Astragaloside IV Synergizes with Ferulic Acid to Alleviate Hepatic Fibrosis in Bile Duct-Ligated Cirrhotic Rats.

BACKGROUND: Due to the multi-factorial etiology of hepatic fibrosis, multi-target therapeutics based on combinatory drugs is known to be a promising strategy for the disease. AIMS: The present study attempted to test the hypothesis that astragaloside IV combined with ferulic acid synergistically inhibits activation of hepatic stellate cells in vivo. METHODS: Bile duct-ligated rats were treated with astragaloside IV or/and ferulic acid for 28 days. Liver fibrosis was measured by histological examination. The oxidative stress-related biomarkers were measured with spectrophotometry. Expressions of mRNA and protein were measured by real-time PCR and Western blotting. RESULTS: Bile duct-ligated rat treatment with astragaloside IV and ferulic acid in combination resulted in synergistic alleviation of hepatic fibrosis. Simultaneously, activation of hepatic stellate cells was significantly inhibited by the combination therapy when compared with astragaloside IV or ferulic acid alone. Interestingly, astragaloside IV, but not ferulic acid, induced accumulation of Nrf2 in the nucleus, synthesized antioxidant enzymes through negative regulation of glycogen synthase kinase-3ß, scavenged reactive oxygen species, and, in turn, suppressed hepatic stellate cells activation in bile duct-ligated rats. Conversely, ferulic acid, but not astragaloside IV, suppressed TGF-ß1 and its receptors expression, which resulted in downregulation of Smad3 and Smad4. CONCLUSIONS: These findings suggest that the combination of astragaloside IV and ferulic acid synergistically induces deactivation of hepatic stellate cells through inhibition of the TGF-ß pathway and activation of the Nrf2 pathway, and suggest that combination of astragaloside IV and ferulic acid is a promising candidate for the treatment of hepatic fibrosis.

[Effect of maternal exposure to Curcumae Rhizoma during pregnancy on neurodevelopment and apoptosis mechanism in offspring].

Curcumae Rhizoma is a Chinese medicinal herb that is contraindicated during pregnancy. Cold-congelation and blood-stasis are corresponding syndromes to Curcumae Rhizoma. Whether syndrome-based treatment is associated with developmental neurotoxicity of Curcumae Rhizoma remains to be unclear. To verify the theory of traditional Chinese medicine of "syndrome-based treatment during pregnancy", the present study induced the mice blood stasis model by immersing mice in ice water. Pregnant C57 BL/6 wild type(WT) mice and pregnant Nrf2 knock out(KO) mice were randomly divided into control groups and Rhizoma Curcumae exposure groups. The mice were exposed to Rhizoma Curcumae during day 5 to day 18 after pregnancy. The neurodevelopment was examined to evaluate the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice exposed to Rhizoma Curcumae. caspase-3 and caspase-9 activity in brain of the offspring were measured by colorimetric assays. Bcl-2 mRNA and protein expression in brain of the offspring were examined by Real-time RT-PCR and Western blot, respectively. According to the findings, C57 BL/6 mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) had a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring, compared with the normal control group, but with no significant change in those of blood-stasis pregnant mice offspring. However, mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) showed no change in Bcl-2 gene expression and p38 MAPK phosphorylation in brain of the offspring. Nrf2 gene knockout using CRISPR/Cas9 resulted in a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring. In conclusion, developmental neurotoxicity of the blood-stasis pregnant mice exposed to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Nrf2 activation involved in the phenomenon of Rhizoma Curcumae of "syndrome-based treatment during pregnancy", but the upstream signal pathway mechanism value shall be further investigated.

Beta-asarone attenuates beta-amyloid-induced apoptosis through the inhibition of the activation of apoptosis signal-regulating kinase 1 in SH-SY5Y cells.

Beta-amyloid (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that beta-asarone improves cognitive function by suppressing neuronal apoptosis in vivo. In this study, we assessed the neuroprotective effects of beta-asarone against the toxicity of Abeta in relation to the mitochondria-mediated cell death process, and to elucidated the role of the ASK1/MKK7/JNK and mitochondrial pathways in beta-asarone-induced neuroprotection in SH-SY5Y cells. Our results show that beta-asarone afforded protection against Abeta-induced toxicity by inhibiting apoptosis in SH-SY5Y cells. This result was also confirmed by caspase-9 and caspase-3 activity assays. Expression of p-ASK1, p-MKK7, p-JNK, Bax, Bad, and cytochrome c release decreased after pretreatment with beta-asarone in SH-SY5Y cells exposed to A1-42. Interestingly, these effects of beta-asarone against Abeta1-42 insult were enhanced by ASK1 siRNA. These findings suggest that beta-asarone prevents Abeta1-42-induced neurotoxicity through attenuating neuronal apoptosis, and might be a potential preventive or therapeutic agent for AD.

A low molecular weight polysaccharide isolated from Agaricus blazei Murill (LMPAB) exhibits its anti-metastatic effect by down-regulating metalloproteinase-9 and up-regulating Nm23-H1.

The components of Agaricus blazei Murill (AbM) have been shown to possess antitumor potentials. Herein, we attempted to explore the anti-metastatic effect and underlying mechanism of a low molecular weight polysaccharide isolated from AbM (LMPAB). Matrigel invasion assay was applied to evaluate the effect of LMPAB on migration of BEL-7402 hepatic cancer cells in vitro. In vivo, the anti-metastatic effect of LMPAB was investigated in mouse B16 melanoma and a double-grafted SW180 tumor models. mRNA and protein levels of metalloproteinase-9 (MMP-9) or nm23-H1 upon LMPAB treatment were detected by real-time PCR and immunohistochemistry assays. LMPAB significantly reduced the invasion of BEL-7402 cells. In vivo, LMPAB was revealed to decrease lung metastatic foci in mouse B16 melanoma model. In the double-grafted SW180 mouse tumor model, we further demonstrated that intratumoral treatment of LMPAB inhibited the growth of tumor on treated side but also suppresses the regression of metastatic tumors on the non-treated side. Moreover, LMPAB reduced MMP-9 but enhanced nm23-H1 mRNA and protein expression. LMPAB displays anti-metastatic activities, indicating the potential of its clinical application for the prevention and treatment of cancer metastasis. Its anti-metastatic effect may relate to the modulation on MMP-9 and nm23-H1.

Immunostimulatory activities of a low molecular weight antitumoral polysaccharide isolated from Agaricus blazei Murill (LMPAB) in Sarcoma 180 ascitic tumor-bearing mice.

LMPAB is a linear beta-(1-3)-glucan we isolated from polysaccharide extract of Agaricus blazei Murill (AbM). Effects of LMPAB on splenic natural killer (NK) cell activity, splenocyte proliferation, index of spleen and thymus, IFN-gamma expression in spleen and the concentration of IL-12, IL-18 and TNF-alpha in serum of S180 ascitic tumor-bearing mice were detected. The results showed that intraperitoneal injection of LMPAB (100 mg x kg(-1) x d(-1)) significantly increased the thymus index. LMPAB augmented splenic NK cell activity in a dose-dependent manner (50-200 mg x kg(-1) x d(-1)). The concanavalin A (3 microg/ ml) stimulated splenocyte proliferation was significantly enhanced by LMPAB at dosages of 50, 100 or 200 mg x kg(-1) x d(-1). Further studies showed that LMPAB (50, 100 or 200 mg x kg(-1) x d(-1), 14d) significantly increased the production of IL-12, TNF-alpha, IL-18 and the expression IFN-gamma as determined by ELISA and immunohistochemistry, respectively. These results clearly indicate that the anti-tumor effects of LMPAB are closely associated with up-regulation of activity of NK cells, expression of IFN-gamma in spleen and the systemic level of IL-12, IL-18 and TNF-alpha in tumor-bearing mice.

Ferulic Acid Protects Against Lead Acetate-Induced Inhibition of Neurite Outgrowth by Upregulating HO-1 in PC12 Cells: Involvement of ERK1/2-Nrf2 Pathway.

Prenatal lead exposure is associated with poor intellectual development in children. However, there are few breakthroughs in therapeutic intervention of developmental lead neurotoxicity. The aim of this study is to evaluate the hypothesis that ferulic acid-mediated promotion of neurite outgrowth following lead exposure might mainly result from its antioxidant capability by extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Exposure of PC12 cells to lead acetate inhibits neurite outgrowth and causes oxidative stress as measured by ROS, LPO, GSH/GSSG, and NAD+/NADH. FA treatment significantly, although not completely, protected the cells against lead acetate-induced neurite outgrowth inhibition. The effects of FA could be blocked by PD98059, zinc protoporphyrin (Zn-PP), and Nrf2 shRNA. In addition, FA induced heme oxygenase 1 (HO-1) gene expression, enhanced antioxidant response element (ARE) promoter activity, promoted ERK1/2 phosphorylation, and Nrf2 translocation in PC12 cells exposed to lead acetate. ERK1/2 locate upstream of Nrf2 and regulate Nrf2-dependent HO-1 expression in antioxidative effects of FA. Our results suggest that FA is a promising candidate for treatment of developmental lead neurotoxicity. These promising findings warrant future investigation evaluating the FA-mediated potentiation of neurite outgrowth following lead exposure in vivo.

Hydroxysafflor yellow A induces apoptosis in activated hepatic stellate cells through ERK1/2 pathway in vitro.

A key feature in the molecular pathogenesis of liver fibrosis requires maintenance of the activated hepatic stellate cells (HSCs) phenotype by inhibition of apoptosis. The induction of apoptosis in activated HSCs has been proposed as an antifibrotic treatment strategy. This study aims at evaluating the effect of hydroxysafflor yellow A (HSYA) on apoptosis of culture-activated HSCs and further elucidating the underlying mechanisms. Primary HSCs were isolated from rats. The analysis of the cell cycle be performed by flow cytometry, detection of apoptosis by Annexin V-FITC/ PI staining, and the results were confirmed by DNA fragmentation, and cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). Real-time polymerase chain reaction and Western blotting were used to analyze the expression of genes. Our results revealed that HSYA significantly induced apoptosis in a dose- and time-dependent manner. HSYA suppresses the activation of ERK1/2 and ERK1/2-regulated gene expression, including Bcl-2, Cytochrome c, caspase-9, and caspase-3, leading to the enhancement of apoptosis. Pharmacological blockade of ERK1/2 kinase abrogation this action of HSYA. Our data provide a molecular basis for the anti-hepatic fibrosis activity of HSYA.

Hydroxysafflor yellow A attenuates carbon tetrachloride-induced hepatic fibrosis in rats by inhibiting ERK5 signaling.

Hepatic stellate cells (HSCs) undergo activation during the development of liver fibrosis. Transcription factor myocyte enhancer factor (MEF2) 2C plays a key role in this process. In the present study, we investigated the effect of hydroxysafflor yellow A (HSYA) on hepatic fibrosis and further investigated potential mechanisms in vivo. Sprague-Dawley rats were administered with CCl(4) together with or without HYSA for 12 weeks. The effect of HYSA on hepatic fibrosis was evaluated using hematoxylin-eosin and Van Gieson staining. Messenger RNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or immunohistochemistry. Our results revealed that CCl(4) treatment induced micronodular hepatic fibrosis with a pronounced deposition of collagen fibers. Treatment with HYSA resulted in a significant decrease in fibrosis, protein expression of α-SMA, and MEF-2C gene expression. This was accompanied by a decreased expression of Tß-RI, Tß-RII, MEKK3, MEK5, and phosphorylation of ERk5. HYSA alone had no effect on the measured parameters. Our findings demonstrate that HSYA protected, at least in part, the rat liver from CCl(4)-caused fibrogenesis through inhibition of hepatic stellate cell (HSC) activation, attenuation of transforming growth factor beta (TGF-ß) signaling. HSYA may become a novel and promising agent for the inhibition of hepatic fibrosis.

Emodin protects rat liver from CCl(4)-induced fibrogenesis via inhibition of hepatic stellate cells activation.

AIM: To investigate the role of emodin in protecting the liver against fibrogenesis caused by carbon tetrachloride (CCl(4)) in rats and to further explore the underlying mechanisms. METHODS: Rat models of experimental hepatic fibrosis were established by injection with CCl(4); the treated rats received emodin via oral administration at a dosage of 20 mg/kg twice a week at the same time. Rats injected with olive oil served as a normal group. Histopathological changes were observed by hematoxylin and eosin staining. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and hepatic hydroxyproline content were assayed by biochemical analyses. The mRNA and protein relevant to hepatic stellate cell (HSC) activation in the liver were assessed using real-time reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, western blotting and enzyme-linked immunosorbent assay. RESULTS: The degree of hepatic fibrosis increased markedly in the CCl(4) group compared to the normal group (P < 0.01), and decreased markedly in the emodin group compared to the CCl(4) group according to METAVIR scale (P < 0.01) compared with those in the normal control group (51.02 +/- 10.64 IU/L and 132.28 +/- 18.14 IU/L). The activities of serum ALT and AST were significantly higher in rats injected with CCl(4) (289.25 +/- 68.84 IU/L and 423.89 +/- 35.67 IU/L, both P < 0.05). The activities of serum ALT and AST were significantly reduced by administration of emodin (176.34 +/- 47.29 IU/L and 226.1 +/- 44.52 IU/L, both P < 0.05). Compared with the normal controls (54.53 +/- 13.46 mg/g), hepatic hydroxyproline content was significantly higher in rats injected with CCl(4) (120.27 +/- 28.47 mg/g, P < 0.05). Hepatic hydroxyproline content was significantly reduced in the rats treated with emodin at 20 mg/kg (71.25 +/- 17.02 mg/g, P < 0.05). Emodin significantly protected the liver from injury by reducing serum AST and ALT activities and reducing hepatic hydroxyproline content. The mRNA levels of transforming growth factor-beta1 (TGF-beta1), Smad4 and alpha-SMA in liver tissues were significantly down-regulated in SD rats that received emodin treatment. Furthermore, significant down-regulation of serum TGF-beta1 protein levels and protein expression of Smad4 and alpha-SMA in liver tissues was also observed in the rats. Emodin inhibited HSC activation by reducing the abundance of TGF-beta1 and Smad4. CONCLUSION: Emodin protects the rat liver from CCl(4)-induced fibrogenesis by inhibiting HSC activation. Emodin might be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.