Autophagy is involved in acetylshikonin ameliorating non-alcoholic steatohepatitis through AMPK/mTOR pathway.

Acetylshikonin (AS), a naphthoquinone constituent derived from Lithospermum erythrorhizon, has been revealed various pharmacological activities including anti-oxidative, anti-inflammatory and antifertility effects. Our previous study has illuminated the effects of AS on preventing obesity and hepatic steatosis in db/db mice. However, the effects of AS and the molecular mechanisms for curing non-alcoholic steatohepatitis (NASH) have not yet been studied. Autophagy has been considered as a lysosomal degradative pathway responsible for the removal of cellular lipid droplets through a process called lipophagy, which is recognized as a potential therapeutic approach for NASH. Here we hypothesize that autophagy is involved in the beneficial effects of AS on methionine-choline deficient (MCD) diet-induced NASH of mice. In this study, we observed that AS treatment ameliorated the pathological signs of NASH, and markedly suppressed the levels of hepatic IL-1ß and TNF-α cytokines, and hepatocyte apoptotic cells in MCD diet-induced mice. Moreover, immunological analyses showed that the elevated expression of the fibrotic markers including α-SMA, collegen I, collegen III and fibronectin in MCD diet-induced mice were notably down-regulated by AS treatment. Nevertheless, the beneficial effects of AS on ameliorating NASH were notably counteracted by co-administration of chloroquine, an autophagy inhibitor. Furthermore, our data suggested that AS treatment increased hepatocyte autophagy in MCD diet-induced mice via AMPK/mTOR pathway. These findings suggest that AS could be therapeutically effective in the development of NASH by ameliorating steatosis, inflammation, liver injury and fibrosis.

Enhancement of scutellarin oral delivery efficacy by vitamin B12-modified amphiphilic chitosan derivatives to treat type II diabetes induced-retinopathy.

BACKGROUND: Diabetic retinopathy is the most common complication in diabetic patients relates to high expression of VEGF and microaneurysms. Scutellarin (Scu) turned out to be effective against diabetes related vascular endothelial cell dysfunction. However, its clinical applications have been limited by its low bioavailability. In this study, we formulated and characterized a novel intestinal target nanoparticle carrier based on amphiphilic chitosan derivatives (Chit-DC-VB12) loaded with scutellarin to enhance its bioavailability and then evaluated its therapeutic effect in experimental diabetic retinopathy model. RESULTS: Chit-DC-VB12 nanoparticles showed low toxicity toward the human colon adenocarcinoma (Caco-2) cells and zebra fish within concentration of 250 µg/ml, owing to good biocompatibility of chitosan. The scutellarin-loaded Chit-DC-VB12 nanoparticles (Chit-DC-VB12-Scu) were then prepared by self-assembly in aqueous solution. Scanning electron microscopy and dynamic light scattering analysis indicated that the Chit-DC-VB12-Scu nanoparticles were spherical particles in the sizes ranging from 150 to 250 nm. The Chit-DC-VB12-Scu nanoparticles exhibited high permeation in Caco-2 cell, indicated it could be beneficial to be absorbed in humans. We also found that Chit-DC-VB12 nanoparticles had a high cellular uptake. Bioavailability studies were performed in Sprague-Dawley rats, which present the area under the curve of scutellarin of Chit-DC-VB12-Scu was two to threefolds greater than that of free scutellarin alone. Further to assess the therapeutic efficacy of diabetic retinopathy, we showed Chit-DC-VB12-Scu down-regulated central retinal artery resistivity index and the expression of angiogenesis proteins (VEGF, VEGFR2, and vWF) of retinas in type II diabetic rats. CONCLUSIONS: Chit-DC-VB12 nanoparticles loaded with scutellarin have better bioavailability and cellular uptake efficiency than Scu, while Chit-DC-VB12-Scu nanoparticles alleviated the structural disorder of intraretinal neovessels in the retina induced by diabetes, and it also inhibited the retinal neovascularization via down-regulated the expression of angiogenesis proteins. In conclusion, the Chit-DC-VB12 nanoparticles enhanced scutellarin oral delivery efficacy and exhibited potential as small intestinal target promising nano-carriers for treatment of type II diabetes induced-retinopathy.

Acetylshikonin from Zicao exerts antifertility effects at high dose in rats by suppressing the secretion of GTH.

Zicao is being highlighted as a promising Chinese medicine due to all the beneficial effects that have been associated with its use. Unfortunately, studies on the toxicity of Zicao in different species are still missing and should be carried out. In this study, we investigated whether Acetylshikonin (AS) from Zicao has an anti-fertility effect through mating experiments and explored its underling mechanism. Sprague-Dawley rats received no treatment or were treated with 120, 360 or 1080 mg/kg AS extract by intragastric administration for 2 weeks. The rat pregnancy rate of the 1080 mg/kg dose group was significantly decreased relative to control group, while it recovered after a month of drug withdrawal, which indicated that the effect of antifertility is reversible. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in rat were significantly decreased by AS. The secretion of FSH in rat anterior pituitary cells was decreased but the synthesis was not affected. AS reduced the number of developing follicle and mature follicle in rat ovarian cortical. Maybe all of these resulted from AS decreased the expression of synaptotagmin-1 and SNAP-25 which were the critical proteins of exocytosis. Our data suggested that AS at high dose can suppress the ability of pregnancy of the rats through decreasing serum FSH and LH levels by affecting exocytosis process of gonadotropic hormone (GTH).

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.

[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].

Scutellarin inhibits translocation of protein kinase C in diabetic thoracic aorta of the rat.

The aims of the present study were to explore the effects of: (i) scutellarin (Scu) on protein kinase C (PKC) translocation caused by diabetic conditions in diabetic rat thoracic aorta; and (ii) phorbol-12-myristate-13-acetate (PMA) treatment of cultured thoracic aortic smooth muscle cells. Diabetes was induced in rats by streptozotocin and diabetic rats were divided into two groups: (i) an Scu-treated group, administered 0.1 g/kg Scu by gavage; and (ii) an aminoquanidine (AG)-treated group, which received dietary supplementation of 0.1% AG from Week 1 of diabetes induction. After 10 weeks, rats were killed and thoracic aortic smooth muscle cells were isolated and cultured. Cell fractions were obtained by ultracentrifugation and PKC activity was assayed by ELISA, whereas the distribution of PKC was verified by western immunoblotting. The PKC activity in the membrane fraction of thoracic aortic smooth muscle cells was significantly increased in diabetic compared with control rats, whereas the administration of Scu significantly inhibited this increase. Phorbol myristate acetate (100 nmol/L, 10 min) induced the translocation of the PKCα, ßI, ßII, δ and ε isoforms, whereas 48 h pretreatment of cells with 1 µmol/L Scu significantly inhibited PMA-induced PKCßI, ßII and δ translocation. The results of the present study suggest that Scu inhibits the translocation of PKC in vivo and in vitro and may have value as a drug in the treatment of diabetic complications via its inhibition of PKC ßI, ßII and δ translocation.

Acetylshikonin stimulates glucose uptake in L6 myotubes via a PLC-ß3/PKCδ-dependent pathway.

Acetylshikonin, a naphthoquinone derivative derived from Lithospermum erythrorhizon, has been shown to have various pharmacological activities; however, its effect on diabetes has rarely been reported. We investigated the hypoglycemic effect of acetylshikonin and found that it decreased blood glucose to a greater extent than insulin and improved glucose tolerance in mice. It also increased glucose uptake in L6 myotubes by inducing the expression and translocation of glucose transporter 4 via decomposition of phosphatidylinositol, increased generation of diacylglycerol, and activation of protein kinase C delta cascades; this is an insulin-, reactive oxygen species-, and AMP-activated protein kinase-independent pathway for glucose uptake. Our findings highlight the antidiabetic potential of acetylshikonin via a possible novel pathway for glucose uptake in L6 myotubes.

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.

Abnormalities of sarcoplasmic reticulum Ca2+ mobilization in aortic smooth muscle cells from streptozotocin-induced diabetic rats.

1. Previously, we found that contractions in response to receptor-dependent (i.e. a(1)-adrenoceptor agonist phenylephrine) and -independent (i.e. cyclopiazonic acid) stimuli are decreased in rat aorta during late diabetes. The aim of the present study was to further investigate the changes of intracellular Ca(2+) homeostasis in diabetic aortic smooth muscle cells. Functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores of the sarcoplasmic reticulum (SR) were evaluated using Fluo-3 acetoxymethyl ester fluorescence, western blot and organ bath techniques. 2. In aortic smooth muscle cells from diabetic rats, the Ca(2+) release and Ca(2+) influx caused by both 10 mmol/L phenylephrine (depletion of IP(3)-sensitive Ca(2+) stores) and 1 mmol/L ryanodine (depletion of ryanodine-sensitive Ca(2+) stores) were both significantly decreased compared with control. Moreover, protein expression levels of IP(3) (260 kDa) and ryanodine receptors (500 kDa) were reduced by 31.8 +/- 7.7 and 69.2 +/- 8.4%, respectively, in aortas from diabetic rats compared with those from control rats. 3. In diabetic rat aorta, phenylephrine-induced contractility was decreased to approximately two-thirds of that in controls, whereas ryanodine alone did not cause obvious contraction in aortas from either control or diabetic rats. 4. The present results suggest that the hyporeactivity of aortic smooth muscle to vasoconstrictors in diabetes results mainly from changes to the IP(3)-sensitive Ca(2+) release pathway. The SR Ca(2+) signalling pathway plays a crucial role in the development of diabetic vascular complications.

Acetylshikonin from Zicao attenuates cognitive impairment and hippocampus senescence in d-galactose-induced aging mouse model via upregulating the expression of SIRT1.

Zicao acts as a pleiotropic medicine in various diseases due to its particular pharmacological properties, including anti-inflammatory, anti-tumor, anti-oxidative, and wound healing effects. However, few studies have focused on the function in neurodegenerative diseases of Zicao. In this study, we investigated the neuroprotective effect of Acetylshikonin (AS) from Zicao on the hippocampus of the d-galactose (d-gal)-induced sub-acute aging mouse model of Alzheimer's disease (AD). The aging model was established in male Kunming mice by subcutaneous injection of d-gal (150 mg/kg/d) for 60 days, and the mice were given AS (270, 540 and 1080 mg/kg/d) or distilled water intragastrically for 30 days after 30 days of d-gal injection. The behavioral results test by Morris Water Maze (MWM) revealed that chronic AS treatment alleviated d-gal-induced learning and memory deficits compared with the d-gal-treated mice. In addition, AS also ameliorated the oxidative stress and neuroinflammation induced by d-gal through decreasing the level of interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), malondialdehyde (MDA) and enhancing the activity of the antioxidant enzymes superoxide dismutase (SOD). Moreover, western blot results showed that AS can up-regulate the expression of Sirtuin 1 (SIRT1) and inhibit d-gal-induced activation of p53/p21 signaling pathway in the hippocampus of mice. These results suggest that AS can execute the prevention and treatment of d-gal-induced brain aging by SIRT1/P53/P21 pathway.

Acetylshikonin from Zicao Prevents Obesity in Rats on a High-Fat Diet by Inhibiting Lipid Accumulation and Inducing Lipolysis.

Various drugs have been developed to treat obesity, but these have undesirable secondary effects, and an efficient but non-toxic anti-obesity drug from natural sources is desired. This study investigated the anti-obesity effects and mechanisms of action of acetylshikonin (AS)-which is used in traditional Chinese medicine-in rats on a high-fat diet (HFD). Rats were fed a normal diet or an HFD; the latter group was received no treatment or were treated with 100, 300, or 900 mg/kg AS extract by intragastric administration for 6 weeks. In addition, 3T3-L1 adipocytes were treated with AS and the effects on adipogenesis and lipolysis were evaluated by western blot analysis of adipogenic transcription factors and lipid-metabolizing enzyme levels and the phosphorylation status of protein kinase (PK) A and hormone-sensitive lipase (HSL). AS prevented HFD-induced obesity including reduction in body weight, white adipose tissue content, liver mass, and serum triglyceride and free fatty acid levels in rats. It also suppressed the expression of adipogenic differentiation transcription factors and decreased the expression of the adipocyte-specific proteins HSL and adipose triglyceride lipase (ATGL). Furthermore, AS treatment induced lipolysis, leading to the release of glycerol and increased in PKA and HSL phosphorylation. These findings demonstrate that AS has anti-obesity effects in a rat model and may be a safe treatment for obesity in humans.

Effect of Salvia miltiorrhiza and ligustrazine injection on myocardial ischemia/reperfusion and hypoxia/reoxygenation injury.

Salvia miltiorrhiza and ligustrazine are traditional Chinese medicines that have been used in combination for treatment of cardiovascular disease, including coronary heart disease, cardiac angina and atherosclerosis in Asia, in particular, China. The present study aimed to determine the effect of S. miltiorrhiza and ligustrazine injection (SLI) on myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R) injuries via the Akt serine/threonine kinase (Akt)­endothelial nitric oxide synthase (eNOS) signaling pathway. Male Sprague­Dawley rats were randomly assigned into six groups: i) Sham group; ii) I/R group; iii) Low­SLI group (6.8 mg/kg/day, i.p.); iv) Medium­SLI group (20.4 mg/kg/day, i.p.); v) High­SLI group (61.2 mg/kg/day, i.p.); vi) verapamil group (6 mg/kg/day, i.p.). Prior to surgery, the aforementioned groups were pretreated with a homologous drug once per day for 3 days. The effect of SLI following 35 min coronary artery occlusion and 2 h reperfusion was evaluated by determining infarct size, hemodynamics, biochemical values and histological observations. Additionally, cell viability, caspase­3 expression, B cell leukemia/lymphoma­2 (Bcl­2)/Bcl­2­associated X protein (Bax) ratio, phosphorylated (p­)Akt and p­eNOS were also investigated following 2 h simulated ischemia and 2 h simulated reperfusion in H9C2 cardiomyocyte cells. Pretreatment with SLI significantly improved cardiac function in a dose­dependent manner and reduced myocardial infarct size, creatine kinase, lactate dehydrogenase and malondialdehyde levels in blood serum. Additionally, myocardial histopathology changes in the rat model were also alleviated in SLI treatment groups. The present in vitro study revealed that treatment with SLI reduced the apoptotic rate of H9C2 cells by inhibiting the activation of caspase­3 and increasing the Bcl­2/Bax ratio. The effect of SLI was associated with increased phosphorylation of the survival kinase Akt at Ser473 and its downstream target eNOS following H/R. The present study determined that SLI may alleviate I/R injury in cardiomyocytes and inhibit apoptosis in rats by the activation of the Akt­eNOS signaling pathway, and downregulation of the expression levels of proapoptotic factors, including caspase-3.

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.

Ginsenoside Rb1 protects rat retinal ganglion cells against hypoxia and oxidative stress.

The current study was designed to investigate the effect of ginsenoside Rb1 (Rb1) on apoptosis induced by hypoxia and oxidative stress in a retinal ganglion cell line (RGC-5). The underlying mechanism was also investigated. RGC-5 cells were pretreated with 10 µmol/l Rb1 for 24 h and exposed to 400 µmol/l cobalt chloride (CoCl2) for 48 h or 600 µmol/l H2O2 for 24 h. The percentage of cells actively undergoing apoptosis was determined by flow cytometry with Annexin V/propidium iodide (PI) double staining. The expression of caspases was determined using western blot analysis. CoCl2 and H2O2 treatments significantly increased the apoptotic percentages to 24.5 and 21.63%, respectively. Pretreatment of Rb1 reduced the total apoptotic percentages to 15.12 and 12.03%, respectively. The expression of cleaved caspase-3, -9 and -8 was increased in the CoCl2-treated group, however, caspase-3 was not increased in the H2O2-treated group. Pretreatment of Rb1 reduced the expression of cleaved caspase-3 and -9 in the CoCl2-treated group, but reduced only cleaved caspase-9 in the H2O2-treated group. These results suggest that Rb1 may prevent RGC-5 cells from apoptosis against hypoxia and oxidative stress via the mitochondrial pathway.

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.

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.

[Study on relationship between leukocytes and early diabetic retinopathy].

PURPOSE: To explore the leukocytic functions in adhesion, stacking and causing microcirculatory disorder in retinal microvessels in early diabetic rats. METHODS: Sixteen healthy adult male Wistar rats were randomly divided into normal control group and 3 months group of diabetes (STZ) . We carried out morphological observations and CD45 (Leukocyte Common Antigen) monoclonal antibody immunohistochemical studies of the retinal digest preparations. RESULTS: Morphological changes including capillary varix, irregularity of capillary caliber, was found in the retinal blood capillaries in diabetic rats at 3 months. Further more, the expression of CD45 was significantly increased in the diabetic retinal microvessels, which were found leukocytes adhering and stacking. CONCLUSION: Changes of retinal microvessels can be found in STZ diabetic rats at 3 months. The leukocytes were important in the onset and development of diabetic retinopathy (DR).