Yiu Wa Kwan

Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Background Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo. Methodology Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting. Conclusion Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.

A review of the pharmacological effects of Arctium lappa (burdock).

Arctium lappa, commonly known as burdock, is being promoted/recommended as a healthy and nutritive food in Chinese societies. Burdock has been used therapeutically in Europe, North America and Asia for hundreds of years. The roots, seeds and leaves of burdock have been investigated in view of its popular uses in traditional Chinese medicine (TCM). In this review, the reported therapeutic effects of the active compounds present in the different botanical parts of burdock are summarized. In the root, the active ingredients have been found to “detoxify” blood in terms of TCM and promote blood circulation to the skin surface, improving the skin quality/texture and curing skin diseases like eczema. Antioxidants and antidiabetic compounds have also been found in the root. In the seeds, some active compounds possess anti-inflammatory effects and potent inhibitory effects on the growth of tumors such as pancreatic carcinoma. In the leaf extract, the active compounds isolated can inhibit the growth of micro-organisms in the oral cavity. The medicinal uses of burdock in treating chronic diseases such as cancers, diabetes and AIDS have been reported. However, it is also essential to be aware of the side effects of burdock including contact dermatitis and other allergic/inflammatory responses that might be evoked by burdock.

The angiogenic effects of Angelica sinensis extract on HUVEC in vitro and zebrafish in vivo

Angiogenesis plays an important role in a wide range of physiological processes such as wound healing and fetal development. Many diseases are associated with imbalances in regulation of angiogenesis, in which it is either excessive or there is insufficient blood vessel formation. Angelica sinensis (AS), commonly used in the prescriptions of Chinese medicine, is a potential candidate for curing such diseases. However, biological effects of AS on angiogenesis and underlying mechanisms are yet to be fully elucidated. This investigation describes the angiogenic effects of AS extract on human endothelial cells (HUVEC) in vitro and zebrafish in vivo. The extract was demonstrated, by XTT assay and microscopic cell counting, to stimulate the proliferation of HUVEC; in addition, flow cytometry analysis indicated that the extract increased the percentage of HUVEC in the S phase. The wound healing migration assay illustrated that a dramatic increase in migration could be measured in AS extract‐treated HUVEC. Meanwhile, the number of invaded cells and the mean tube length were significantly increased in AS extract treatment groups. The extract was also demonstrated to promote changes in subintestinal vessels (SIVs) in zebrafish, one feature of angiogenesis. In addition, AS extract was found by real‐time PCR to enhance vascular endothelial growth factor (VEGF) mRNA expression. In a bead‐based immunoassay, higher levels of p38 and JNK 1/2 expression were also observed in effusions compared with control cells. All results suggest that Angelica sinensis extract can promote angiogenesis, and that the angiogenic effects involve p38 and JNK 1/2 phosphorylation. J. Cell. Biochem. 103: 195–211, 2008.

Angiogenic effect of saponin extract from Panax notoginseng on HUVECs in vitro and zebrafish in vivo

Angiogenesis plays an important role in a wide range of physiological processes such as wound healing and fetal development. In fact, many diseases are associated with imbalance in the regulation of angiogenesis in which there is either excessive or insufficient blood vessel formation. Panax notoginseng, a blood circulation invigorating herb, is commonly used in traditional Chinese medicine to treat circulation‐related diseases. However, the biological effects of saponin extract from Panax notoginseng (PNS) on angiogenesis and the underlying mechanisms are yet to be fully elucidated. This investigation describes the angiogenic effects of PNS on human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish in vivo. The 2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)5[(phenylamino)carbonyl]2H‐tetrazolium hydroxide (XTT) assay and microscopic cell counting demonstrated that the extract was able to stimulate the proliferation of HUVECs. Meanwhile, the numbers of invaded cells and tube branches were significantly increased in PNS treatment groups. PNS was also shown to promote changes in the subintestinal vessels, a feature of angiogenesis, in zebrafish. In addition, by using real‐time polymerase chain reaction (PCR), PNS was found to enhance vascular endothelial growth factor (VEGF) and kinase‐domain region/fetal liver kinase‐1 in mice (KDR/Flk‐1) mRNA expression, and the PNS‐induced HUVECs proliferation could be abolished by a KDR/Flk‐1 inhibitor. Furthermore, the proliferation of HUVECs induced by PNS was significantly attenuated by inhibitors of PI3K‐Akt‐eNOS. All the results suggest that PNS can promote angiogenesis, and that the proangiogenic effects involve the VEGF‐KDR/Flk‐1 and PI3K‐Akt‐eNOS signaling pathways. Copyright

Identification of histone methylation multiplicities patterns in the brain of senescence-accelerated prone mouse 8

Histone post-translational modifications (PTMs) are involved in diverse biological processes and methylation was regarded as a long-term epigenetic mark. Though aging represented one of the major risk factors for neurodegenerative diseases, no systematic investigations had correlated the patterns of histone PTMs in the brain with aging and the roles of such concerted histone PTMs in brain aging are still unknown. In this study, enzyme digestion, nano-LC, MALDI-TOF/TOF MS analysis and Western blotting were combined to investigate the defined methylation of core histones (H2A, H2B, H3 and H4) in the brain of 12-month-old senescence accelerated mouse prone 8 (SAMP8). The expression of several modified histones in the brain of 3-, and 12-month-old SAMP8 mice as well as that of the age-matched control senescence accelerated-resistant mouse (SAMR1) was compared. In the brain of 12-month-old SAMP8 mice, seven methylation sites (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2A R89) were detected and most PTMs sites were located on histone H3. Mono-methylated H4K20 decreased significantly in the brain of 12-month-old SAMP8 mice. Methylated H3K27 and H3K36 coexisted in the aged brain with different methylation multiplicities. Di-methylated H3K79 expressed in the neurons of cerebral cortex and hippocampus. This study showed histone methylation patterns in the aged SAMP8 mice brain and provided the experimental evidences for further research on histone PTMs in the aged brain. We hope these results could initiate a platform for the exchange of comprehensive information concerning aging or neurodegenerative disease and help us interpret the change of gene expression and DNA repair ability at epigenetic level.

Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 microM) and methylene blue (10 microM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS(Ser1177) protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 microM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 microM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 microM, an estrogen receptor (ER alpha/ER beta) antagonist) or mifepristone (10 microM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K(+) (K(ATP)) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK(Ca) and K(ATP) channels.

Mechanisms of the relaxant effect of a Danshen and Gegen formulation on rat isolated cerebral basilar artery

AIM OF THE STUDY Danshen (root of Salvia miltiorrhiza) and gegen (root of Pueraria lobata) are two herbs used in traditional Chinese medicine, most commonly for their putative cardioprotective and anti-atherosclerotic effects. In this study, the actions of a danshen and gegen formulation (DG; ratio 7:3) were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium; K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS DG produced concentration-dependent relaxation of the artery rings with an IC(50) of 895±121 μg/ml. Mechanical removal of the endothelium or pretreatment with the BK(Ca) channel inhibitor iberiotoxin (100 nM), the K(V) channel inhibitor 4-aminopyridine (1 mM), or the K(IR) channel inhibitor barium chloride (100 μM), all had no effect on the DG-induced response (P>0.05 for all). However, pretreatment with the K(ATP) channel inhibitor glibenclamide (1 μM), the non-selective K(+) channel inhibitor tetraethylammonium (TEA, 100 mM), or a combination of all the K(+) channel inhibitors (iberiotoxin+4-aminopyrindine+barium chloride+glibenclamide+TEA) produced significant inhibition on the DG-induced response (P<0.01 for all); its maximum vasorelaxant effect (Imax) was reduced by 37, 24, and 30%, respectively. Preincubation of the artery rings with DG for 10 min produced concentration-dependent (1, 3 and 7 mg/ml) and total inhibition on the CaCl(2)-induced vasoconstriction. CONCLUSIONS These findings suggest the vasorelaxant effect of DG on rat basilar artery is independent of endothelium-derived mediators, whereas, inhibition of Ca(2+) influx in the vascular smooth muscle cells is important, and a minor component is mediated by the opening of K(ATP) channels. DG could be a useful cerebroprotective agent in some patients with occlusive cerebrovascular disease.

Radix Astragali extract promotes angiogenesis involving vascular endothelial growth factor receptor-related phosphatidylinositol 3-kinase/Akt-dependent pathway in human endothelial cells

Angiogenesis plays an important role in a wide range of physiological processes and many diseases are associated with dysregulation of angiogenesis. Radix Astragali, commonly used in traditional Chinese medicine, is a potential candidate for treating such diseases. However, the biological effects of Radix Astragali on angiogenesis and its underlying mechanisms are yet to be elucidated fully. This study describes the angiogenic effects of Radix Astragali extract (RAE) on human umbilical vein endothelial cells (HUVEC) in vitro. It was shown that RAE treatment stimulated HUVEC to proliferate. A significant increase in migration was observed in RAE‐treated HUVEC using the wound healing migration assay. In addition, a significant increase in the number of branching points was observed during endothelial cell capillary formation after RAE treatment. It was shown that RAE enhances vascular endothelial growth factor (VEGF) mRNA expression, and that a specific blocker of VEGF receptor 2 (KDR/Flk) inhibited the RAE‐induced HUVEC proliferation. In addition, a decrease in the RAE‐induced HUVEC proliferation was observed after treatment with inhibitors of phosphatidylinositol 3‐kinase (PI3K), Akt and endothelial nitric oxide synthase (eNOS). Taken together, these data suggest that RAE is a potent stimulator of angiogenesis and that its pro‐angiogenic effects involve the VEGF‐KDR/Flk and PI3K‐Akt‐eNOS pathways. Copyright

Mechanisms responsible for the in vitro relaxation of ligustrazine on porcine left anterior descending coronary artery

In this study, we have evaluated the underlying mechanisms responsible for the relaxation response of ligustrazine (2,3,5,6-tetra-methyl-pyrazine; 2,3,5,6-MP) and its structural analogues (2-methyl-pyrazine (2-MP); ethyl-pyrazine (EP); 2,3-di-methyl-pyrazine (2,3-MP); 2,5-di-methyl-pyrazine (2,5-MP); 2,6-di-methyl-pyrazine (2,6-MP) and 2,3,5-tri-methyl-pyrazine (2,3,5-MP)) in porcine left anterior descending coronary artery (tertiary branch, O.D. </=1 mm). In 5-hydroxytryptamine (3 microM) precontracted preparations, cumulative administration (0.1-300 microM) of all pyrazine analogues caused an endothelium-independent, concentration-dependent relaxation. The relative inhibitory potency, as compared at concentration with which 50% relaxation occurred, was 2,3,5,6-MP>2,3,5-MP>EP>2,5-MP>/=2,6-MP>/=2,3-MP>2-MP. Besides, salbutamol and forskolin caused an endothelium-independent relaxation. The relaxation response of ligustrazine, salbutamol and forskolin was blunted in the presence of cis-N-(2-phenylcyclopentyl) azacyclotridec-1-en-2-amine (MDL 12330A) (10 microM, an adenylate cyclase inhibitor) and N-[2-((bromocinnamyl)amino)ethyl]-5-isoquinoline-sulphonamide (H-89, a protein kinase A inhibitor, 3 microM). Patch-clamp, whole-cell electrophysiological studies using single smooth muscle cells of the left anterior descending coronary artery revealed that ligustrazine (300 microM), salbutamol (30 microM) and forskolin (1 microM) inhibited the nifedipine-sensitive L-type Ca(2+) channels, and the inhibitory effect was eradicated by MDL 12330A (10 microM) and H-89 (1 microM). However, neither the Ca(2+)-dependent K(+) channel nor the ATP-dependent K(+) channel was modified by ligustrazine (300 microM). In conclusion, our results indicate that ligustrazine-mediated left anterior descending coronary artery relaxation is due to the activation of adenylate cyclase/protein kinase A cascade and the subsequent inhibition of nifedipine-sensitive, voltage-dependent L-type Ca(2+) channels. However, opening of K(+) channels seems to play no role in mediating the relaxation effect of ligustrazine.

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms.

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.