[Retracted] Arnebin­1 promotes angiogenesis by inducing eNOS, VEGF and HIF­1α expression through the PI3K­dependent pathway.

Following the publication of the above paper, it was drawn to the Editor's attention by a concerned reader that the α­tubulin protein bands shown in Fig. 2A on p. 689 were strikingly similar to data appearing in different form in the following paper: Tian R, Li Y and Gao M: Shikonin causes cell­cycle arrest and induces apoptosis by regulating the EGFR­NF­κB signalling pathway in human epidermoid carcinoma A431 cells. Biosci Rep 35: e00189, 2015. Moreover, there were a pair of overlapping data panels shown in the cell invasion and migration assay data in Fig. 5B on p. 692, one identified instance of western blot data being shared between Figs. 3D and 4F, and a pair of overlapping data panels in Fig. 5D, such that all these data, which were intended to have shown the results from differently performed experiments, may have been derived from a smaller number of original sources. Owing to the fact that the contentious data in the above article were already under consideration for publication prior to its submission to International Journal of Molecular Medicine and an overall lack of confidence in the presented data, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 36: 685­697, 2015; DOI: 10.3892/ijmm.2015.2292].

Efficacy of Acetylshikonin in Preventing Obesity and Hepatic Steatosis in db/db Mice.

Zicao (Lithospermum erythrorhizon) has been used in clinics as a traditional Chinese medicine for thousands of years. Acetylshikonin (AS) is the main ingredient of Zicao, Xinjiang, China. The objective of this study was to investigate the anti-obesity and anti-nonalcoholic fatty liver disease (NAFLD) efficacy of AS in a model of spontaneous obese db/db mice. Mice were divided into Wild Type (WT) groups and db/db groups, which received no treatment or treatment with 100 mg/kg/day clenbuterol (CL) hydrochloride or 540 mg/kg/day AS by oral gavage for eight weeks. The results provided the evidence that AS prevented obesity and NAFLD including reduction in body weight, food efficiency ratio, serum triglyceride (TG) and free fatty acid (FFA) levels in db/db mice. Administration of AS markedly suppressed the levels of hepatic alanine aminotransferase (ALT), aspartate aminotransferase (AST) and pro-inflammatory cytokines in treated groups when compared with that of db/db groups. Further investigation of the lipid synthesis-related protein using Western blotting revealed that hepatic protein expression of sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthetase (FAS) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were significantly downregulated by AS treatment. These findings suggest that AS exerts anti-obesity and anti-NAFLD effects through the regulation of lipid metabolism and anti-inflammatory effects.

Arnebin-1 promotes angiogenesis by inducing eNOS, VEGF and HIF-1α expression through the PI3K-dependent pathway.

Arnebin-1, a naphthoquinone derivative, plays a crucial role in the wound healing properties of Zicao (a traditional wound healing herbal medicine). It has been noted that Arnebin-1, in conjunction with vascular endothelial growth factor (VEGF), exerts a synergistic pro-angiogenic effect on human umbilical vein endothelial cells (HUVECs) and accelerates the healing process of diabetic wounds. However, the mechanisms responsible for the pro-angiogenic effect of arnebin­1 on HUVECs and its healing effect on diabetic wounds have not yet been fully elucidated. In this study, in an aim to elucidate these mechanisms of action of arnebin­1, we investigated the effects of arnebin­1 on the VEGF receptor 2 (VEGFR2) and the phosphoinositide 3-kinase (PI3K)­dependent signaling pathways in HUVECs treated with VEGF by western blot analysis. The pro­angiogenic effects of arnebin­1 on HUVECs, including its effects on proliferation and migration, were evaluated by MTT assay, Transwell assay and tube formation assay in vitro. The expression levels of hypoxia-inducible factor (HIF)­1α, endothelial nitric oxide synthase (eNOS) and VEGF were determined by western blot analysis in the HUVECs and wound tissues obtained from non­diabetic and diabetic rats. CD31 expression in the rat wounds was evaluated by immunofluorescence staining. We found that the activation of the VEGFR2 signaling pathway induced by VEGF was enhanced by arnebin­1. Arnebin­1 promoted endothelial cell proliferation, migration and tube formation through the PI3K­dependent pathway. Moreover, Arnebin­1 significantly increased the eNOS, VEGF and HIF­1α expression levels in the HUVECs and accelerated the healing of diabetic wounds through the PI3K­dependent signaling pathway. CD31 expression was markedly enhanced in the wounds of diabetic rats treated with arnebin­1 compared to the wounds of untreated diabetic rats. Therefore, the findings of the present study indicate that arnebin-1 promotes the wound healing process in diabetic rats by eliciting a pro-angiogenic response.

Arnebin-1 promotes the angiogenesis of human umbilical vein endothelial cells and accelerates the wound healing process in diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Zicao is a traditional wound healing herbal medicine that has been used for several hundred years in China. A survey of the published literatures revealed that arnebin-1, one of the naphthoquinone derivatives, played the most important role in wound healing property of this plant. However, whether arnebin-1 affects angiogenesis in vitro and has an effect on wound healing process in diabetic rats remains enigmatic. To investigate the effect of arnebin-1 with or without VEGF on proliferation, migration and tube formation of HUVECs in vitro and the effect of its topical application in the form of ointment on wound healing in a cutaneous punch wound model of alloxan-induced diabetic rats in vivo. MATERIALS AND METHODS: The pro-angiogenic functions of arnebin-1 on HUVECs including proliferation, migration and angiogenesis were evaluated through MTT assay, wound healing assay, transwell assay and tube formation assay in vitro. Male Sprague-Dawley rats were injected intraperitoneally with alloxan to induce type І diabetic rats. Three wounds were created in each rat on the dorsal surface, and then divided to be basement treated, arnebin-1 ointment treated and untreated group correspondingly. The indicators including wound closure rate and histological evaluation were investigated on day 4 and 7 post-wounding. RESULTS: Without VEGF, arnebin-1 did not affect the proliferation of HUVECs significantly, but had a positive effect on cell migration and tube formation. However, in the presence of minimal VEGF, Arnebin-1 could increase the proliferation, enhance the migration and promote the tube formation of HUVECs significantly. The wound closure rate was increased significantly in arnebin-1 treated group compared to that of untreated and basement treated groups in diabetic rats, and the histological evaluation also showed well organized dermal layer, reduced number of macrophages, increased number of fibroblasts, remarkable degree of neovascularization and epithelization in arnebin-1 treated group. CONCLUSION: These findings suggest that arnebin-1 has a pro-angiogenic effect, and a synergetic effect with VEGF promotes the wound healing process in diabetic rats.

Translocation of protein kinase C δ contributes to the moderately high glucose-, but not hypoxia-induced proliferation in primary cultured human retinal endothelial cells.

Diabetic retinopathy is one of the most common complications in patients with diabetes and affects ~75% of them within 15 years of the onset of the disease. Activation of protein kinase C (PKC) is a key feature of diabetes mellitus and may be involved in the pathogenesis of diabetic retinopathy. The present study aimed to examine the translocation of protein kinase C (PKC) isoforms, which are triggered by high an moderately high glucose levels as well as hypoxic conditions. The underlying cell mechanisms of PKC translocation in primary cultured human retinal endothelial cells (HRECs) were also investigated. The expression levels of PKC isoforms were assessed using western blot analysis. Cell proliferation was determined using the MTT assay and DNA synthesis was assessed by bromodeoxyuridine incorporation. Translocation of PKC isoforms was examined by western blot analysis and immunofluorescence. The expression of PKC α, ßI, ßII, δ and ε was detected, while PKC ζ was not detected in HRECs. The results of the present study were consistent with the findings of a previous study by our group, reporting that moderately high glucose levels and hypoxia, but not high glucose levels, significantly increased cell proliferation. It was demonstrated that the PKC δ isoform was translocated from the cytosol to the membrane only under moderately high glucose conditions, while PKC α and ε isoforms were translocated from the cytosol to the membrane at high glucose conditions. In addition, PKC ßI was translocated under all three conditions. Translocation of PKC ßII was comparable among all groups. Furthermore, rottlerin, an inhibitor of PKC δ, blocked cell proliferation, which was induced by moderately high glucose levels, but not by hypoxia. Ro32-0432, an inhibitor of PKC α, ßI and ε, did not significantly affect proliferation of HRECs in all treatment groups. In conclusion, the present study suggested that PKC α, ßI, ßII, δ and ε were expressed in primary cultured HRECs, whereas PKC ζ was not. Cell proliferation induced by moderately high glucose concentrations was associated with translocation of the PKC δ isoform; however, hypoxic conditions did not induce translocation.

High-fat diet-induced obesity leads to resistance to leptin-induced cardiomyocyte contractile response.

Levels of the obese gene product leptin are often elevated in obesity and may contribute to obesity-induced cardiovascular complications. However, the role of leptin in obesity-associated cardiac abnormalities has not been clearly defined. This study was designed to determine the influence of high-fat diet-induced obesity on cardiac contractile response of leptin. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix system in cardiomyocytes from adult rats fed low- and high-fat diets for 12 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were examined including peak shortening, duration and maximal velocity of shortening/relengthening (TPS/TR(90), +/-dl/dt), Fura-2-fluorescence intensity change (DeltaFFI), and intracellular Ca(2+) decay rate (tau). Expression of the leptin receptor (Ob-R) was evaluated by western blot analysis. High-fat diet increased systolic blood pressure and plasma leptin levels. PS and +/-dl/dt were depressed whereas TPS and TR(90) were prolonged after high-fat diet feeding. Leptin elicited a concentration-dependent (0-1,000 nmol/l) inhibition of PS, +/-dl/dt, and DeltaFFI in low-fat but not high-fat diet-fed rat cardiomyocytes without affecting TPS and TR(90). The Janus kinase 2 (JAK2) inhibitor AG490, the mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the nitric oxide synthase (NOS) inhibitor L-NAME abrogated leptin-induced cardiomyocyte contractile response in low-fat diet group without affecting the high-fat diet group. High-fat diet significantly downregulated cardiac expression of Ob-R. Elevation of extracellular Ca(2+) concentration nullified obesity-induced cardiomyocyte mechanical dysfunction and leptin-induced depression in PS. These data indicate presence of cardiac leptin resistance in diet-induced obesity possibly associated with impaired leptin receptor signaling.

Scutellarin induced Ca(2+ ) release and blocked KCl-induced Ca(2+ ) influx in smooth muscle cells isolated from rat thoracic artery.

This study was designed to investigate the effect of scutellarin (1) on the modulation of intracellular Ca(2+ ) concentration in thoracic smooth muscle cells of rat. Single smooth muscle cells were obtained enzymatically. Fluo-3 AM was used to determine the alteration of intracellular-free Ca(2+ )([Ca(2+ )](i)) and the changes in fluorescence intensity under different agonists were recorded. Compound 1 induced Ca(2+ ) transients in the medium with and/or without Ca(2+ ). In the Ca(2+ )-free medium, after pretreatment of 1, thapsigargin failed to cause the elevation of [Ca(2+ )](i). However, 1 still caused the elevation of [Ca(2+ )](i) after pretreatment of thapsigargin. The infusion of 1 blocked KCl-induced Ca(2+ ) entry and this effect was hardly reversible. The results of present study suggested that 1 increased [Ca(2+ )](i) by blocking sarcoplasmic reticulum Ca(2+ )/ATPase and blocked voltage-dependent Ca(2+ ) channels in smooth muscle cells of the rat thoracic aortic artery.

Scutellarein inhibits hypoxia- and moderately-high glucose-induced proliferation and VEGF expression in human retinal endothelial cells.

AIM: This study was designed to examine the effect of scutellarein on high glucose- and hypoxia-stimulated proliferation of human retinal endothelial cells (HREC). METHODS: HREC were cultured under normal glucose (NG), moderate, and high glucose (NG supplemented with 10 or 25 mmol/L D-glucose) and/or hypoxic (cobalt chloride treated) conditions. Cell proliferation was evaluated by a cell counting kit. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. RESULTS: The proliferation of HREC was significantly elevated in response to moderately-high glucose and hypoxic conditions. The combination of high glucose and hypoxia did not have any additive effects on cell proliferation. Consistent with the proliferation data, the expression of VEGF was also upregulated under both moderately-high glucose and hypoxic conditions. The treatment with scutellarein (1x10(-11) to 1x10(-5) mol/L) significantly inhibited high glucose- or hypoxia-induced cell proliferation and VEGF expression. CONCLUSION: Both hypoxia and moderately-high glucose were potent stimuli for cell proliferation and VEGF expression in HREC without any significant additive effects. Scutellarein is capable of inhibiting the proliferation of HREC, which is possibly related to its ability to suppress the VEGF expression.