Salvianolic acid B protects against acute and chronic liver injury by inhibiting Smad2C/L phosphorylation.

Salvianolic acid B (Sal B) has strong antioxidant and anti-fibrosis effects, which are related to the transforming growth factor ß/Smad signaling pathway. However, how Sal B affects this antioxidant pathway and the phosphorylation (p-) of Smad2 at both the COOH-terminal (pSmad2C) and linker region (pSmad2L) are unknown. The aims of the present study were to investigate the underlying mechanisms of Sal B on acute and chronic liver injury induced by CCl4 and H2O2, and its effects on p-Smad2C/L. In in vivo experiments, acute and chronic liver injury models were induced by CCl4, and the oxidative damage cell model was established in vitro with H2O2. Liver histopathology was assessed using hematoxylin and eosin and Van Gieson's staining. Moreover, serum biochemical indicators were analyzed using specific assay kits. Furthermore, the present study evaluated the oxidant/antioxidant status in acute and chronic liver injury models by oxidative stress parameters such as malondialdehyde, glutathione and superoxide dismutase. In addition, western blot analysis was performed to analyze the protein expression levels of pSmad2C, pSmad2L, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). It was found that Sal B improved liver histology, decreased the levels of aminotransferase and attenuated oxidative stress in acute and chronic liver injury models. Additionally, the protein expression levels of pSmad2C and pSmad2L were decreased, but Nrf2 and HO-1 expression levels were increased both in vivo and in vitro. Collectively, the present results suggested that Sal B may protect against acute and chronic liver injury via inhibition of Smad2C/L phosphorylation, and the Nrf2/HO-1 signaling pathway may play an important role in this process.

Smad3 C-terminal phosphorylation site mutation attenuates the hepatoprotective effect of salvianolic acid B against hepatocarcinogenesis.

Smad3 phosphorylation is implicated in hepatic fibro-carcinogenesis. Moreover, Smad3 phospho-isoform pSmad3L and pSmad3C are reversible and antagonistic, and the balance could shift from carcinogenesis to tumor-suppression. pSmad3C has recently assigned to perform a preventative effect against primary liver injury. Salvianolic acid B (Sal B), a component derived from Salvia miltiorrhiza, is empirically used for hepatic diseases. Our prior study clarified that Sal B could delay hepatic fibrosis-carcinoma progression by converting pSmad3L/3C in mice. However, the roles of Smad3 phospho-isoform conversion and antagonism in the anti-hepatocarcinogenic effects of Sal B in pSmad3C- or/and pSmad3L-mutated mice/cells remain vague. Currently, corresponding doses/concentrations of Sal B was co-administrated to pSmad3C+/- mutational mice/plasmids-transfected HepG2 cells. Notably, in vivo functional studies revealed that pSmad3C mutation attenuates Sal B-induced ameliorative effects on histopathological characteristics and decreased serological biomarkers, and potential mechanism involves attenuation of increases in pSmad3C/p21 and decreases in pSmad3L/PAI-1/c-Myc expression. Expectedly, in vitro results showed that up-regulating pSmad3C enhances the inhibitory effects on proliferation, migration and contributes to apoptosis accompanied by a shift of pSmad3L/PAI-1/c-Myc oncogenic to pSmad3C/p21 tumour-suppressive signalling; however, opposite effects occur when upregulated pSmad3L. This study is the first to identify pSmad3C as a key target by which Sal B prevents hepatocarcinogenesis.

Smad3 gene C-terminal phosphorylation site mutation aggravates CCl4 -induced inflammation in mice.

The expression of C-terminal phosphorylated Smad3 (pSmad3C) is down-regulated with the progression of liver disease. Thus, we hypothesized that pSmad3C expression may be negatively related to liver disease. To develop novel therapeutic strategies, a suitable animal model is required that will allow researchers to study the effect of Smad3 domain-specific phosphorylation on liver disease progression. The current study aimed to construct a new mouse model with the Smad3 C-terminal phosphorylation site mutation and to explore the effects of this mutation on CCl4 -induced inflammation. Smad3 C-terminal phosphorylation site mutant mice were generated using TetraOne™ gene fixed-point knock-in technology and embryonic stem cell microinjection. Resulting mice were identified by genotyping, and the effects on inflammation were explored in the presence or absence of CCl4 . No homozygous mice were born, indicating that the mutation is embryonic lethal. There was no significant difference in liver phenotype and growth between the wild-type (WT) and heterozygous (HT) mice in the absence of reagent stimulation. After CCl4 -induced acute and chronic liver damage, liver pathology, serum transaminase (ALT/AST) expression and levels of inflammatory factors (IL-6/TNF-α) were more severely altered in HT mice than in WT mice. Furthermore, pSmad3C protein levels were lower in liver tissue from HT mice. These results suggest that Smad3 C-terminal phosphorylation may have a protective effect during the early stages of liver injury. In summary, we have generated a new animal model that will be a novel tool for future research on the effects of Smad3 domain-specific phosphorylation on liver disease progression.

Salvianolic acid B exerts anti-liver fibrosis effects via inhibition of MAPK-mediated phospho-Smad2/3 at linker regions in vivo and in vitro.

AIM: To investigate anti-liver fibrosis effects of Salvianolic acid B (Sal B) from Salvia miltiorrhiza Bunge involved mitogen-activated protein kinase (MAPK)-mediated transforming growth factor-beta (TGF-ß) signaling. MAIN METHODS: Diethylnitrosamine (DEN)-induced liver fibrosis in mice and TGF-ß1-activated hepatic stellate cells (HSCs) were established and treated with dosage/concentration-graded Sal B and/or MAPK activator (Vacquinol-1: MKK4-specific activator)/inhibitors (PD98059: ERK-specific inhibitor; SP600125: JNK-specific inhibitor; SB203580: p38-specific inhibitor). Histopathological characteristics and cell migration were assessed, α-SMA, Collagen I and members of TGF-ß/MAPK/Smad signal transduction pathway were measured. KEY FINDINGS: Results in vivo showed that Sal B alleviated DEN-caused liver fibrosis embodied in ameliorative histopathological characteristics and decreased protein levels of hepatic fibrosis related markers (α-SMA, Collagen I, TGF-ß1), its molecular mechanisms of action were correlative with inhibited activation of MAPK and phosphorylation of Smad2/3 at linker regions (P-Smad2/3L) and Smad2 at C-terminal (P-Smad2C) while increased phosphorylation of Smad3 at C-terminal (P-Smad3C). Results in vitro showed that Sal B restrained TGF-ß1-induced HSCs activation, Collagen I production and cell migration; Sal B inhibited activation of MAPK and markedly decreased protein levels of P-Smad2/3L and P-Smad2C while slightly increased P-Smad3C in TGF-ß1-stimulated HSCs, the expression of PAI-1 was inhibited by Sal B; activating MAPK receded inhibitory effects of Sal B on α-SMA, Collagen I, P-Smad2L and P-Smad3L expression while inhibited activation of MAPK reinforced those. SIGNIFICANCE: Sal B attenuates liver fibrosis via mediation of TGF-ß/Smad and MAPK pathways, especially inhibition of MAPK-mediated P-Smad2/3L signaling, which maybe provides theoretical foundation of Sal B for treating clinically liver fibrosis.

Comparison of Demographic Parameters and Predation Rates of Orius strigicollis (Hemiptera: Anthocoridae) Fed on Eggs of Tetranychus urticae (Acari: Tetranychidae) and Cadra cautella (Lepidoptera: Pyralidae).

Orius strigicollis (Poppius) is an anthocorid bug with high foraging ability on thrips as well as on mites, and the bug has been considered as a potential biological control agent in Taiwan. Life table and predation studies of O. strigicollis fed on Cadra cautella (Walker) and Tetranychus urticae (Koch) eggs were conducted at 25 ± 1°C. Data were analyzed and compared using TWOSEX-MSChart and CONSUME-MSChart software. O. strigicollis fed on eggs of C. cautella, a substitute prey, showed significantly higher survival rate and developmental rate than individuals fed on their natural prey, T. urticae eggs. The fecundity of O. strigicollis fed on C. cautella eggs was, on average, 13.2 times higher than that of those fed on T. urticae eggs, despite of the fact that during the entire nymphal stage, the consumption rate of O. strigicollis on T. urticae eggs was ca. 9 times higher than on almond moth eggs The conversion rate (i.e., number of prey eggs needed to produce one predator egg) for this predatory bug reared on T. urticae eggs and almond moth eggs were 604.6 and 6.0, respectively, indicating that almond moth eggs served as an effective alternative prey for ensuring the predator's reproduction. This is the first study pertaining to the population parameters and predation rates of O. strigicollis using the age-stage two-sex approach to describe differences between O. strigicollis populations reared on natural and alternative preys. This information may be useful in mass rearing programs and field application involving this biological control agent.