaule P, Axel R. A novel ras-related gene family. Cell. 1985; 41(1):31-40.
• [21]Pawlak G, Helfman DM. MEK mediates v-Src-induced disruption of the actin cytoskeleton via inactivation of the Rho-ROCK-LIM kinase pathway. J Biol Chem. 2002; 277(30):26927-33.
• [22]Funakoshi Y, Ichiki T, Shimokawa H, Egashira K, Takeda K, Kaibuchi K, Takeya M, Yoshimura T, Takeshita A. Rho-Kinase mediates angiotensin II-Induced monocyte chemoattractant protein-1 expression in rat vascular smooth muscle cells. Hypertension. 2001; 38(1):100-4.
• [23]Takeda K, Ichiki T, Tokunou T, Iino N, Fujii S, Kitabatake A, Shimokawa H, Takeshita A. Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression. Arterioscler Thromb Vasc Boil. 2001; 21(5):868-73.
• [24]Wilkinson- Berka JL. Vasoactive factors and diabetic retinopathy: vascular endothelia growth factor, cycoloxygenase-2 and nitric oxide. Curr Pharm Des. 2004; 10(27):3331-48.
• [25]Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva I, Garcia JG, Verin AD. Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms. J Cell Physiol. 2004; 201(1):55-70.
• [26]Gorovoy M, Niu J, Bernard O, Profirovic J, Minshall R, Neamu R, Voyno-Yasenetskaya T. LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells. J Biol Chem. 2005; 280(28):26533-42.
• [27]Essler M, Retzer M, Bauer M, Heemskerk JW, Aepfelbacher M, Siess W. Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase. J Biol Chem. 1999; 274(43):30361-4.
• [28]Bolz SS, Vogel L, Sollinger D, Derwand R, de Wit C, Loirand G, Pohl U. Nitric oxide-induced decrease in calcium sensitivity of resistance arteries is attributable to activation of the myosin light chain phosphatase and antagonized by the RhoA/Rho kinase pathway. Circulation. 2003; 107(24):3081-7.
• [29]Chandrasekar N, Mohanam S, Gujrati M, Olivero WC, Dinh DH, Rao JS. Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells. Oncogene. 2003; 22(3):392-400.
• [30]Okamura N, Saito M, Mori A, Sakamoto K, Kametaka S, Nakahara T, Ishii K. Vasodilator effects of fasudil, a Rho-kinase inhibitor, on retinal arte-rioles in stroke-prone spontaneously hypertensive rats. J Ocul Pharmacol Ther. 2007; 23(3):207-12.
• [31]Song Y, Zhao XP, Song K, Shang ZJ. Ephrin-A1 is up-regulated by hypoxia in cancer cells and promotes angiogenesis of HUVECs through a coordinated cross-talk with eNOS. PLoS ONE. 2013; 8(9): Article ID e74464