Methyl helicterilate ameliorates alcohol-induced hepatic fibrosis by modulating TGF-ß1/Smads pathway and mitochondria-dependent pathway.

This study aimed to investigate the effect and underlying mechanism of Methyl helicterilate from Helicteres angustifolia (MHHA) on alcohol-induced hepatic fibrosis. The results showed that MHHA treatment markedly alleviated alcohol-induced liver injury and notably reduced collagen deposition in liver tissue. It significantly enhanced the activity of alcohol dehydrogenase and aldehyde dehydrogenase. Moreover, MHHA treatment markedly decreased the content of inflammatory cytokines, alleviated collagen accumulation, and inhibited the expression of TGF-ß1 and Smad2/3 in liver tissue. The experiments in cells showed that MHHA significantly inhibited HSC activation by blocking TGF-ß1/Smads signaling pathway. Additionally, it notably induced HSC apoptosis by modulating the mitochondria-dependent pathway. The present study demonstrates that MHHA treatment significantly ameliorates alcoholic hepatic fibrosis and the underlying mechanism may be ascribed to the inhibition of the TGF-ß1/Smads pathway and regulation of the mitochondria-mediated apoptotic pathway.

Role of RKIP in human hepatic stellate cell proliferation, invasion, and metastasis.

The purpose of this study was to investigate the effect of Raf kinase inhibitor protein (RKIP) on the growth, apoptosis, invasion, and metastasis of human hepatic stellate cell line (LX-2). A recombinant plasmid (pcDNA3.1-RKIP) or RKIP-targeting small interfering RNA (siRNA) vector (siRNA-RKIP) was transfected into LX-2 cells to interfere with the RKIP expression. The results demonstrated that increased RKIP expression significantly reduced cell viability, clonogenic growth, and invasion. Further, it promoted cell apoptosis and induced cell cycle arrest in the G1 phase. Overexpression of RKIP led to inactivation of LX-2 cells, as evidenced by the decrease in the expression levels of collagen I and α-smooth muscle actin (α-SMA). In addition, increased RKIP expression significantly reduced the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), the transcriptional activity of nuclear factor-κB (NF-κB), and the levels of matrix metalloproteinases-1 and -2. In conclusion, these findings clearly demonstrate that RKIP inhibits LX-2 cell growth, metastasis, and activation, primarily by downregulating the ERK/MAPK and NF-κB signaling pathways.

[Knockdown of Raf kinase inhibitor protein promotes the proliferation of LX-2 human hepatic stellate cells].

Objective To investigate the role of Raf kinase inhibitor protein (RKIP) in the proliferation of LX-2 human hepatic stellate cells. Methods The recombinant plasmid siRNA-RKIP was transfected into LX-2 cells. Five days later, the stably transfected cells were screened and cultured. MTT assay was used to detect cell proliferation after RKIP was silenced. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. The expressions of α-smooth muscle actin (α-SMA) and collagen type 1 (Col1) mRNA were detected by quantitative real-time PCR. The expressions of RKIP, α-SMA, Col1 and extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK/MAPK) signaling pathway related proteins were assessed by Western blot analysis. Results Compared with the control group, knockdown of RKIP significantly induced LX-2 cell proliferation, reduced cell apoptosis, raise cell number in G2, and increased the proteins and mRNA expressions of Col1 and α-SMA. Moreover, low-expression of RKIP significantly enhanced the phosphorylation of ERK/MAPK. Conclusion Knockdown of RKIP promotes LX-2 cell proliferation; its mechanism is related to the activation of ERK/MAPK signaling pathway.

[Over-expression of Raf kinase inhibitor protein (RKIP) inhibits the proliferation of LX-2 human hepatic stellate cells].

Objective To investigate the effect of Raf kinase inhibitor protein (RKIP) over-expression on the proliferation of LX-2 human hepatic stellate cells. Methods Recombinant plasmid pcDNA3.1-RKIP was transfected into LX-2 cells. G418 was used to screen and culture stably infected cells. MTT assay and colony formation assay were used to examine the effect of RKIP over-expression on cell proliferation and colony formation, respectively. Western blotting was performed to assess the expressions of RKIP, α-smooth muscle actin (α-SMA), type 1 collagen (Col1) and matrix metalloproteinase 1(MMP-1) and MMP-2 as well as extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK/MAPK) signaling pathway-related proteins. Results Compared with the control cells, RKIP over-expression significantly inhibited LX-2 cell proliferation and colony formation, and reduced the protein expressions of Col1, α-SMA, MMP-1 and MMP-2. Moreover, RKIP over-expression remarkably inhibited the phosphorylation of ERK/MAPK. Conclusion Over-expressed RKIP inhibits LX-2 cell proliferation and the mechanism is related to the inhibition of ERK/MAPK signaling pathway.

Loss of Raf kinase inhibitor protein is associated with malignant progression in hepatic fibrosis.

Raf kinase inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. But its role in hepatic fibrogenesis remains unclear. In the present study, we found that the absence of RKIP expression significantly enhanced the proliferation of HSC-T6 cells. Reduced RKIP expression promoted the activation of HSCs and the accumulation of collagen, as evidenced by the increases in the levels of collagen I and α-smooth muscle actin. Moreover, down-regulating RKIP expression led to severe histopathological changes and collagen accumulation in hepatic tissues of rats with liver fibrosis. Furthermore, the absence of RKIP promoted the activation of ERK/MAPK pathway in vitro and in vivo. Our findings clearly demonstrate an inverse correlation between RKIP level and the degree of the liver injury and fibrosis. Loss of RKIP may be associated with malignant progression in hepatic fibrosis.

Isoorientin from Gypsophila elegans induces apoptosis in liver cancer cells via mitochondrial-mediated pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gypsophila elegans has been used as a traditional herbal medicine for treating immune disorders and chronic liver diseases in China. The aim of this study is to isolate an active ingredient from this herb and investigate its anti-tumor activity. MATERIALS AND METHODS: An active ingredient was isolated from the ethanol extract using bioassay-guided screening. And its anti-tumor activity was analyzed by testing the cytotoxicity, lactate dehydrogenase (LDH) release, clonogenecity and migration in HepG2 cells. To investigate its potential mechanism, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS), cytochrome c, mitochondria membrane potential (MMP) and caspase level were determined in liver cancer cell line HepG2. RESULTS: A flavonoid glycoside, i.e., G. elegans isoorientin (GEI), was isolated from this herb and identified as Isoorientin-2″-O-α-l-arabinopyranosyl. Our results showed that GEI significantly inhibited the viability and proliferation of HepG2 cells in a dose- and time-dependent manner, and its cytotoxic effect was also confirmed by the elevated level of LDH. GEI treatment could markedly inhibit the clonogenicity and migration of HepG2 cells. Moreover, GEI induced remarkable apoptotic death of HepG2 cells through cell cycle arrest at G1 phase via the regulation of cell cycle-related genes, such as cyclin D, cyclin E and CDK2. Further study showed that GEI treatment significantly elevated ROS formation, followed by attenuation of MMP via up-regulation of Bax and down-regulation of Bcl-2, accompanied by cytochrome c release to the cytosol. In addition, GEI treatment resulted in a significant dose-dependent increase in caspase-3 and -9 proteolytic activities. CONCLUSION: The present study demonstrates that the ability of GEI to induce apoptosis against HepG2 cells mediated by mitochondrial-mediated pathway.

Gypsophila elegans isoorientin attenuates CCl4-induced hepatic fibrosis in rats via modulation of NF-κB and TGF-ß1/Smad signaling pathways.

The hepatoprotective effect of Gypsophila elegans isoorientin (GEI) was evaluated using a hepatic fibrosis model induced by CCl4 in rats. The results revealed that GEI significantly prevented CCl4-induced liver injury and fibrosis, as evidenced by the attenuation of histopathological changes, the decrease in serum aminotransferase, and the inhibition of collagen accumulation. GEI strongly inhibited lipid peroxidation and recruited anti-oxidative defense system. Moreover, GEI alleviated pro-inflammatory cytokines such as TNF-α, IL-1ß and IL-6 via inhibiting nuclear factor-κB (NF-κB) activation. In addition, GEI down-regulated the phosphorylation of Smad2/3 and up-regulated the level of hepatic Smad7, thereby inhibiting TGFß1/Smad signaling pathway. In conclusion, our findings indicate that GEI can inhibit CCl4-induced hepatic fibrosis, which may be ascribed to its radical scavenging action, antioxidant activity, and modulation of NF-κB and TGF-ß1/Smad signaling pathways.

Pratensein ameliorates ß-amyloid-induced cognitive impairment in rats via reducing oxidative damage and restoring synapse and BDNF levels.

This study was designed to investigate the protective effect of pratensein against cognitive impairment induced by amyloid beta (1-42) (Aß1-42) in rats. Aß1-42 peptide was injected bilaterally in the hippocampus of rat. Next, pratensein was administered orally for 3 weeks. Our findings demonstrated that treatment with pratensein ameliorated learning and memory deficits in Aß1-42 rat model of AD. Pratensein treatment significantly attenuated neuronal degeneration and apoptosis in hippocampus. Moreover, the over-expression in IL-1ß and TNF-α as well as the extensive astrogliosis and microgliosis in hippocampus induced by Aß1-42 were significantly reduced following administration of pratensein. Concomitantly, pratensein treatment significantly suppressed the activation of NF-κB in hippocampus. In addition, pratensein was able to increase the levels of synaptophysin and brain-derived neurotrophic factor (BDNF). These results indicate that pratensein could significantly ameliorate Aß1-42-induced spatial learning and memory impairment through reducing neuroinflammation via inhibition of glial activation and NF-κB activation, and restoring synapse and BDNF levels, suggesting that administration of pratensein could likely provide a therapeutic approach for AD.

Protective effect of madecassoside against cognitive impairment induced by D-galactose in mice.

This study was designed to investigate the protective effect of madecassoside from Hydrocotyle sibthorpioides against cognitive impairment induced by D-galactose (D-gal) in mice. The result revealed that treatment with madecassoside significantly reversed D-gal-induced learning and memory impairments, as measured by the Morris water-maze test. Studies on the potential mechanisms of this action showed that madecassoside significantly reduced oxidative stress and suppress inflammatory responses via blocking NF-κB and ERK/p38 MAPK pathways. Moreover, madecassoside markedly attenuated the content and deposition of ß-amyloid peptide by inducing a decrease in the expression of amyloid protein precursor, ß-site amyloid cleaving enzyme-1 and cathepsin B and an increase in the levels of neprilysin and insulin-degrading enzyme. Madecassoside significantly increased the expression of synapse plasticity-related proteins in the hippocampus, such as postsynaptic density 95, long-term potentiation, N-methyl-D-aspartic acid receptors, Ca(2+)/calmodulin-dependent protein kinase II, NMDA receptor subunit 1, protein kinase C, protein kinase A, cAMP-response element binding protein, and brain-derived neurotrophic factor. In addition, madecassoside significantly increased the levels of acetylcholine but decreased cholinesterase activity. In conclusion, the protective effect of madecassoside against d-gal-induced cognitive impairment was mainly due to its ability to reduce oxidative damage, improve synaptic plasticity and restore cholinergic function. These findings suggest that madecassoside can be considered as a potential agent for preventing cognitive impairment.

Helenalin attenuates alcohol-induced hepatic fibrosis by enhancing ethanol metabolism, inhibiting oxidative stress and suppressing HSC activation.

A compound was isolated from Centipeda minima using bioassay-guided screening. The structure of this compound was elucidated based on its spectral data, and it was identified as helenalin. The hepatoprotective effect of helenalin was evaluated using a liver fibrosis model induced by intragastric administration with alcohol within 24 weeks in rats. The results revealed that helenalin significantly prevented alcohol-induced hepatic injury and fibrogenesis, as evidenced by the decrease in serum aminotransferase, the attenuation of histopathological changes, and the inhibition of the hepatic fibrosis indicators, such as hyaluronic acid, type III precollagen, laminin, hydroxyproline and collagen α type I. Mechanistically, studies showed that helenalin expedited ethanol metabolism by enhancing the alcohol and aldehyde dehydrogenase activities. Furthermore, helenalin alleviated lipid peroxidation, recruited the antioxidative defense system, inhibited CYP2E1 activity, and reduced the inflammatory mediators, including TGF-ß1, TNF-α, IL-6 and IL-1ß and myeloperoxidase, via down-regulation of NF-κB. Helenalin significantly decreased collagen deposition by reducing the profibrotic cytokines like transforming growth factor-ß, platelet-derived growth factor-ß and connective tissue growth factor, and promoted extracellular matrix degradation by modulating the levels of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9. In addition, helenalin inhibited HSC activation as evidenced by the down-regulation of α-SMA and TGF-ß levels. In conclusion, helenalin had a significant protective effect on chronic ethanol-induced hepatic fibrosis and may be a major bioactive ingredient of C. minima.

Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/D-galactosamine induced fulminant hepatic failure in mice.

A compound was isolated from Potentilla chinensis, and it was identified as tormentic acid (TA) based on its physicochemical properties and spectral data. The hepatoprotective effect of TA was evaluated using an acute liver failure model induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN). The results revealed that TA significantly prevented LPS/D-GalN-induced fulminant hepatic failure, as evidenced by the decrease in serum aminotransferase and total bilirubin activities and the attenuation of histopathological changes. TA alleviated the pro-inflammatory cytokines including TNF-α and NO/iNOS by inhibiting nuclear factor-κB (NF-κB) activity. Moreover, TA strongly inhibited lipid peroxidation, recruited the anti-oxidative defense system, and increased HO-1 activity. In addition, TA significantly attenuated increases in TUNEL-positive hepatocytes through decreasing the levels of cytochrome c, as well as caspases-3, 8 and 9, while augmenting the expression of Bcl-2. In conclusion, TA protects hepatocytes against LPS/D-GalN-induced injury by blocking NF-κB signaling pathway for anti-inflammatory response and attenuating hepatocellular apoptosis. Consequently, TA is a potential agent for preventing acute liver injury and may be a major bioactive ingredient of Potentilla chinensis.

Isolation and identification of an anti-hepatitis B virus compound from Hydrocotyle sibthorpioides Lam.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle sibthorpioides (Apiaceae) have been used as a folk remedy for the treatment of fever, edema, detoxication, throat pain, psoriasis and hepatitis B virus infections in China. The aim of this study is to isolate and identify an anti-HBV compound from this herb. MATERIALS AND METHODS: A compound (saponin) was isolated from the active ethanol extract using bioassay-guided screening. The structure of the saponin was elucidated by spectroscopic methods and compared with published data. The anti-HBV activity of the saponin was evaluated by detecting the levels of HBV antigens, extracellular HBV DNA, nuclear covalent closed circular DNA (cccDNA) and five HBV promoters in HepG2.2.15 cells. In addition, the levels of serum HBsAg/HBeAg, DHBV DNA, ALT/AST and hepatic pathological changes were analyzed in DHBV-infected ducks. RESULTS: The chemical analysis indicated that the saponin isolated from Hydrocotyle sibthorpioides is asiaticoside. The pharmacodynamics experimental studies showed that asiaticoside effectively suppressed the levels of HBsAg/HBeAg, extracellular HBV DNA and intracellular cccDNA in a dose-dependent manner. Furthermore, experiments demonstrated that asiaticoside markedly reduced viral DNA transcription and replication by inhibiting the activities of core, s1, s2, and X gene promoters. In addition, asiaticoside markedly reduced DHBV replication without any obvious signs of toxicity. The levels of serum DHBV DNA, HBsAg/HBeAg were increased 3 days after drug withdrawal, but the levels rebounded slightly in the asiaticoside treatment groups compared with the 3TC treatment group. Moreover, analysis of the serum ALT/AST levels and the liver pathological changes indicated that asiaticoside could alleviate liver damage. CONCLUSIONS: Our results show that asiaticoside could efficiently inhibit HBV replication both in vitro and in vivo, and asiaticoside may be a major bioactive ingredient in Hydrocotyle sibthorpioides.