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

Jiang YH, Sun W, Li W, Hu HZ, Zhou L, Jiang HH, Xu JX - BMC Complement Altern Med (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Affiliated Hospital of Shandong University of Traditional Chinese Medicine, West Wenhua road #42, Jinan, Shandong, 250011, PR China. jiang_yuehua@hotmail.com.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

The content of cNOS, tNOS, iNOS and NO in cell supernatants. The content of cNOS, tNOS, iNOS and NO in cell supernatants was determined by nitrate reductase method. a The content of cNOS in the supernatant was assayed after HUVECs were incubated with calycosin in different concentration (0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml or 20 μg/ml) for 24 h; b The content of cNOS in the supernatant was assayed after HUVECs were incubated with 10 μg/ml calycosin for different time (0 h, 4 h, 8 h, 12 h, 24 h or 48 h); c The content of cNOS, tNOS and iNOS in cell supernatants; d The content of NO in cell supernatants. △P < 0.05, compared with normal HUVECs; ★P < 0.05, compared with LPS-induced inflammatory and injured HUVECs
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Fig2: The content of cNOS, tNOS, iNOS and NO in cell supernatants. The content of cNOS, tNOS, iNOS and NO in cell supernatants was determined by nitrate reductase method. a The content of cNOS in the supernatant was assayed after HUVECs were incubated with calycosin in different concentration (0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml or 20 μg/ml) for 24 h; b The content of cNOS in the supernatant was assayed after HUVECs were incubated with 10 μg/ml calycosin for different time (0 h, 4 h, 8 h, 12 h, 24 h or 48 h); c The content of cNOS, tNOS and iNOS in cell supernatants; d The content of NO in cell supernatants. △P < 0.05, compared with normal HUVECs; ★P < 0.05, compared with LPS-induced inflammatory and injured HUVECs

Mentions: HUVECs demonstrated eminent dose dependent and time dependent changes in the content of cNOS with the change of calycosin. HUVECs generated the highest cNOS level after incubation with 10 μg/ml calycosin for 24 h (Fig. 2 a and b). After treatment of HUVECs with LPS for 24 h, the level of cNOS was decreased, whereas iNOS and its product NO in the culture supernatant were elevated, indicating inflammatory endothelial activation. Calycosin intervention significantly increased cellular cNOS level, whereas it suppressed iNOS (P < 0.05, Fig. 2 c and d). These observations indicate Calycosin effectively alleviated the inflammatory endothelial injury. Interestingly, the overall concentration of NO was increased, which might be caused by the elevated cNOS level. Y27632 also increased the levels of cNOS and tNOS significantly (P < 0.05), but it had little effect on iNOS and NO. Valsartan effectively reduced iNOS and NO level (P < 0.05), but it failed to affect cNOS and tNOS.Fig. 2


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

Jiang YH, Sun W, Li W, Hu HZ, Zhou L, Jiang HH, Xu JX - BMC Complement Altern Med (2015)

The content of cNOS, tNOS, iNOS and NO in cell supernatants. The content of cNOS, tNOS, iNOS and NO in cell supernatants was determined by nitrate reductase method. a The content of cNOS in the supernatant was assayed after HUVECs were incubated with calycosin in different concentration (0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml or 20 μg/ml) for 24 h; b The content of cNOS in the supernatant was assayed after HUVECs were incubated with 10 μg/ml calycosin for different time (0 h, 4 h, 8 h, 12 h, 24 h or 48 h); c The content of cNOS, tNOS and iNOS in cell supernatants; d The content of NO in cell supernatants. △P < 0.05, compared with normal HUVECs; ★P < 0.05, compared with LPS-induced inflammatory and injured HUVECs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562353&req=5

Fig2: The content of cNOS, tNOS, iNOS and NO in cell supernatants. The content of cNOS, tNOS, iNOS and NO in cell supernatants was determined by nitrate reductase method. a The content of cNOS in the supernatant was assayed after HUVECs were incubated with calycosin in different concentration (0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml or 20 μg/ml) for 24 h; b The content of cNOS in the supernatant was assayed after HUVECs were incubated with 10 μg/ml calycosin for different time (0 h, 4 h, 8 h, 12 h, 24 h or 48 h); c The content of cNOS, tNOS and iNOS in cell supernatants; d The content of NO in cell supernatants. △P < 0.05, compared with normal HUVECs; ★P < 0.05, compared with LPS-induced inflammatory and injured HUVECs
Mentions: HUVECs demonstrated eminent dose dependent and time dependent changes in the content of cNOS with the change of calycosin. HUVECs generated the highest cNOS level after incubation with 10 μg/ml calycosin for 24 h (Fig. 2 a and b). After treatment of HUVECs with LPS for 24 h, the level of cNOS was decreased, whereas iNOS and its product NO in the culture supernatant were elevated, indicating inflammatory endothelial activation. Calycosin intervention significantly increased cellular cNOS level, whereas it suppressed iNOS (P < 0.05, Fig. 2 c and d). These observations indicate Calycosin effectively alleviated the inflammatory endothelial injury. Interestingly, the overall concentration of NO was increased, which might be caused by the elevated cNOS level. Y27632 also increased the levels of cNOS and tNOS significantly (P < 0.05), but it had little effect on iNOS and NO. Valsartan effectively reduced iNOS and NO level (P < 0.05), but it failed to affect cNOS and tNOS.Fig. 2

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Affiliated Hospital of Shandong University of Traditional Chinese Medicine, West Wenhua road #42, Jinan, Shandong, 250011, PR China. jiang_yuehua@hotmail.com.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus