[Show abstract][Hide abstract] ABSTRACT: To determine if growth arrest-specific 6 (Gas6) plays an important role in the regulation of angiogenesis in human retinal microvascular endothelial cells (HRMECs) and in vessel development of zebrafish.
Proliferation, wound-healing cell migration, and tube formation were measured in HRMECs treated with recombinant human Gas6 (rhGas6). Sprague-Dawley rat aortas in Matrigels were treated with rhGas6, and microvessel sprouting emanating from arterial rings was analyzed. Transgenic zebrafish embryos (flk:GFP) were microinjected with rhGas6 at 50 hours post-fertilization (hpf), and ectopic sprouting of subintestinal vessels (SIVs) was observed under a confocal microscope. Morpholino oligonucleotides (MOs) were microinjected to knockdown gas6 in zebrafish embryos, and intersegmental vessel impairment was observed. The effect of the extracellular signal-regulated kinase (ERK1/2) inhibitor on the migration of HRMECs and on vessel development in zebrafish embryos was tested.
rhGas6 stimulated proliferation, migration, and tube formation in HRMECs in a dose-dependent manner. In rat aortas, rhGas6 induced vessel outgrowth, and the sprouting length was longer than that of controls. The rhGas6-microinjected zebrafish embryos had significantly increased vessel outgrowth in the SIVs. Recombinant human vascular endothelial growth factor (rhVEGF) served as a positive control. Knockdown of gas6 inhibited angiogenesis in the developing vessels of zebrafish. The ERK1/2 inhibitor inhibited HRMEC migration and intersegmental vessel formation in zebrafish embryos.
These data suggest that Gas6 plays a pivotal role in proliferation, migration, and sprouting of angiogenic endothelial cells in the retina and in zebrafish embryos. Furthermore, Gas6 induced angiogenic processes are induced via phosphorylation of ERK1/2.
PLoS ONE 01/2014; 9(1):e83901. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic hyperglycemia leads to the formation of advanced glycation end products (AGEs), which accelerates the development of diabetic complications. Previous studies have shown that extract of Cassiae semen (CS), the seed of Cassia tora, has inhibitory activity on AGEs formation in vitro and reduces transforming growth factor-beta1 (TGF-β1) and extracellular matrix protein expression via inhibition of AGEs-mediated signaling in glomerular mesangial cells. In this study, to examine the preventive effects of CS extract on the development of diabetic nephropathy in vivo, streptozotocin (STZ)-injected diabetic rats were orally administered CS extract (200mg/kg body weight/day) for 12 weeks. Serum glucose, triglycerides, and total cholesterol in diabetic rats were significantly higher compared to control rats. CS or aminoguanidine (AG) treatment significantly reduced these factors. Proteinuria and creatinine clearance were also significantly decreased in the CS-treated group compared with the untreated diabetic group. The CS-treated group had significantly inhibited COX-2 mRNA and protein, which mediates the symptoms of inflammation in the renal cortex of diabetic rats. Furthermore, histopathological studies of kidney tissue showed that in diabetic rats, AGEs, the receptor for AGEs, TGF-β1, and collagen IV were suppressed by CS treatment. Our data suggest that oral treatment of CS can inhibit the development of diabetic nephropathy via inhibition of AGEs accumulation in STZ-induced diabetic rats.
Phytomedicine: international journal of phytotherapy and phytopharmacology 12/2013; · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidences suggest that aldose reductase (AR) inhibitors and advanced glycation end product (AGE) formation inhibitors may prevent chronic hyperglycemia-induced long-term complication in diabetes. Transforming growth factor-beta1 (TGF-beta1) plays an important role in the development of diabetic nephropathy. Allium species have been utilized in folk medicine throughout the world for the treatment of various physical disorders. However, the benefits of Allium victorialis (A. victorialis) against diabetic complications, especially nephropathy, have yet to be explored. In the present study, we investigated the protective effect of the compounds isolated from A. victorialis leaf on diabetic nephropathy.
In vitro AR activity, AGEs formation, and AGE-receptor for AGEs (RAGE) binding in human RAGE (hRAGE)-overexpressing cells were tested. High glucose-induced transforming growth factor-beta1 (TGF-beta1) expression was also examined in mouse kidney mesangial cells (MMCs) cultured under high glucose.
Of the isolated eight compounds from A. victorialis leaf extracts tested, quercitrin exhibited the most pronounced inhibitory effects on AR activity (IC50 value of 0.17 muM) and AGEs formation (IC50 value of 4.20 muM). Furthermore, quercitrin disrupted AGE-RAGE binding in a concentration-dependent manner in hRAGE-overexpressing cells. Additionally, of the eight compounds tested, ferulic acid significantly reduced high glucose-induced TGF-beta1 expression and secretion in MMCs.
Our results suggest that active compounds isolated from A. victorialis leaf exhibit inhibitory effects on AR activity in rat lenses and AGE formation. Further, ferulic acid reduces TGF-beta1 mRNA expression and secretion in MMCs under diabetic conditions. Thus, A. victorialis is a good candidate for the development of treatments for diabetic nephropathy.
BMC Complementary and Alternative Medicine 10/2013; 13(1):251. · 2.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.
Experimental Cell Research 01/2012; 318(2):152-9. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Low-density lipoprotein (LDL) is subjected to glycoxidation in diabetes, and a novel signalling mechanism by which glycoxidised LDL functions in glomerular mesangial cells remains to be ascertained.
We performed gene expression analysis in mouse glomerular mesangial cells treated with LDL modified by glycation and oxidation (GO-LDL, 100 µg/ml) for 48 h by using DNA microarray analysis and quantitative real-time PCR. We examined the GO-LDL-specific changes in gene and protein expression in mesangial cells and glomeruli of type 2 diabetic Zucker diabetic fatty (ZDF) rats.
By microarray profiling, we noted that GO-LDL treatment increased Axl receptor tyrosine kinase (Axl) mRNA expression (∼2.5-fold, p<0.05) compared with normal LDL (N-LDL) treatment in mesangial cells. Treatment with GO-LDL also increased the protein levels of Axl and its ligand Gas6 as measured by Western blotting. These increases were inhibited by neutralising Axl receptor-specific antibody. Silencing Gas6 by siRNA inhibited GO-LDL-induced Axl expression in mesangial cells. Axl and Gas6 protein were also increased in cells cultured in high glucose (30 mM) or methylglyoxal (200 µM). Gas6 treatment increased the expression and secretion of TGF-β1 protein, a key regulator of extracellular matrix expression in the glomeruli of diabetic kidneys. Immunohistochemical analyses of glomeruli from 20-week-old ZDF rats exhibited increased Axl protein expression. Rottlerin, a selective PKC-δ inhibitor, completely blocked Gas6-induced TGF-β1 expression.
These data suggest that LDL modified by glycoxidation may mediate Axl/Gas6 pathway activation, and this mechanism may play a significant role in the pathogenesis of diabetic nephropathy.
PLoS ONE 01/2012; 7(11):e50297. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding.
[Show abstract][Hide abstract] ABSTRACT: Non-enzymatic glycation reactions between reducing sugar and free reactive amino groups of protein lead to the formation of advanced glycation end products, which increase under conditions of aging or diabetes. A previous study showed that extracts of Cassiae Semen (CS), the seed of Cassia tora, had inhibitory activity on advanced glycation end products formation in vitro. To examine the pharmacological effects of a butanol-soluble extract of CS under conditions of diabetic nephropathy, we evaluated the expression of transforming growth factor-beta1 (TGF-beta1) and fibronectin, key mediators of diabetic nephropathy, in mouse glomerular mesangial cells cultured in the presence of S100b (a specific ligand for receptor of advanced glycation end products). CS inhibited S100b-induced TGF-beta1 and fibronectin expression in mouse mesangial cells by suppressing activation of Smad2/3, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), and oxidative stress. Moreover, CS suppressed nuclear factor-kappa B (NF-kappaB) activation in S100b-stimulated mouse mesangial cells. To identify the active compounds of CS, three major compounds, rubrofusarin-6-O-beta-d-gentiobioside (CS-A), toralactone-9-O-beta-d-gentiobioside (CS-B), and cassiaside (CS-C), were tested in cells. Of these compounds, CS-A significantly decreased the expression of TGF-beta1 and fibronectin and NF-kappaB DNA binding activity. These findings suggest that CS, especially CS-A, has potential as a preventive agent for advanced glycation end products-related diabetic complications.
European journal of pharmacology 09/2010; 641(1):7-14. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the effects of KIOM-79 in preventing the development of diabetic complications, such as cataracts.
The inhibitory effects of KIOM-79 were assessed in a model of xylose-induced lens opacity and on changes mediated by high levels of glucose in human lens epithelial (HLE-B3) cells.
In lenses treated with KIOM-79, opacity was significantly improved and glutathione (GSH) was increased compared to controls. In HLE-B3 cells treated with KIOM-79, high glucose-mediated increases in TGF-beta2, alphaB-crystallin, and fibronectin were significantly inhibited in a dose-dependent manner. KIOM-79 decreased the phosphorylation of p-Smad2/3, pp38MAPK, pp44/42, and NF-kappaB signaling in cells grown under high glucose conditions.
KIOM-79 is protective against lens opacity and protects HLE-B3 cells from the toxic effects of high glucose. Therefore, KIOM-79 may provide a potential therapeutic approach for preventing diabetic complications, such as cataracts.
Journal of ethnopharmacology 08/2010; 130(3):599-606. · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A trace uranium (U) detection method was developed with a handheld voltammetric analyzer that was the size of a mobile phone, with working sensors made of simple graphite pencil electrode (PE). The optimum stripping voltammetric conditions were sought, and the following results were obtained: 0.0 to 0.08 ng/L working ranges and a statistically relative standard deviation of 1.78% (RSD; n=15) at a 10.0 microg/L U spike. The experiment accumulation time used was only 150 s. Under this condition, the diagnostic detection limit approached 0.007 ng/L. The method was applied to soil of a natural rock in a radioactive mineralogy site. Earthworms that resided at this site were assayed. The method was found to be applicable in biological diagnosis or in real-time in vivo survey.
Environmental Toxicology and Chemistry 05/2010; 29(5):1025-30. · 2.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Puerarin is a natural product isolated from Puerarin lobata and has various pharmacological effects, including anti-hyperglycemic and anti-allergic properties. In the present study, we investigated the effect of puerarin against advanced glycation end products (AGEs)-induced inflammation in mouse mesangial cells. Puerarin acts by inducing the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Puerarin was able to enhance phosphorylation of protein kinase C (PKC) delta, but not PKC alpha/beta II, in a time-dependent manner. Induction of HO-1 expression by puerarin was suppressed by GF109203X, a general inhibitor of PKC, and by rottlerin, a specific inhibitor of PKC delta. However, induction was not suppressed by Gö6976, a selective inhibitor for PKC alpha/beta II. Additionally, the knockdown of endogenous PKC delta by small interfering RNA (siRNA) resulted in the inhibition of HO-1 expression and Akt phosphorylation. Puerarin increased antioxidant response element (ARE)-Luciferase activity in a dose- and time-dependent manner in transfected mouse mesangial cells. Mutation of the ARE sequence abolished puerarin-induced HO-1 expression. Furthermore, puerarin treatments resulted in a marked increase in NF-E2 related factor-2 (Nrf-2) translocation, leading to up-regulation of HO-1 expression. However, transfection of Nrf-2 specific siRNA abolished HO-1 expression. Pretreatment with puerarin inhibited the expressions of COX-2, MMP-2 and MMP-9. But, these effects were reversed by ZnPP, an inhibitor of HO-1. Taken together, our results demonstrate that puerarin-induced expression of HO-1 is mediated by the PKC delta-Nrf-2-HO-1 pathway and inhibits N-carboxymethyllysine (CML)-induced inflammation in mouse mesangial cells.
Toxicology and Applied Pharmacology 04/2010; 244(2):106-13. · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated whether KIOM-79 inhibits transforming growth factor-beta 1 (TGF-beta1) and fibronectin expression in mouse mesangial cells cultured under S100b, a specific ligand of the receptor for advanced glycation end products (RAGE).
Cell counting kit (CCK-8) assay was employed to evaluate the viability of KIOM-79-treated mesangial cells. The effect of KIOM-79 on S100b-induced TGF-beta1 and fibronectin expression was investigated using RT-PCR, ELISA, and Western blot on mesangial cells.
KIOM-79 (up to 50 microg/ml) appeared to have no effect on cell viability. S100b induced an increase in the expression TGF-beta1 and fibronectin. Expression of TGF-beta1 and fibronectin was inhibited significantly by KIOM-79 treatment in mesangial cells. KIOM-79 also inhibited the expression of NF-kB and inactivated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 in mesangial cells. KIOM-79 pretreatment inhibited increased malondialdehyde (a product of lipid peroxidation and a marker for oxidative stress) levels in S100b-induced mesangial cells.
These data demonstrate that KIOM-79 inhibits expression of TGF-beta1 and fibronectin through inactivation of MAPK/ERK1/2 signaling, reduction in malondiadehyde levels, and inhibition of NF-kB in mesangial cells cultured under diabetic conditions. KIOM-79 could be beneficial for preventing of the development of diabetic complications such as nephropathy.
Journal of ethnopharmacology 09/2009; 125(3):374-9. · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aldose reductase (AR) and TGF-beta have been implicated in the development of diabetic complications, such as cataracts. In an attempt to obtain potential agents for the prevention of diabetic cataracts from natural products, we purified genistein from the roots of Pueraria lobata and investigated its inhibitory effects upon AR activity and its antioxidant effects on rat lenses. The inhibition of AR activity by genistein increased in a dose-dependent manner and the opacities of lenses were significantly improved when treated with genistein. In addition, we determined the effects of genistein on mechanisms induced by exposure to high glucose in human lens epithelial (HLE-B3) cells. We found that genistein was able to reduce the expression of TGF-beta2, alphaB-crystallin, and fibronectin mRNAs in HLE-B3 cells that were cultured in high glucose conditions. In addition, a reduction in glutathione (GSH) levels and thiobarbituratic acid-reactive substances was observed. These results show that genistein is protective against lens opacity and also inhibits high glucose-mediated toxic effects in HLE-B3 cells. These effects are likely achieved by preventing AR and cellular oxidation; therefore, genistein may be a potential therapeutic agent for preventing and treating complications associated with diabetes mellitus, such as diabetic cataracts.
European Journal of Pharmacology 11/2008; 594(1-3):18-25. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated whether KIOM-79, a mixture of extracts obtained from Puerariae lobata, Magnolia officinalis, Glycyrrhiza uralensis and Euphorbia pekinensis, could inhibit vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells cultured under high glucose (HG, 25mM) or S100b (a specific ligand of the receptor for advance glycation end products (RAGE), 5microg/ml). In this study, the effect of KIOM-79 on HG or S100b-induced VEGF expression was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, RT-PCR, ELISA, and Western blot on human RPE cells. The MTT assay (p<0.01) revealed that KIOM-79 (up to 1mg/ml) had no effect on cell growth. HG or S100b induced an increase in expression of VEGF at both mRNA and protein levels (p<0.05; p<0.01 versus control). The increase in VEGF expression by HG or S100b was dose- and time-dependently prevented by KIOM-79 (p<0.05 versus 25mM glucose; p<0.01 versus S100b). Also, KIOM-79 inhibited protein kinase C (PKC)-alpha/beta(alpha) and p38 mitogen-activated protein kinase (MAPK) activation. Our results demonstrate that KIOM-79 can inhibit VEGF expression via inhibition of the MAPK and PKC pathway.
Journal of Ethnopharmacology 06/2007; 112(1):166-72. · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the effect of KIOM-79, 80% ethanolic extract of a new herbal prescription, on non-obese type 2 diabetic Goto-Kakizaki (GK) rats. The rats were treated orally with KIOM-79 (500 mg/kg body weight) once a day for 13 weeks to examine the long-term effects on hyperglycemia and glomerular histology as well as biochemical and functional abnormalities in kidney. As the results, we found that KIOM-79 reduced hyperglycemia (p<0.01), ameliorated insulin resistance (p<0.001), urinary protein excretion (p<0.01) and creatinine clearance (Ccr) (p<0.001), and inhibited glomerular AGE formation (p<0.001) in diabetic GK rats. We also found that KIOM-79 prevented the glomeruli enlargement, overexpression of type IV collagen (p<0.001), PKC protein (p<0.01), TGF-beta mRNA (p<0.05) and VEGF mRNA (p<0.05). Thus, based on our finding, KIOM-79 could reduce the hyperglycemia, and prevent or retard the development of diabetic nephropathy.
Journal of Ethnopharmacology 06/2007; 111(2):240-7. · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Magnolol, a natural product isolated from Magnolia officinalis, has various pharmacological effects, such inhibition of effect on inflammation and tumor metastasis, protection against cerebral ischaemic injury, and potent antioxidant activity. In this present study, we evaluated the inhibitory effects of magnolol on transforming growth factor-beta1 (TGF-beta1) and fibronectin expression induced by high concentrations of glucose or S100b (a specific receptor of advance glycation end products ligand) in human retinal pigment epithelial cells (human RPE cells). No effect on cell growth was found with magnolol (up to 20 microg/ml) using a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay. High glucose (25 mM) or S100b (5 microg/ml) induced increases in expression of TGF-beta1 and fibronectin. The increases in TGF-beta1 and fibronectin expression with high glucose or S100b were prevented by magnolol in a dose-dependent manner. Also, magnolol inhibited extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)/Akt activation. The present study demonstrates that high glucose- or S100b-induced TGF-beta1 and fibronectin expression, but this increased expression is inhibited by magnolol via the ERK/MAPK/Akt signaling pathway in human RPE cells.
European Journal of Pharmacology 06/2007; 562(1-2):12-9. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the effect of magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl), a marker compound isolated from the cortex of Magnolia officinalis, in non-obese type 2 diabetic Goto-Kakizaki (GK) rats. The rats were treated orally with magnolol (100 mg/kg body weight) once a day for 13 weeks. In magnolol-treated GK rats, fasting blood glucose and plasma insulin were significantly decreased, and the pancreatic islets also showed strong insulin antigen positivity. Urinary protein and creatinine clearance (Ccr) were significantly decreased. Pathological examination revealed the prevention of the glomeruli enlargement in magnolol-treated GK rats. The overproduction of renal sorbitol, advanced glycation endproducts (AGEs), type IV collagen, and TGF-beta1 mRNA were significantly reduced in magnolol-treated GK rats. Thus based on our findings, the use of magnolol could result in good blood glucose control and prevent or retard development of diabetic complications such as diabetic nephropathy.
Life Sciences 02/2007; 80(5):468-75. · 2.56 Impact Factor