Osthole inhibits GSK-3ß/AMPK/mTOR pathway-controlled glycolysis and increases radiosensitivity of subcutaneous transplanted hepatocellular carcinoma in nude mice.

PURPOSE: Osthole possesses anti-tumor activities. However, whether osthole can have a radiosensitization effect on hepatic cancer remains unclear. Here, an HCC-LM3 cells-inoculated subcutaneous transplanted tumor was adopted to explore the effect of osthole. METHODS: The tumor-bearing mice were treated with 100 mg/kg osthole for 12 days, 4 Gy irradiation twice, or their combination. The tumor volume and weight, lactic acid content, glycolytic enzyme activities, and protein expression of glycogen synthase kinase 3ß (GSK-3ß), p­GSK-3ß, mammalian target of rapamycin (mTOR), p­mTOR, AMP-activated protein kinase (AMPK), p­AMPK, glucose transporter 1/3, and pyruvate kinase M2 were determined. The GSK-3ß-overexpressed HCC-LM3 or SK-Hep­1 cell models were also adopted to verify the effects of osthole on expression of these proteins. RESULTS: The tumor volume and weight, lactic acid content, and glycolytic enzyme activities in tumor tissues were lower in the osthole + radiation group than in the radiation group. Moreover, osthole could reverse the radiation-induced increments of p­GSK-3ß/GSK-3ß and p­mTOR/mTOR protein ratios and the expression of glucose transporter 1/3 and pyruvate kinase M2 proteins in tumor tissues, and increase the protein ratio of p­AMPK/AMPK. The effects of osthole on these glycolysis-related proteins were also observed in GSK-3ß-overexpressed HCC-LM3 or SK-Hep­1 cell models. CONCLUSION: Osthole has a radiosensitizing effect on subcutaneous transplanted hepatocellular carcinoma, and its mechanism may be related to inhibition of GSK-3ß/AMPK/mTOR pathway-controlled glycolysis.

Osthole increases the radiosensitivity of hepatoma cells by inhibiting GSK-3ß/AMPK/mTOR pathway-controlled glycolysis.

Osthole is a natural coumarin substance that has an inhibitory effect on hepatic cancer, but its radiosensitization effect on hepatoma cells has not been reported. This study aimed to investigate the effect of osthole. Human HCC-LM3 and SK-Hep-1 hepatoma cells were used and treated with or without osthole, irradiation, or their combination; the cell survival, migration, colony formation, DNA damage repair, intracellular lactic acid content, and glycolysis-related glycogen synthase kinase-3ß (GSK-3ß), p-GSK-3ß, AMP-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), p-mTOR, glucose transporter-1 (GLUT-1), GLUT-3, and pyruvate kinase isozyme type M2 (PKM2) protein expressions were determined. Compared with the irradiation group, the osthole plus irradiation group could further decrease the survival rate, migration, colony formation, and DNA damage repair of both hepatoma cells, indicating a synergistic effect of the combination treatment. Moreover, the combination of osthole and irradiation could decrease the content of intracellular lactic acid, ratios of intracellular p-GSK-3ß/GSK-3ß and p-mTOR/mTOR proteins, and expressions of intracellular GLUT-1/3 and PKM2 proteins, and increase the ratio of intracellular p-AMPK/AMPK proteins. Osthole can increase the radiosensitivity of hepatoma cells, and its radiosensitization mechanisms may be related to glycolytic inhibition by attenuating the GSK-3ß/AMPK/mTOR pathway.

Osthole Increases the Sensitivity of Liver Cancer to Sorafenib by Inhibiting Cholesterol Metabolism.

Osthole is a natural product that has an inhibitory effect on liver cancer, but its effect on the sensitivity of liver cancer to sorafenib is poorly understood. Here, we investigated the effect of osthole and possible sensitization mechanisms. Our results showed that the combination of 2.5 µM sorafenib and 10 µM osthole had significantly synergistic inhibitory effects on proliferation, colony formation, and migration of HCCLM3, sorafenib-resistant HCCLM3 (HCCLM3-SR), and SK-Hep-1 cells. After treatment of HCCLM3 cells-inoculated subcutaneous xenotransplanted tumor mice with 100 mg/kg osthole, 70 mg/kg sorafenib or their combination for 24 day, the tumor volume, tumor weight, and tumor weight coefficient were significantly lower in the osthole + sorafenib group than in the sorafenib group. Compared with the control group, the total cholesterol and low density lipoprotein-cholesterol contents in serum and tumor tissue were significantly decreased in the osthole or osthole + sorafenib groups, the sterol regulatory element binding protein (SREBP)-2c, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and low-density lipoprotein receptor (LDLR) protein expressions in tumor tissue were significantly downregulated as well. In conclusion, osthole can increase the sensitivity of liver cancer to sorafenib, and the mechanism is related to the downregulations of SREBP-2c, HMGCR, and LDLR protein expressions and subsequent inhibition of cholesterol metabolism.

Stevioside inhibits lipopolysaccharide-induced epithelial-to-mesenchymal transition of NRK-52E cells by PPARγ activation.

BACKGROUND: Stevioside is a natural diterpenoid compound that possesses anti-inflammatory, immunomodulatory, anti-diabetic, anti-hypertensive, and renal protective effects, but its effect on lipopolysaccharide (LPS)-induced epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells, an important immune pathological mechanism of renal fibrosis, remains unknown. This study employed the renal proximal tubular cells NRK-52E to investigate the effect of stevioside. METHODS: The LPS-stimulated renal NRK-52E cells were treated with 50, 100, or 200 µM stevioside in the presence or absence of peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9662, the expression levels of intracellular E-cadherin, vimentin, α-smooth muscle actin (α-SMA), PPARγ, nuclear factor kappa B (NF-κB) p65, transforming growth factor-ß1 (TGF-ß1), signal transducer and activator of transcription 3 (STAT3), p-STAT3, Smad2/3, and p-Smad2/3 proteins were detected by Western blot analysis. RESULTS: In LPS-stimulated NRK-52E cells, stevioside treatment could reverse the expressions of EMT-related E-cadherin, vimentin, and α-SMA proteins, increase the expression of PPARγ protein, and decrease the expressions of NF-κB p65, TGF-ß1, p-STAT3, Smad2/3, and p-Smad2/3 proteins, especially in the 200 µM stevioside-treated group. However, these beneficial effects of stevioside were attenuated or canceled by pretreatment with PPARγ antagonist GW9662. CONCLUSIONS: Stevioside can inhibit the LPS-induced EMT via the reductions of NF-κB, TGF-ß1, Smad2/3, p-Smad2/3, and p-STAT3 protein expressions by PPARγ activation in NRK-52E cells, which may provide a pharmacological basis for the potential application of stevioside in the prevention and treatment of renal fibrosis.

Apigenin increases radiosensitivity of glioma stem cells by attenuating HIF-1α-mediated glycolysis.

Apigenin, a natural flavonoid compound present in a variety of edible plants and health foods, has an anti-tumor effect and inhibits hypoxia inducible factor-lα (HIF-1α) expression in hypertrophic cardiac tissues. However, whether or not apigenin has a radiosensitization effect on glioma stem cells (GSCs) is unknown. Our present study aimed to investigate the effect of apigenin and its possible mechanisms. The human GSCs SU3 and its radioresistance line SU3-5R were treated with apigenin, radiation, or their combination, and the cell proliferation, migration, colony formation, and intracellular lactic acid and glycolytic related protein expressions were determined. Additionally, a cell model with hypoxia-induced HIF-1α expression was used and treated with apigenin. The current results displayed that the combination of apigenin and radiation could synergically reduce the viability, colony formation, and migration of the both GSCs. Moreover, this combination could also decrease the radiation-induced increments of glycolytic production lactic acid in the both GSCs and related protein expressions, including HIF-1α, glucose transporter (GLUT)-1/3, nuclear factor kappa B (NF-κB) p65, and pyruvate kinase isozyme type M2 (PKM2). Further study confirmed that after treatment of hypoxia-cultured SU3 or SU3-5R cells with apigenin, the expression levels of HIF-1α, GLUT-1/3, NF-κB p65, and PKM2 proteins were reduced. These results demonstrated that apigenin could increase the radiosensitivity of GSCs and its radiosensitization mechanisms were attributable to the attenuation of glycolysis, which might result from the inhibition of HIF-1α expression and subsequent reductions of GLUT-1/3, NF-κB, and PKM2 expressions.

Apigenin attenuates TGF-ß1-stimulated cardiac fibroblast differentiation and extracellular matrix production by targeting miR-155-5p/c-Ski/Smad pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Apigenin is a natural flavonoid compound present in chamomile (Matricaia chamomilla L.) from the Asteraceae family, which is used in the treatment of cardiovascular diseases by traditional healers, but its effects on differentiation and extracellular matrix (ECM) production of cardiac fibroblasts (CFs) induced by transforming growth factor beta 1 (TGF-ß1) are poorly understood. AIM OF THE STUDY: This study aimed to examine these effects and potential molecular mechanisms and to provide a new application of apigenin in the prevention and treatment of cardiac fibrosis. MATERIALS AND METHODS: The TGF-ß1-stimulated CFs or the combination of TGF-ß1-stimulated and microRNA-155-5p (miR-155-5p) inhibitor- or mimic-transfected CFs were treated with or without apigenin. The expression levels of intracellular related mRNA and proteins were detected by real-time polymerase chain reaction and Western blot methods, respectively. The luciferase reporter gene containing cellular Sloan-Kettering Institute (c-Ski) wild or mutant type 3'-UTR was used and the luciferase activity was examined to verify the direct link of miR-155-5p and c-Ski. RESULTS: After treatment of TGF-ß1-stimulated CFs with 6-24 µM apigenin, the expression of c-Ski was increased, while levels of miR-155-5p, α-smooth muscle actin, collagen Ⅰ/Ⅲ, Smad2/3, and p-Smad2/3 were decreased. After transfection of CFs with the miR-155-5p inhibitor or mimic, the similar or inverse results were respectively observed as well. The combination of TGF-ß1 and miR-155-5p inhibitor or mimic might cause an antagonistical or synergistic effect, respectively, and apigenin addition could enhance the effects of the inhibitor and antagonize the effects of the mimic. Luciferase reporter gene assay demonstrated that c-Ski was a direct target of miR-155-5p. CONCLUSION: These findings suggested that apigenin could inhibit the differentiation and ECM production in TGF-ß1-stimulated CFs, and its mechanisms might partly be attributable to the reduction of miR-155-5p expression and subsequent increment of c-Ski expression, which might result in the inhibition of Smad2/3 and p-Smad2/3 expressions.

Protective effect of apigenin on d-galactosamine/LPS-induced hepatocellular injury by increment of Nrf-2 nucleus translocation.

Apigenin has a protective effect on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced mouse liver injury through the increments of hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) and peroxisome proliferator-activated receptor γ (PPARγ) expressions, but its exact mechanisms are still uncertain. This study aimed to further verify its protective effect on hepatocytes and to determine its target of action. The results showed that after treatment of D-GalN/LPS-stimulated hepatocytes with 2.5-20 µM apigenin, the supernatant alanine aminotransferase, aspartate aminotransferasein, tumor necrosis factor-α, and malondialdehyde levels and intracellular nuclear factor-κB protein expression were decreased, while the supernatant superoxide dismutase (SOD) and catalase (CAT) levels, intracellular PPARγ and inhibitor of kappa B-alpha protein expressions, and nucleus Nrf-2 protein expression were increased. After pretreatment with BML-111 or GW9662, the apigenin-induced nucleus Nrf-2 or intracellular PPARγ protein expressions were completely inhibited, respectively, but the both pretreatment differently affected the protective effect of apigenin on hepatocytes. The former completely canceled the protective effect, whereas the latter did not. These findings further demonstrate that apigenin can exert a protective effect on D-GalN/LPS-induced hepatocellular injury via the increment of Nrf-2 nucleus translocation, which may increase the SOD and CAT levels and PPARγ protein expression and subsequently inhibit the inflammatory response.

Apigenin-induced HIF-1α inhibitory effect improves abnormal glucolipid metabolism in Angâ ¡/hypoxia-stimulated or HIF-1α-overexpressed H9c2 cells.

BACKGROUND: Apigenin, a natural flavonoid compound, can improve the myocardial abnormal glucolipid metabolism and down-regulate the myocardial hypoxia inducible factor-1α (HIF-1α) in hypertensive cardiac hypertrophic rats. However, whether or not the ameliorative effect of glucolipid metabolism is from the reduction of HIF-1α expression remains uncertain. PURPOSE: This study aimed to investigate the exact relationship between them in angiotensin Ⅱ (Ang Ⅱ)/hypoxia-stimulated or HIF-1α overexpressed H9c2 cells. METHODS: Two cell models with Ang Ⅱ/hypoxia-induced hypertrophy and HIF-1α overexpression were established. After treatment of the cells with different concentrations of apigenin, the levels of total protein, free fatty acids (FFA), and glucose were detected by the colorimetric method, the level of atrial natriuretic peptide (ANP) was detected by the ELISA method, and the expressions of HIF-1α, peroxisome proliferator-activated receptor α/γ (PPARα/γ), carnitine palmitoyltmnsferase-1 (CPT-1), pyruvate dehydrogenase kinase-4 (PDK-4), glycerol-3-phosphate acyltransferase genes (GPAT), and glucose transporter-4 (GLUT-4) proteins were detected by the Western blot assay. RESULTS: Following treatment of the both model cells with apigenin 1-10 µM for 24 h, the levels of intracellular total protein, ANP, and FFA were decreased, while the level of cultured supernatant glucose was increased. Importantly, apigenin treatment could inhibit the expressions of HIF-1α, PPARγ, GPAT, and GLUT-4 proteins, and increase the expressions of PPARα, CPT-1, and PDK-4 proteins. CONCLUSION: Apigenin could exert an ameliorative effect on abnormal glucolipid metabolism in AngⅡ/hypoxia-stimulated or HIF-1α-overexpressed H9c2 cells, and its mechanisms were associated with the inhibition of HIF-1α expression and subsequent upregulation of PPARα-mediated CPT-1 and PDK-4 expressions and downregulation of PPARγ-mediated GPAT and GLUT-4 expressions.

Stevioside attenuates isoproterenol-induced mouse myocardial fibrosis through inhibition of the myocardial NF-κB/TGF-ß1/Smad signaling pathway.

Stevioside, a natural glycoside compound, has many beneficial biological activities, but its protective effect on myocardial fibrosis has not been reported yet. This study aimed to investigate the effect of stevioside. The isoproterenol-induced model mice were orally given stevioside 75-300 mg kg-1 for 40 days. The results showed that after the administration of stevioside, the myocardial hydroxyproline, collagen accumulation, and protein expressions of collagen I/III, α-smooth muscle actin, transforming growth factor-ß1 (TGF-ß1), nuclear factor kappa B p65 (NF-κB p65), Smad2/3, and P-Smad2/3 were decreased, while the glutathione peroxidase and superoxide dismutase levels in serum and myocardial tissues and protein expressions of myocardial peroxisome proliferator-activated receptor γ (PPARγ) and Smad7 were increased. After the preincubation of isoproterenol-stimulated cardiac fibroblasts with the PPARγ antagonist GW9662, stevioside-reduced protein expressions were decreased, but stevioside-induced Smad7 was not affected. These findings demonstrated that stevioside could exert a protective effect on mouse myocardial fibrosis, and its mechanisms were associated with the increments of antioxidant ability, PPARγ activation, and Smad7 expression, which caused a synergistic inhibition of the NF-κB/TGF-ß1/Smad signaling pathway.

Apigenin inhibits d-galactosamine/LPS-induced liver injury through upregulation of hepatic Nrf-2 and PPARγ expressions in mice.

Apigenin is a natural flavonoid compound widely distributed in a variety of vegetables, medicinal plants and health foods. This study aimed to examine the protective effect of apigenin against d-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced mouse liver injury and to investigate the potential biochemical mechanisms. The results showed that after oral administration of apigenin 100-200 mg/kg for 7 days, the levels of serum alanine aminotransferase and aspartate aminotransferase were decreased, and the severity of liver injury was alleviated. Importantly, apigenin pretreatment increased the levels of hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) and peroxisome proliferator-activated receptor γ (PPARγ) protein expressions as well as superoxide dismutase, catalase, glutathione S-transferase and glutathione reductase activities, decreased the levels of hepatic nuclear factor-κB (NF-κB) protein expression and tumor necrosis factor-α. These findings demonstrated that apigenin could prevent the D-GalN/LPS-induced liver injury in mice, and its mechanisms might be associated with the increments of Nrf-2-mediated antioxidative enzymes and modulation of PPARγ/NF-κB-mediated inflammation.

Apigenin protects against alcohol-induced liver injury in mice by regulating hepatic CYP2E1-mediated oxidative stress and PPARα-mediated lipogenic gene expression.

Alcohol is a major cause of liver injury, and there are currently no ideal pharmacological reagents that can prevent or reverse this disease. Apigenin is one of the most common flavonoids present in numerous plants and has many beneficial effects. But whether or not apigenin may protect against alcohol-induced liver injury remains unknown. Our aim was to examine the effect and potential mechanisms. The experimental mice were given 56% erguotou wine or simultaneously given apigenin 150-300 mg/kg by gavage for 30 days. The results showed that in the apigenin-treated mice, the expression of hepatic cytochrome P450 2E1 (CYP2E1) and nuclear factor kappa B proteins as well as contents of hepatic malondialdehyde and tumor necrosis factor-alpha were reduced, while the levels of hepatic reduced glutathione, glutathione reductase, glutathione peroxidase, and glutathione S-transferase were increased, especially in the 300 mg/kg group. A significant change in hepatic steatosis was also observed in the apigenin 300 mg/kg group. Apigenin pretreatment could increase the expression of hepatic peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase-1 proteins, and decrease the expression of hepatic sterol regulatory element binding protein-1c, fatty acid synthase, and diacylglycerol acyltransferase proteins. These findings demonstrated that apigenin might exert a protective effect on alcohol-induced liver injury, and its mechanisms might be related to the regulations of hepatic CYP2E1-mediated oxidative stress and PPARα-mediated lipogenic gene expression.

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway after treatment with TGF-ß1 in mouse cardiac fibroblasts.

BACKGROUND: Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-ß1 (TGF-ß1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-ß1. METHODS: The mouse CFs stimulated with TGF-ß1 were cultured and treated with osthole 1.25-5µg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-ß receptor I (TßRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively. RESULTS: After treatment with TGF-ß1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TßRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole. CONCLUSION: The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-ß1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms.

Chrysanthemum morifolium extract improves hypertension-induced cardiac hypertrophy in rats by reduction of blood pressure and inhibition of myocardial hypoxia inducible factor-1alpha expression.

CONTEXT: Chrysanthemum morifolium Ramat. (Asteraceae) extract (CME) possesses a vasodilator effect in vitro. However, the use of polyphenol-rich CME in the treatment of hypertension-induced cardiac hypertrophy has not been reported. OBJECTIVE: We investigated the effect of polyphenol-rich CME on hypertension-induced cardiac hypertrophy in rats and its possible mechanism of action. MATERIALS AND METHODS: The Sprague-Dawley rat model with cardiac hypertrophy was induced by renovascular hypertension. The blood pressure, cardiac weight index, free fatty acids (FFA) in serum and myocardium, and protein expressions of myocardial hypoxia inducible factor-1α (HIF-1α), peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1a (CPT-1a), pyruvate dehydrogenase kinase-4 (PDK-4) and glucose transporter-4 (GLUT-4) were measured after treating hypertensive rats with polyphenol-rich CME of anthodia 75-150 mg/kg once daily for 4 weeks. A myocardial histological examination was also conducted. RESULTS: After CME treatment, the blood pressure, cardiac weight and cardiac weight index decreased by 5.7-9.6%, 9.2-18.4% and 10.9-20.1%, respectively, and the cardiomyocyte cross-sectional area also decreased by 8.3-30.4%. The CME treatment simultaneously decreased the FFA in serum and myocardium and protein expressions of myocardial HIF-1α and GLUT-4, and increased the protein expressions of myocardial PPARα, CPT-1a and PDK-4, especially in the CME 150 mg/kg group (p < 0.05 or p < 0.01). DISCUSSION AND CONCLUSION: Polyphenol-rich CME may alleviate hypertensive cardiac hypertrophy in rats. Its mechanisms may be related to the reduction of blood pressure and amelioration of the myocardial energy metabolism. The latter may be attributed to the inhibition of HIF-1α expression and subsequent modulation of PPARα-mediated CPT-1a, PDK-4 and GLUT-4 expressions.

Apigenin ameliorates hypertension-induced cardiac hypertrophy and down-regulates cardiac hypoxia inducible factor-lα in rats.

Apigenin is a natural flavonoid compound that can inhibit hypoxia-inducible factor (HIF)-1α expression in cultured tumor cells under hypoxic conditions. Hypertension-induced cardiac hypertrophy is always accompanied by abnormal myocardial glucolipid metabolism due to an increase of HIF-1α. However, whether or not apigenin may ameliorate the cardiac hypertrophy and abnormal myocardial glucolipid metabolism remains unknown. This study aimed to examine the effects of apigenin. Rats with cardiac hypertrophy induced by renovascular hypertension were treated with apigenin 50-100 mg kg(-1) (the doses can be achieved by pharmacological or dietary supplementation for an adult person) by gavage for 4 weeks. The results showed that after treatment with apigenin, the blood pressure, heart weight, heart weight index, cardiomyocyte cross-sectional area, serum angiotensin II, and serum and myocardial free fatty acids were reduced. It is important to note that apigenin decreased the expression level of myocardial HIF-1α protein. Moreover, apigenin simultaneously increased the expression levels of myocardial peroxisome proliferator-activated receptor (PPAR) α, carnitine palmitoyltransferase (CPT)-1, and pyruvate dehydrogenase kinase (PDK)-4 proteins and decreased the expression levels of myocardial PPARγ, glycerol-3-phosphate acyltransferase genes (GPAT), and glucose transporter (GLUT)-4 proteins. These findings demonstrated that apigenin could improve hypertensive cardiac hypertrophy and abnormal myocardial glucolipid metabolism in rats, and its mechanisms might be associated with the down-regulation of myocardial HIF-1α expression and, subsequently increasing the expressions of myocardial PPARα and its target genes CPT-1 and PDK-4, and decreasing the expressions of myocardial PPARγ and its target genes GPAT and GLUT-4.

Osthole improves glucose and lipid metabolism via modulation of PPARα/γ-mediated target gene expression in liver, adipose tissue, and skeletal muscle in fatty liver rats.

CONTEXT: Osthole may be a dual agonist of peroxisome proliferator-activated receptors (PPAR) α/γ and ameliorate the insulin resistance (IR), but its mechanisms are not yet understood completely. OBJECTIVE: We investigated the effects of osthole on PPARα/γ-mediated target genes involved in glucose and lipid metabolism in liver, adipose tissue, and skeletal muscle in fatty liver and IR rats. MATERIALS AND METHODS: The rat model was established by orally feeding high-fat and high-sucrose emulsion for 9 weeks. The experimental rats were treated with osthole 5-10 mg/kg by gavage after feeding the emulsion for 6 weeks, and were sacrificed 4 weeks after administration. RESULTS: After treatment with osthole 5-10 mg/kg for 4 weeks, the lipid levels in serum and liver were decreased by 37.9-67.2% and 31.4-38.5% for triglyceride, 33.1-47.5% and 28.5-31.2% for free fatty acid, respectively, the fasting blood glucose, fasting serum insulin, and homeostasis model assessment of IR were also decreased by 17.2-22.7%, 25.9-26.7%, and 37.5-42.8%, respectively. Osthole treatment might simultaneously decrease the sterol regulatory element binding protein-1c, diacylglycerol acyltransferase, and fatty acid synthase mRNA expressions in liver and adipose tissue, and increase the carnitine palmitoyltransferase-1A mRNA expression in liver and glucose transporter-4 mRNA expression in skeletal muscle, especially in the osthole 10 mg/kg group (p < 0.01). DISCUSSION AND CONCLUSION: Osthole can improve glucose and lipid metabolism in fatty liver and IR rats, and its mechanisms may be associated with synergic modulation of PPARα/γ-mediated target genes involved in glucose and lipid metabolism in liver, adipose tissue, and skeletal muscle.

PPARα/γ agonists and antagonists differently affect hepatic lipid metabolism, oxidative stress and inflammatory cytokine production in steatohepatitic rats.

Peroxisome proliferator-activated receptor (PPAR) α/γ may control lipid metabolism and inflammatory response by regulating the downstream target genes, and play a crucial role in the process of non-alcoholic steatohepatitis (NASH) formation, but the difference and interaction between PPARα and PPARγ are poorly understood. The rat model with NASH was established by orally feeding high-fat and high-sucrose emulsion for 6weeks. The results shown that after the model rats were simultaneously treated with PPARα/γ agonists, the total cholesterol (TC), triglyceride (TG) and inflammatory cytokine levels in serum and hepatic tissue, the hepatic steatosis and inflammatory cellular infiltration were decreased, and were consistent with the results of hepatic lipogenic gene and nuclear factor (NF)-κB protein expressions. Conversely, these indexes were increased by PPARα/γ antagonist treatment. Compared with the model group, the serum free fatty acid (FFA) level was increased in the PPARα agonist-treated group, decreased in the PPARγ agonist-treated group, and unchanged in the PPARα/γ agonists-treated group. The hepatic FFA level was low in the PPARα/γ agonists-treated groups, but no significant variation in the PPARα/γ antagonists-treated groups. The increments of hepatic reduced glutathione (GSH) and superoxide dismutase (SOD) contents in the PPARα/γ agonists-treated groups were accompanied by decreased hepatic malondialdehyde (MDA) content. These findings demonstrated that PPARα/γ activation might decrease the hepatic lipid accumulation, oxidative stress and inflammatory cytokine production, and PPARγ could counterbalance the adverse effect of PPARα on circulating FFA. It was concluded that the integrative application of PPARα and PPARγ agonists might exert a synergic inhibitory effect on NASH formation through the modulation of PPARα/γ-mediated lipogenic and inflammatory gene expressions.

Osthole inhibits inflammatory cytokine release through PPARα/γ-mediated mechanisms in LPS-stimulated 3T3-L1 adipocytes.

CONTEXT: Peroxisome proliferator-activated receptor (PPAR) α/γ may control inflammatory response by regulating the nuclear factor-kappa B (NF-κB). Osthole may be a dual agonist of PPARα/γ, but whether or not osthole may inhibit inflammatory cytokines in cultured 3T3-L1 adipocytes is unclear. OBJECTIVE: We investigated the action of osthole and its potential mechanisms in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes. MATERIALS AND METHODS: The 3T3-L1 adipocytes stimulated with LPS were cultured and treated with different concentrations of osthole. The inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cultured supernatants were detected by the enzyme-linked immunosorbent assay (ELISA) method, and the protein expressions of PPARα/γ and NF-κB p65 in adipocytes were detected by the Western blot method, respectively. RESULTS: Following treatment of adipocytes with osthole 0.1-1.6 µM, the TNF-α and IL-6 levels in cultured supernatants were decreased, and the NF-κB p65 protein expression in adipocytes was also decreased, while the PPARα/γ protein expressions were increased. After pretreatment of adipocytes with specific inhibitor(s) of PPARα and /or PPARγ, the inhibitory effects of osthole on TNF-α and IL-6 were decreased or almost cancelled, and the effects on NF-κB p65 protein expression also exhibited similar variations. CONCLUSION: Osthole could inhibit the TNF-α and IL-6 production in LPS-stimulated adipocytes, and its mechanism might be related to reduction of NF-κB expression via activation of PPARα/γ.

Involvement of hepatic peroxisome proliferator-activated receptor α/γ in the therapeutic effect of osthole on high-fat and high-sucrose-induced steatohepatitis in rats.

Our previous studies have indicated that osthole may be a dual agonist of peroxisome proliferator-activated receptor (PPAR) α/γ and decrease the hepatic lipid accumulation. But there has been no report about therapeutic effect on steatohepatitis. In the present study, we investigated the action of osthole and its potential mechanisms. The rats with steatohepatitis induced by orally feeding high-fat and high-sucrose emulsion were given osthole 5-20 mg/kg for 4 weeks. The results showed that after treatment with osthole, the serum alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceride (TG), and free fatty acid (FFA) levels, the hepatic TG, FFA, tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6, and interleukin-8 contents, and the hepatic weight and liver index were lowered, especially in the osthole 20 mg/kg group. The histological evaluation of liver specimens demonstrated that osthole might improve the hepatic steatosis and inflammation. At the same time, osthole treatment increased the hepatic protein expressions of PPARα/γ and lipoprotein lipase, and decreased the hepatic protein expressions of nuclear factor-κB, sterol regulatory element-binding protein-1c, fatty acid synthase, and diacylglycerol acyltransferase. These findings demonstrate that osthole is effective in treating rat steatohepatitis, and the PPARα/γ may be involved in the osthole-induced modulation of hepatic lipogenic gene expressions and inflammatory cytokine production.

Protective effect of apigenin on mouse acute liver injury induced by acetaminophen is associated with increment of hepatic glutathione reductase activity.

Apigenin, a natural plant flavone, has many beneficial effects, but there is no report about treatment of acetaminophen-induced liver injury. Our aim was to examine the protective effect of apigenin on acetaminophen-induced mouse acute liver injury and to investigate the potential mechanisms. A mouse model with acute liver injury was induced by intraperitoneally given acetaminophen 350 mg kg(-1) after oral administration of apigenin 100 and 200 mg kg(-1) for 7 days. The results showed that after treatment with apigenin, the levels of serum alanine aminotransferase and aspartate aminotransferase were gradually decreased, and the severity of liver injury was decreased. In particular, significant changes in liver necrosis were observed in the apigenin 200 mg kg(-1) group. Apigenin could gradually increase the hepatic glutathione reductase (GR) activity and reduced glutathione (GSH) content, and decrease the hepatic malondialdehyde content, but the activities of glutathione peroxidase and glutathione S-transferase in hepatic tissues between the model group and the apigenin-treated groups were not significantly different. It was concluded that apigenin could protect against acetaminophen-induced acute liver injury in mice, and the mechanisms might be associated with enhancing hepatic GSH content via increment of GR activity.

Inhibitory effects of Qushuanling Capsule () on thrombus formation and platelet aggregation in rats.

OBJECTIVE: To investigate the effects of Qushuanling Capsule ( QSLC) on thrombus formation and platelet aggregation in rats. METHODS: Arteriovenous bypass, venous thrombosis, and middle cerebral artery thrombosis models were used in rats to investigate the anti-thrombotic effects of QSLC, a compound of nine Chinese herbs. The platelet aggregation induced by adenosine diphosphate (ADP), thrombin or arachidonic acid (AA), as well as the contents of thromboxane B(2) (TXB(2)) and 6-keto-prostaglandin F1α (6-keto-PGF1α) in rat plasma and aortic walls, were determined to investigate the possible mechanisms of the anti-thrombotic effects of QSLC. RESULTS: After oral administration with QSLC for 7 days, arteriovenous bypass thrombosis was obviously suppressed compared with the model group, venous thrombosis was also obviously suppressed, rat behaviors were obviously improved, and brain infarct size as well as water content were also reduced. The platelet aggregation induced by ADP or thrombin was inhibited by QSLC, but the drug had no effect on AA-induced platelet aggregation and content of TXB(2) and 6-keto-PGF1α in plasma and the aortic wall. CONCLUSION: These results suggest that QSLC can be used in the prevention and treatment of thrombotic diseases, and that its mechanism of action may be related to inhibition of platelet aggregation.