Mitogen-activated protein kinase inhibitors reduce the nuclear accumulation of phosphorylated Smads by inhibiting Imp 7 or Imp 8 in HepG2 cells.

The transforming growth factor (TGF)-ß/Smad signaling pathway is involved in hepatocellular carcinoma development. Smad2 and Smad3 are phosphorylated following TGF-ß1 stimulation and subsequently oligomerize with Smad4 to form the Smad2/3/4 complex, which translocates into the nucleus and regulates target genes, including plasminogen activator inhibitor type 1 (PAI1). Importin (Imp)7 and Imp8 are responsible for transporting phosphorylated (p)Smad2/3 and Smad4 into the nucleus. In our previous study, it was demonstrated that mitogen-activated protein kinase (MAPK) inhibitors, including inhibitors of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 could inhibit the transcription of PAI1, but ERK inhibitor had no significant effect on the phosphorylation of Smad2/3, and the formation of Smad2/3/4 complexes, which was different from the effect of JNK or p38 inhibitor. We hypothesized that MAPK inhibitors, particularly ERK inhibitor, reduced the transport of Smads into the nucleus by affecting Imp7 and Imp8. To confirm this hypothesis, HepG2 cells were incubated with different MAPK inhibitors for 5 h and subsequently stimulated with TGF-ß1 for 1 h. Next, the intracellular locations of Smads (pSmad2C, pSmad2L, pSmad3C, pSmad3L and Smad4) and Imp7/8 were detected using immunofluorescence staining assays, and the expression of Imp7/8 was investigated using immunoblotting. It was revealed that JNK or p38 inhibitor decreased the phosphorylation of Smad2C, Smad2L and Smad3L, and affected their nuclear accumulation. Although only inhibiting the phosphorylation of Smad2C, ERK inhibitor affected the nuclear accumulation of pSmad2C, pSmad2L, pSmad3C and pSmad3L. The three MAPK inhibitors attenuated the nuclear distribution of Smad4, and the expression and nuclear accumulation of Imp7. ERK and JNK inhibitors attenuated the expression and nuclear accumulation of Imp8. Thus, the results of the present study suggest that MAPK inhibitors, particularly ERK inhibitor, modulate the nuclear accumulation of Smads via the inhibition of Imp 7/8.

Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocarcinogenesis via modulating TGF-ß/TßR and Imp7/8.

Compound Astragalus and Salvia miltiorrhiza extract (CASE) is a Chinese herbal formula consisting of astragalosides, astragalus polysaccharide and salvianolic acids extracted from Astragalus membranaceus and Salvia miltiorhiza. Previous studies by our group have demonstrated that CASE effectively suppresses diethylinitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats via modulating transforming growth factor ß/Mothers against decapentaplegic (TGFß/Smad) signaling. To further elucidate the mechanism of CASE, the effects of CASE on TGF-ß1, the serine/threonine kinase receptors of TGF-ß [TGF-ß receptor type-I (TßRI) and TßRII] and karyopherins [Importin 7 (Imp7) and Imp8], which are crucial for TGF-ß/Smad signaling in fibro-hepatocarcinogenesis, were assessed in the present study using in vivo (DEN-induced HCC in rats) and in vitro [TGF-ß1-stimulated rat myofibroblasts (MFBs) and HepG2 cells] models of fibro-hepatocarcinogenesis. Hematoxylin and eosin staining revealed that CASE may suppress inflammatory reactions and fibrosis in HCC as well as increasing the differentiation of HCC cells. Positive TGF-ß1 staining was increased in HCC nodule areas and in adjacent normal liver tissues in DEN-treated rats, while TßRI staining was increased only in normal adjacent liver tissues. The elevated expression of TGF-ß1, TßRI and TßRII was suppressed by CASE. CASE treatment also reduced glutathione S-transferase P 1 and Imp7/8 protein expression in fibro-hepatocarcinogenesis. In vitro experiments confirmed that CASE was able to decrease the expression of TßRI and TßRII in TGF-ß1-stimulated MFBs and HepG2 cells. These results indicate that the anti-HCC effect of CASE may be achieved by mediating TGF-ß/TßR and Imp7/8 protein expression, suggesting that CASE has multiple targets in HCC treatment.

Bioactive compounds from Paecilomyces tenuipes regulating the function of the hypothalamo-hypophyseal system axis in chronic unpredictable stress rats.

BACKGROUND: A bioactive compound from Paecilomyces tenuipes (BCPT) has an inhibitory effect on monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in vitro and in vivo, which indicates BCPT may be a potential antidepressant. In this study we aimed to study the antidepressant effects of BCPT in the chronic unpredictable stress (CUS) model in rats and explore underlying mechanisms in the hypothalamic-pituitary-adrenal (HPA) axis. METHODS: The antidepressant effects of BCPT were studied in the chronic unpredictable stress model in rats. Animals were housed isolated, except the control group. Rats were exposed daily to different random stressors from day 1 to 21. Awarding response was detected by calculating the 24-hour consumption of sucrose water. Cortisol (CORT) and adrenocorticotropic hormone (ATCH) contents in serum and arginine vasopressin (AVP) contents in the pituitary body were detected by radio immunoassays. Total RNA of hippocampus or hypothalamus was extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the measurement of corticotrophin releasing hormone (CRH) mRNA or mineralocorticoid receptor (MR) mRNA and glucocorticoid receptor (GR) mRNA levels. Statistical analyses were performed using one way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) test. RESULTS: Chronic unpredictable stress resulted in reduction of sensitivity to reward and abnormality in the HPA axis in the animal model. BCPT improved the reward reaction as measured by increasing sucrose consumption, remarkably reduced serum CORT and ACTH levels and the AVP content in the pituitary body in the CUS-treated rats, decreased the expression of CRH mRNA, enhanced the expression of hippocampus MR mRNA, GR mRNA and decreased the ratio of MR/GR. CONCLUSIONS: BCPT has potentially antidepressant-like activity and normalized the HPA axis hyperactivity in a CUS model of depression in rats. This may be an important mechanism of its antidepressant effect.

NADPH oxidase-derived production of reactive oxygen species is involved in learning and memory impairments in 16-month-old female rats.

Women undergoing the natural menopause can experience progressive cognitive dysfunction, particularly in the form of memory impairment. However, the mechanisms underlying memory impairments in the menopause remain to be elucidated. There is increasing evidence that oxidative damage caused by excessive reactive oxygen species (ROS) production may correlate with age­associated cognitive impairment. The nicotinamide adenosine dinucleotide phosphate oxidase (NOX) family is important in the generation of ROS in the brain. It has been hypothesized that the accumulation of ROS, derived from NOX, may be involved in menopause­associated learning and memory impairments. The present study investigated whether NOX­derived ROS generation affected the learning and memory ability in 3­month and 16­month­old female rats. The results of a morris water maze assessment revealed that there were significant learning and memory impairments in the 16­month­old female rats. Furthermore, the activity of superoxide dismutase (SOD), level of malondialdehyde (MDA), production of ROS and expression levels of NOX2, p47phox, Ras­related C3 botulinum toxin substrate 1 (RAC1) and protein kinase C α (PKCα) were investigated in the cortex and hippocampus of 3­month and 16­month old female rats. The results demonstrated that the activity of SOD was significantly decreased, whereas the levels of MDA, production of ROS and expression levels of NOX2, p47phox, RAC1 and PKCα were significantly increased in the 16­month old female rats. These results suggested that NOX­mediated oxidative stress may be important in menopause­associated learning and memory impairments.

Grape seed proanthocyanidins prevent DOCA-salt hypertension-induced renal injury and its mechanisms in rats.

Renal dysfunction is one of the major effects of DOCA (deoxycorticosterone acetate)-salt hypertension and there is an increasing amount of evidence that oxidative stress damages the function of the kidney. Grape seed proanthocyanidins (GSPE) have been reported to be potent anti-oxidants and free radical scavengers. The present study sought to investigate the ability of GSPE to prevent renal injury in DOCA-salt hypertensive rats and to explore the molecular mechanisms underlying its protective effects. A total of 54 Sprague Dawley (SD) rats were randomly divided into 7 groups: Sham group (n = 7), UnX-sham group (n = 8), DOCA-salt group (n = 8), GSPE150 group (150 mg kg(-1), n = 7), GSPE240 group (240 mg kg(-1), n = 8), GSPE384 group (384 mg kg(-1), n = 8) and ALM (amlodipine besylate tablets) group (5 mg kg(-1), n = 8), and treated for 4 weeks. Compared to sham group rats, renal injury was observed in DOCA-salt hypertensive group rats as the urine protein, KW/BW (kidney weight/body weight), degree of renal fibrosis, renal MDA (malondialdehyde) and Hyp (hydroxyproline) contents significantly increased (P < 0.01). Moreover, SOD (Superoxide Dismutase) activities decreased in the model group (P < 0.01). In contrast, DOCA-salt hypertensive rats treated with different dose of GSPE or ALM showed a significant improvement of renal injury with decreased urine protein, KW/BW, degree of renal fibrosis, renal total MDA and Hyp contents compared to the untreated group. In addition, SOD activities increased in the treatment group. Since the experimental modeling time was short, kidney damage occurs to a lesser extent. BUN (Blood Urea Nitrogen), Scr (Serum Creatinine) and UA (Uric Acid) contents did not appear significantly changed in all groups. Finally, the activation of JNK and p38 kinases in the kidney was suppressed in rats treated with GSPEs or ALM compared to the untreated group, suggesting that the inhibition of these kinase pathways by GSPE contributes to the improvement of renal function. Taking these results together, we conclude that the anti-hypertensive and anti-oxidative stress beneficial effects of GSPE on renal injury in rats with DOCA-salt hypertension occur via the attenuation of JNK and p38 activity.

Protective effects of ginsenoside Rg1 on chronic restraint stress induced learning and memory impairments in male mice.

Alzheimer's disease (AD) is one of the major neurological diseases of the elderly. Chronic stress, which can induce atrophy and functional impairments in several key brain areas such as the frontal cortex and hippocampus, plays an important role in the generation and progression of AD. Currently, there are no effective drug treatment options for preventing chronic stress induced learning and memory impairments and neuronal damage. Ginsenoside Rg1 (Rg1) is a steroidal saponin abundantly contained in ginseng. This study explored the neuroprotective effects of Rg1 on chronic restraint stress (CRS) induced learning and memory impairments in a mouse model. Our results showed that Rg1 (5mg/kg) significantly protected against learning and memory impairments induced by CRS in a Morris water maze. Besides, Rg1 (2, 5mg/kg) was able to decrease ROS generation and attenuate the neuronal oxidative damage in the frontal cortex and hippocampus CA1 in mice. Additionally, the inhibition of NOX2, p47phox and RAC1 expression is also involved in the action mechanisms of Rg1 in this experimental model. This study provided an experimental basis for the clinical application of Rg1 in chronic stress induced neuronal oxidative damage.

Protective effects of astragalosides on dexamethasone and Aß25-35 induced learning and memory impairments due to decrease amyloid precursor protein expression in 12-month male rats.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder of the elderly characterized by learning and memory impairment. Stress level glucocorticoids (GCs) and ß-amyloid (Aß) peptide deposition are found to be correlated with dementia progression in patients with AD. The astragalosides (AST) was extracted from traditional Chinese herb Astragalus membranaceous. In this study, 12 months male rats were treated with Aß(25-35) (10 µg/rat, hippocampal CA1 injection) and dexamethasone (DEX, 1.5mg/kg, ig) and AST (8, 16 and 32 mg/kg, ig) or ginsenoside Rg1 (Rg1, 5 mg/kg, ig) for 14 days. We investigated the protective effect of AST against DEX+Aß(25-35) injury in rats and its mechanisms of action. Our results indicate that DEX+Aß(25-35) can induce learning and memory impairments and increase APP and Aß(1-40) expression. AST (16, 32 mg/kg) or Rg1 (5mg/kg) treatment significantly improve learning and memory, down-regulate the mRNA levels of APP and ß-secretase, decrease expression of APP and Aß(1-40) in hippocampus. The results indicated that DEX might increase hippocampal vulnerability to Aß(25-35) and highlight the potential neuronal protection of AST.

Dexamethasone and Aß25-35 accelerate learning and memory impairments due to elevate amyloid precursor protein expression and neuronal apoptosis in 12-month male rats.

Alzheimer's disease (AD) is an irreversible, progressive brain disorder of the elderly characterized by learning and memory impairment. Stress level glucocorticoids (GCs) and ß-amyloid (Aß) peptides deposition are found to be correlated with dementia progression in patients with AD. However, little is known about the simultaneous effects of glucocorticoids and Aß on learning and memory impairment and its mechanism. In this study, 12-month-old male rats were chronically treated with Aß(25-35) (10 µg/rat, hippocampal CA1 injection) and dexamethasone (DEX, 1.5mg/kg) for 14 days to investigate the effects of DEX and Aß(25-35) treatment on learning and memory impairments, pathological changes, neuronal ultrastructure, amyloid precursor protein (APP) processing and neuronal cell apoptosis. Our results showed that DEX or Aß(25-35) treatment alone for 14 days had caused slight damage on learning and memory impairments and hippocampal neurons, but damages were significantly increased with DEX+Aß(25-35) treatment. And the mRNA levels of the APP, ß-secretase and caspase 3 were significantly increased after DEX+Aß(25-35) treatment. The immunohistochemistry demonstrated that APP, Aß(1-40), caspase 3 and cytochrome c in hippocampus CA1 were significantly increased. Furthermore, Hoechst 33258 staining and Aß(1-40) ELISA results showed that DEX+Aß(25-35) treatment induced hippocampus CA1 neuron apoptosis and increased the level of Aß(1-40). The results suggest that the simultaneous effects of GCs and Aß may have important roles in the etiopathogenesis of AD, and demonstrate that stressful life events and GC therapy may increase the toxicity of Aß and have cumulative impacts on the course of AD development and progression.