Ting Bai

Thymoquinone attenuates liver fibrosis via PI3K and TLR4 signaling pathways in activated hepatic stellate cells

Thymoquinone (TQ) is the major active compound derived from the medicinal Nigella sativa. In the present study, we investigated the anti-fibrotic mechanism of TQ in lipopolysaccharide (LPS)-activated rat hepatic stellate cells line, T-HSC/Cl-6. T-HSC/Cl-6 cells were treated with TQ (3.125, 6.25 and 12.5μM) prior to LPS (1μg/ml). Our data demonstrated that TQ effectively decreased activated T-HSC/Cl-6 cell viability. TQ significantly attenuated the expression of CD14 and Toll-like receptor 4 (TLR4). TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase-protein kinase B (Akt) phosphorylation. The expression of α-SMA and collagen-I were significantly decreased by TQ. Furthermore, TQ decreased X linked inhibitor of apoptosis (XIAP) and cellular FLIP (c-FLIPL) expression, which are related with the regulation of apoptosis. Furthermore, TQ significantly increased the survival against LPS challenge in d-galactosamine (d-GlaN)-sensitized mice, and decreased the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were in line with in vitro results. Our data demonstrated that TQ attenuates liver fibrosis partially via blocking TLR4 expression and PI3K phosphorylation on the activated HSCs. Therefore, TQ may be a potential candidate for the therapy of hepatic fibrosis.

Thymoquinone alleviates thioacetamide-induced hepatic fibrosis and inflammation by activating LKB1-AMPK signaling pathway in mice.

The current study was conducted to investigate the anti-fibrotic effect and its possible underlying mechanisms of thymoquinone (TQ) against hepatic fibrosis in vivo. TQ is the major active compound derived from the medicinal Nigella sativa. Liver fibrosis was induced in male Kunming mice by intraperitoneal injections of thioacetamide (TAA, 200 mg/kg). Mice were treated concurrently with TAA alone or TAA plus TQ (20 mg/kg or 40 mg/kg) given daily by oral gavage. Our data demonstrated that TQ treatment obviously reversed liver tissue damage compared with TAA alone group, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM) proteins. TQ significantly attenuated TAA-induced liver fibrosis, accompanied by reduced protein and mRNA expression of α-smooth muscle actin (α-SMA), collagen-І and tissue inhibitor of metalloproteinase-1 (TIMP-1). TQ downregulated the expression of toll-like receptor 4 (TLR4) and remarkably decreased proinflammatory cytokine levels as well. TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) phosphorylation. Furthermore, TQ enhanced the phosphorylation adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B (LKB)-1. In conclusion, TQ may reduce ECM accumulation, and it may be at least regulated by phosphorylation of AMPK signaling pathways, suggesting that TQ may be a potential candidate for the therapy of hepatic fibrosis.

Gentiana manshurica Kitagawa reverses acute alcohol-induced liver steatosis through blocking sterol regulatory element-binding protein-1 maturation.

This study was undertaken to investigate the protective effects of Gentiana manshurica Kitagawa (GM) on acute alcohol-induced fatty liver. Mice were treated with ethanol (5 g/kg of body weight) by gavage every 12 h for a total of three doses to induce acute fatty liver. Methanol extract of GM (50, 100, or 200 mg/kg) or silymarin (100 mg/kg) was gavaged simultaneously with ethanol for three doses. GM administration significantly reduced the increases in serum ALT and AST levels, the serum and hepatic triglyceride levels, at 4 h after the last ethanol administration. GM was also found to prevent ethanol-induced hepatic steatosis and necrosis, as indicated by liver histopathological studies. Additionally, GM suppressed the elevation of malondialdehyde (MDA) levels, restored the glutathione (GSH) levels, and enhanced the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities. The concurrent administration of GM efficaciously abrogated cytochrome P450 2E1 (CYP2E1) induction. Moreover, GM significantly reduced the nuclear translocation of sterol regulatory element-binding protein-1 (nSREBP-1) in ethanol-treated mice. These data indicated that GM possessed the ability to prevent ethanol-induced acute liver steatosis, possibly through blocking CYP2E1-mediated free radical scavenging effects and SREBP-1-regulated fatty acid synthesis. Especially, GM may be developed as a potential therapeutic candidate for ethanol-induced oxidative damage in liver.

Cryptotanshinone inhibits LPS-induced proinflammatory mediators via TLR4 and TAK1 signaling pathway.

Cryptotanshinone (CTN), one of the major constituents of tanshinones, was investigated for anti-inflammatory activity in the murine macrophage cell line RAW 264.7. CTN inhibited the production of nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. Since CTN was considered as inhibiting LPS-triggered phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation, we consequently evaluated the expression of toll-like receptor 4 (TLR4) and CD14, as well as phosphorylation of TGF-β-activated kinase 1 (TAK1). CTN reduced the expression of CD14 and TLR4, and suppressed LPS-induced phosphorylation of TAK1. Furthermore, CTN significantly increased the survival rate against LPS challenge in D-galactosamine-sensitized mice, which was in line with in vitro results. These results suggested that CD14/TLR4 and TAK1 might be the potential molecular targets for addressing the protective effects of CTN on LPS-induced inflammatory effects in macrophages.

Hepatoprotective effect of cryptotanshinone from Salvia miltiorrhiza in d-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Cryptotanshinone from Salvia miltiorrhiza Bunge was investigated for hepatoprotective effects in d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Cryptotanshinone (20 or 40 mg/kg) was orally administered 12 and 1h prior to GalN (700 mg/kg)/LPS (10 μg/kg) injection. The increased mortality and TNF-α levels by GalN/LPS were declined by cryptotanshinone pretreatment. In addition, cryptotanshinone attenuated GalN/LPS-induced apoptosis, characterized by the blockade of caspase-3, -8, and -9 activation, as well as the release of cytochrome c from the mitochondria. In addition, cryptotanshinone significantly suppressed JNK, ERK and p38 phosphorylation induced by GalN/LPS, and phosphorylation of TAK1 as well. Furthermore, cryptotanshinone significantly inhibited the activation of NF-κB and suppressed the production of proinflammatory cytokines. These findings suggested that hepatoprotective effect of cryptotanshinone is likely associated with its anti-apoptotic activity and the down-regulation of MAPKs and NF-κB associated at least in part with suppressing TAK1 phosphorylation.

Betulinic acid and betulin ameliorate acute ethanol-induced fatty liver via TLR4 and STAT3 in vivo and in vitro

Ethanol consumption leads to many kinds of liver injury and suppresses innate immunity, but the molecular mechanisms have not been fully delineated. The present study was conducted to determine whether betulinic acid (BA) or betulin (BT) would ameliorate acute ethanol-induced fatty liver in mice, and to characterize whether Toll like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) were involved in ethanol-stimulated hepatic stellate cells (HSCs). EtOH (5mg/kg) and BA or BT (20 or 50mg/kg) were applied in vivo, while EtOH (50mM) and BA or BT (12.5 or 25μM) were applied in vitro. Administration of BA or BT significantly prevented the increases of serum ALT and AST caused by ethanol, as well as serum TG. Supplement of BA or BT prevented ethanol-induced acidophilic necrosis, increased hepatocyte nuclei and stromal inflammation infiltration as indicated by liver histopathological studies. Administration of BA or BT significantly decreased CYP2E1 activities and expression of SREBP-1caused by ethanol, however, lower dosage of BA or BT showed slight effects on CYP2E1 activity or expression of SREBP-1c. BA or BT administration significantly decreased the expression of TLR4, and increased the phosphorylation of STAT3. In vitro, BA or BT treatment reduced the expressions of α-SMA and collagen-I in ethanol-stimulated HSCs via regulation of TLR4 and STAT3, coincided with in vivo. All of these findings demonstrated that BA or BT might ameliorate acute ethanol-induced fatty liver via TLR4 and STAT3 in vivo and in vitro, promising agents for ethanol-induced fatty liver therapies.

The anti-fibrotic effect of betulinic acid is mediated through the inhibition of NF-κB nuclear protein translocation

The purpose of the study was to investigate the anti-fibrotic effect and the potential mechanisms of action of betulinic acid (BA) against hepatic fibrosis in vivo and in vitro. BA is an active compound isolated from the bark of the birch tree Betula spp. (Betulaceae). Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200mg/kg) twice weekly for 6weeks in Wistar rats. The administration of BA (20 or 50mg/kg) was started following TAA injections and was continued for 6 or 8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver tissue hydroxyproline and α-smooth muscle actin (α-SMA). In vitro, BA effectively decreased the HSC-T6 cell viability induced by TNF-α and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of α-SMA and tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner. This study provides evidence that BA exerts a significant anti-fibrosis effect by modulating the TLR4/MyD88/NF-κB signaling pathway.

25-OCH3-PPD induces the apoptosis of activated t-HSC/Cl-6 cells via c-FLIP-mediated NF-κB activation.

25-OCH(3)-PPD is a dammarane-type triterpene sapogenin isolated from the roots, leaves and seeds of Panax notoginseng, which has shown anti-tumor effects in several human cancer lines. In this study, we evaluated the effects of 25-OCH(3)-PPD on apoptosis of activated t-HSC/Cl-6 cells induced by tumor necrosis factor-α (TNF-α). The inhibitory effects of eleven compounds isolated from Panax ginseng and P. notoginseng were detected in activated t-HSC/Cl-6 cells. 25-OCH(3)-PPD produced a significant inhibitory effect on activated t-HSC/Cl-6 cells. However, 25-OCH(3)-PPD showed almost no effect on the cell viability of Chang liver cells, a type of normal human hepatic cell line. Therefore, we aimed to determine the anti-fibrotic potential of 25-OCH(3)-PPD and to characterize the signal transduction pathways involved in activated HSCs. 25-OCH(3)-PPD decreased the fibrosis markers, including α-smooth muscle actin (α-SMA), transforming growth factor β-1 (TGF-β1) and tissue inhibitors of metalloproteinases-1 (TIMP-1). 25-OCH(3)-PPD elevated the level of cellular GSH in activated HSCs, which demonstrated that 25-OCH(3)-PPD might inhibit HSC activation by its antioxidant capacity. Further analyses revealed that 25-OCH(3)-PPD increased the levels of cleaved caspase-3, decreased the ratio of Bcl-2/Bax and the expression of survivin via c-FLIP-mediated NF-κB activation and shed light on the regulation of apoptosis. Therefore, 25-OCH(3)-PPD may prove to be an excellent candidate agent for the therapy of hepatic fibrosis.

Acanthoic acid, a diterpene in Acanthopanax koreanum, ameliorates the development of liver fibrosis via LXRs signals

Liver X receptors (LXRs)-mediated signals in acanthoic acid (AA) ameliorating liver fibrosis were examined in carbon tetrachloride (CCl4)-induced mice and TGF-β stimulated hepatic stellate cells (HSCs). AA was isolated from the root of Acanthopanax koreanum Nakai (Araliaceae). CCl4-treated mice were intraperitoneally injected with 10% CCl4 in olive oil (2 mL/kg for 8 weeks). In AA treated groups, mice were intragastrically administrated with AA (20 mg/kg or 50 mg/kg) 3 times per week for 8 weeks. Administration of AA reduced serum aminotransferase and tissue necrosis factor-α (TNF-α) levels evoked by CCl4, and the reverse of liver damage was further confirmed by histopathological staining. Administration of AA reduced the expression of fibrosis markers and regulated the ratio of MMP-13/TIMP-1, further reversed the development of liver fibrosis. TGF-β (5 ng/ml) was added to activate HSC-T6 cells for 2 h, and then treated with AA (1, 3, or 10 μmol/l) for 24 h before analysis. Cells were collected and proteins were extracted to detect the expressions of LXRs. AA could inhibit the expression of α-SMA stimulated by TGF-β and increase the expression of LXRβ. In vivo and in vitro experiments, AA could modulate liver fibrosis induced by CCl4-treatment via activation of LXRα and LXRβ, while inhibit HSCs activation only via activation of LXRβ. Acanthoic acid might ameliorate liver fibrosis induced by CCl4 via LXRs signals.

Ginsenoside Rh2 Downregulates LPS-Induced NF-κB Activation through Inhibition of TAK1 Phosphorylation in RAW 264.7 Murine Macrophage

The present study was carried out to evaluate the inhibitory effects of ginsenoside Rh2 on nuclear-factor- (NF-) κB in lipopolysaccharide- (LPS-) activated RAW 264.7 murine macrophages. RAW 264.7 cells were pretreated with indicated concentrations of ginsenoside Rh2 for 1 h prior to the incubation of LPS (1 μg/mL) for indicated time period. Ginsenoside Rh2 reduced CD14 and Toll-like receptor 4 (TLR4) expressions 24 h after LPS stimulation. Furthermore, ginsenoside Rh2 significantly inhibited TGF-beta-activated kinase 1 (TAK1) phosphorylation 30 min after LPS stimulation. Ginsenoside Rh2 was further shown to inhibit NF-κB p65 translocation into the nucleus by suppressing IκB-α degradation. Also, LPS increased mRNA expression of TNF-α and IL-1α time-dependently, while TQ reduced TNF-α within 3 h and IL-1α within 1 h. And we firstly found that pretreatment of ginsenoside Rh2 successively inhibited hypoxia-inducible factor- (HIF-) 1α expression increased by LPS. In conclusion, ginsenoside Rh2 may inhibit LPS-induced NF-κB activation and reduce HIF-1α accumulation, suggesting that ginsenoside Rh2 may be considered as a potential therapeutic candidate for chronic inflammatory diseases.