ß-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction.

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of ß-catenin, one with ß-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived ß-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of ß-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of ß-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or ß-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel ß-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

The liver has an incredible capacity to repair itself or 'regenerate' ­ that is, it has the ability to replace damaged tissue with new tissue. In order to do this, the organ relies on hepatocytes (the cells that form the liver) and bile duct cells (the cells that form the biliary ducts) dividing and transforming into each other to repair and replace damaged tissue, in case the insult is dire. During long-lasting or chronic liver injury, bile duct cells undergo a process called 'ductular reaction', which causes the cells to multiply and produce proteins that stimulate inflammation, and can lead to liver scarring (fibrosis). Ductular reaction is a hallmark of severe liver disease, and different diseases exhibit ductular reactions with distinct features. For example, in cystic fibrosis, a unique type of ductular reaction occurs at late stages, accompanied by both inflammation and fibrosis. Despite the role that ductular reaction plays in liver disease, it is not well understood how it works at the molecular level. Hu et al. set out to investigate how a protein called ß-catenin ­ which can cause many types of cells to proliferate ­ is involved in ductular reaction. They used three types of mice for their experiments: wild-type mice, which were not genetically modified; and two strains of genetically modified mice. One of these mutant mice did not produce ß-catenin in biliary duct cells, while the other lacked ß-catenin both in biliary duct cells and in hepatocytes. After a short liver injury ­ which Hu et al. caused by feeding the mice a specific diet ­ the wild-type mice were able to regenerate and repair the liver without exhibiting any ductular reaction. The mutant mice that lacked ß-catenin in hepatocytes showed a temporary ductular reaction, and ultimately repaired their livers by turning bile duct cells into hepatocytes. On the other hand, the mutant mice lacking ß-catenin in both hepatocytes and bile duct cells displayed sustained ductular reactions, inflammation and fibrosis, which looked like that seen in patients with liver disease associated to cystic fibrosis. Further probing showed that ß-catenin interacts with a protein called CTFR, which is involved in cystic fibrosis. When bile duct cells lack either of these proteins, another protein called NF-B gets activated, which causes the ductular reaction, leading to inflammation and fibrosis. The findings of Hu et al. shed light on the role of ß-catenin in ductular reaction. Further, the results show a previously unknown interaction between ß-catenin, CTFR and NF-B, which could lead to better treatments for cystic fibrosis in the future.

The electrostimulation and scar inhibition effect of chitosan/oxidized hydroxyethyl cellulose/reduced graphene oxide/asiaticoside liposome based hydrogel on peripheral nerve regeneration in vitro.

The application of hollow nerve conduits in the repair of peripheral nerve defects is effected by inferior recovery, and nerve extension is hampered by the scar tissue generated during the repair process. In this study, the filler in hollow nerve conduit, chitosan/oxidized hydroxyethyl cellulose (CS/OHEC) hydrogel loaded asiaticoside liposome and the conductive reduced graphene oxide (rGO) were developed and used to reform the microenvironment for peripheral nerve regeneration. The physiochemical properties of CS/OHEC/rGO/asiaticoside liposome hydrogel were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and compressive modulus, porosity, swelling ratio, degradation and conductivity. In addition, the asiaticoside release profiles in vitro were investigated. The hydrogel had a continuous porous network structure with pore size distribution in the range of 50-250 µm. The majority of the hydrogels had porosities above 70%, and a compressive modulus of 0.45 MPa. The weight loss rate of hydrogel reached 76.14 ± 4.45% within 8 weeks. The conductivity of the hydrogel was 5.27 ± 0.42 × 10-4 S/cm. The hydrogel was non-toxic and suitable for adhesion and proliferation of nerve cells in vitro. In addition, the application of electrical stimulation after the addition of rGO can promote the differentiation and proliferation of nerve cells, accelerating nerve regeneration. The asiaticoside released from the hydrogel had a significant inhibitory effect on the growth and collagen secretion of fibroblasts, eliminating scars for regenerative nerves, which can promote the function recovery of defected peripheral nerve. Together, these positive results indicate that the hydrogel would be a promising candidate for peripheral nerve regeneration.

Eupafolin Suppresses Esophagus Cancer Growth by Targeting T-LAK Cell-Originated Protein Kinase.

Eupafolin is the main bioactive component extracted from the traditional Chinese medicine Ay Tsao (Artemisia vulgaris L.), and its anti-tumor activity has had been studied in previous researches. T-LAK cell-originated protein kinase (TOPK) belongs to serine/threonine protein kinase and is highly expressed in several cancer cells and tissues, such as colon cancer, lung cancer, esophagus cancer, and so on. Therefore, it was recognized as an important target for treating tumors. Nowadays, we found that eupafolin suppressed TOPK activities at the first time in vitro and in vivo. The cells study indicated that eupafolin suppressed TOPK activities in JB6 Cl41 and KYSE450 cells. Furthermore, knockdown of TOPK in KYSE450 cells decreased their sensitivities to eupafolin. The animal study showed that the injection of eupafolin in patient-derived xenograft (PDX) mouse effectively suppressed tumor growth. Histone H3 and Ki67 were reduced, and cleaved caspase 3 was increased in tumor tissues after eupafolin treatment. To sum up, eupafolin as an TOPK inhibitor can suppress growth of esophagus cancer in vitro and in vivo. The TOPK downstream signaling molecule histone H3 in tumor tissues was also reduced after eupafolin treatment. In short, eupafolin can suppress growth of esophagus cancer cells as an TOPK inhibitor both in vitro and in vivo.

Corrigendum to "Ethanol Extract from Ampelopsis sinica Root Exerts Anti-Hepatitis B Virus Activity via Inhibition of p53 Pathway In Vitro".

[This corrects the article DOI: 10.1093/ecam/neq011.].

Ethanol Extract from Ampelopsis sinica Root Exerts Anti-Hepatitis B Virus Activity via Inhibition of p53 Pathway In Vitro.

Ampelopsis sinica root is widely used in Chinese folk medicine for treating liver disorders caused by the hepatitis B virus (HBV). The present study was performed in order to investigate the anti-HBV activity and mechanisms of the ethanol extract from A. sinica root (EASR) in vitro. The antiviral activity of EASR was examined by detecting the levels of HBsAg, HBeAg and extracellular HBV DNAs in stable HBV-producing human hepatoblastoma HepG2 2.2.15 cells. We found that EASR effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, and it also suppressed the amount of extracellular HBV DNA. After EASR treatment, the percentage of apoptotic cells was found to be significantly higher than that of control by flow cytometric analysis. A luciferase reporter gene assay was used to determine the effects of EASR on the activities of HBV promoters and intracellular signaling pathways. The results showed that EASR selectively inhibited the activities of HBV promoters (Cp, S1p and Fp) and the p53 signaling pathway in HepG2 cells significantly. These data indicate that EASR exerts anti-HBV effects via inhibition of HBV promoters and the p53-associated signaling pathway, which helps to elucidate the mechanism underlying the potential therapeutic value of EASR.

In vitro antiviral activity of lutein against hepatitis B virus.

Despite the availability of an effective vaccine, the hepatitis B virus (HBV) infection and its treatment remains one of the foremost public health problems in the world. The present study was performed in order to investigate the anti-HBV activity of lutein in vitro. The antiviral activity of lutein was examined by detecting the levels of HBsAg, HBeAg and extracellular HBV DNA in stable HBV-producing human hepatoblastoma HepG2 2.2.15 cells. It was found that lutein effectively suppressed the secretion of HBsAg from HepG2 2.2.15 cells in a dose-dependent manner, and it also suppressed the amount of extracellular HBV DNA. A luciferase reporter gene assay was used to determine the effects of lutein on the activities of HBV promoters. The results showed that lutein inhibited the activity of HBV full-length promoter (Fp). These data indicate that lutein possesses an anti-HBV activity and exerts its antivirus effects via inhibition of HBV transcription.

In vitro anti-hepatitis B virus effect of Hypericum perforatum L.

The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.

N-butanol Extract from Melilotus Suaveolens Ledeb Affects Pro- and Anti-Inflammatory Cytokines and Mediators.

Melilotus suaveolens Ledeb is a traditional medicinal plant for treating inflammation-related disease. This explores the inner anti-inflammatory mechanism of n-butanol extract from M. suaveolens Ledeb. Inflammatory cellular model was established by lipopolysaccharide intervention on RAW264.7 cell line. Levels of secreted cytokines TNF-α, IL-1ß, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-α, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, activation of NF-κB and ingredients in the extract were assayed by ELISA, real time quantitative PCR, western blot, immunocytochemical test and HPLC fingerprint test, respectively. As a result, the extract could not only markedly reduce the production of pro-inflammatory mediators to different extents by blocking NF-κB activation but also promote the release of anti-inflammatory mediator HO-1 significantly. Each 1 g extract contained 0.023531 mg coumarin and another two high polar ingredients, probably saponins. It can be concluded that the extract has similar effects on antagonizing pro-inflammatory mediators and cytokines like Dexamethasone, and has effects on promoting the production of anti-inflammatory mediators.

Anti-inflammatory effects of ethyl acetate fraction from Melilotus suaveolens Ledeb on LPS-stimulated RAW 264.7 cells.

AIM OF THE STUDY: This paper aimed to elucidate the anti-inflammatory effects of EtOAc fraction prepared from Melilotus suaveolens Ledeb ethanol extract with a cellular model of LPS-stimulated RAW 264.7 cell. MATERIALS AND METHODS: Some key pro-inflammatory cytokines and mediators including IL-1 beta, IL-6, NO, iNOS, COX-2 and TNF-alpha, two important anti-inflammatory cytokines and mediators IL-10 and HO-1, I-kappaB and NF-kappaB were studied by sandwich ELISA, real-time PCR, western blot analysis and immunocytochemistry. At last a HPLC fingerprint was taken to evaluate the fraction. RESULTS: The EtOAc fraction could significantly inhibit the production of IL-1 beta, IL-6, NO, TNF-alpha, COX-2 in LPS-stimulated cell than that of single LPS-stimulated cell (p<0.01 or p<0.05), and the extract could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p<0.01 or p<0.05). Meanwhile, the extract also could inhibit the production of NF-kappaB compared to single LPS-stimulated cell. All the results showed that the extract had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell. CONCLUSIONS: Taken together, the anti-inflammatory actions of M. suaveolens Ledeb EtOAc fraction might be due to the down-regulation of IL-1 beta, IL-6, NO, TNF-alpha and COX-2 via the suppression of NF-kappaB activation, and another pathway was up regulating the production of IL-10 and HO-1. Meanwhile, the EtOAc fraction might be further studied to isolate the active anti-inflammatory ingredients besides coumarin.

[Effect of petroleum ether extract from Melilotus suaveolens Ledeb on the expression of NF-kappaB and Heme oxygenase 1].

AIM: To study the anti-inflammatory effect of petroleum ether extract from Melilotus suaveolens Ledeb. METHODS: Inflammatory cell model was constructed by LPS acting on the RAW264.7 cell line. The expression and distribution of NF-kappaB were detected using immunocytochemical method. The expression of mRNA and protein of Heme oxygenase 1(HO-1) were detected by real-time PCR and Western blot. RESULTS: The immunocytochemical analysis showed that the cytoplasm stained to brown presented NF-kappaB inactivation after the intervention of petroleum ether extract while the cell nucleus stained to brown presented NF-kappaB activation after the only intervention of LPS. The expression of HO-1 mRNA was significantly enhanced by the extract in a dose-dependent manner, and the expression of HO-1 protein was markedly enhanced too. CONCLUSION: The petroleum ether extract from Melilotus suaveolens Ledeb can resist inflammation by inhibiting the activation of proinflammatory factor NF-kappaB and enhancing the expression of anti-inflammatory factor HO-1.

Anti-inflammatory mechanism of a folk herbal medicine, Duchesnea indica (Andr) Focke at RAW264.7 cell line.

This study is to explore the anti-inflammatory mechanism of the ethanol extract of Duchesnea indica (Andr) Focke. An inflammatory cellular model was established by addition of lipopolysaccharide (LPS) on RAW264.7 cell line. The cellular secretion of TNF-alpha, IL-1beta, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-alpha, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, and activation of NF-kappaB were assayed by ELISA, the Griess method, real-time quantitative PCR, and Western blot and immunocytochemistry method, respectively. The ethanol extract of D. indica not only reduced production of pro-inflammatory cytokines and mediators and blocked NF-kappaB activation, but also slightly promoted release of the anti-inflammatory mediator HO-1 and suppressed IL-10 secretion. In conclusion, the anti-inflammatory effects of the extract of D. indica are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-kappaB activation. The extract of D. indica can also slightly promote HO-1 production to reduce inflammation.

Preliminary exploration on anti-inflammatory mechanism of Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) in vitro.

Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) is a novel member of the tannin family which has been discovered from many medicinal plants and has been confirmed in many pharmacological activities. However, the purified Corilagin that was used in experiment is rare, and the anti-inflammatory mechanism of Corilagin has not been investigated clearly. This study is to explore the inner anti-inflammatory mechanism of Corilagin. Inflammatory cellular model was established by lipopolysaccharide (LPS) interfering on RAW264.7 cell line. Levels of TNF-alpha, IL-1beta, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-alpha, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, translocation of NF-kappaB were assayed by ELISA or Griess method, real-time quantitative PCR, western blot and immunocytochemistry method, respectively. As a result, Corilagin could significantly reduce production of pro-inflammatory cytokines and mediators TNF-alpha, IL-1beta, IL-6, NO (iNOS) and COX-2 on both protein and gene level by blocking NF-kappaB nuclear translocation. Meanwhile Corilagin could notably promote release of anti-inflammatory factor HO-1 on both protein and gene level, but suppress the release of IL-10. In conclusion, the anti-inflammatory effects of Corilagin are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-kappaB activation. Corilagin also can promote HO-1 production to induce regression of inflammation but can inhibit IL-10 production like Dexamethasone. Corilagin possesses a potential anti-inflammatory effect by not only abating inflammatory impairment but also promoting regression of inflammation and has a good prospect to be used in many inflammation-related diseases.

Anti-inflammatory effects of ethanol extract from Kummerowia striata (Thunb.) Schindl on lps-stimulated RAW 264.7 cell.

Kummerowia striata (Thunb.) Schindl has long been used as a fork herb in inflammation-related therapy. This study was undertaken to determine the anti-inflammatory effect of the plant. High performance liquid chromatography (HPLC) was used for evaluating the extract. While dexamethasone (DM) was used as a positive control, the effects of ethanol extract on the production of IL-1beta, IL-6, NO, COX-2 and TNF-alpha, the expression of iNOS mRNA, TNF-alpha mRNA, COX-2 mRNA, protein production of COX-2 and HO-1, NF-kappaB and I-kappaB of LPS-stimulated RAW 264.7 cells were studied by sandwich ELISA, real-time PCR, Western blot analysis and immunocytochemistry assay respectively. The results showed that K. striata (Thunb.) Schindl had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell. On one hand, it could significantly inhibit the production of IL-1beta, IL-6, NO, TNF-alpha, COX-2 in LPS-stimulated cell than that of single LPS stimulated cell (p < 0.01 or p < 0.05). On the other hand, it could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p < 0.01 or p < 0.05). Furthermore, the extract also could inhibit the production of NF-kappaB and I-kappaB compared to single LPS stimulated cell. In a word, it suggested that the anti-inflammatory actions of K. striata (Thunb.) Schindl ethanol extract might be due to the down-regulation of IL-1beta, IL-6, NO, TNF-alpha and COX-2 via the suppression of NF-kappaB activation and conversation of I-kappaB production, and another pathway was up regulating the production of IL-10 and HO-1.

Anti-inflammatory mechanism of Rungia pectinata (Linn.) Nees.

This study is to explore the inner anti-inflammatory mechanism of the ethanol extract of Rungia pectinata (Linn.) Nees. As a result, the ethanol extract of Rungia pectinata (Linn.) Nees could not only strongly reduce production of pro-inflammatory cytokines and mediators via blocking NF-kappaB activation but slightly promote release of anti-inflammatory mediator HO-1 and suppress IL-10 secretion. In conclusion, compared to Dexamethasone, Rungia pectinata (Linn.) Nees has not only similar effects on antagonizing pro-inflammatory mediators and cytokines but also mild effects on promoting production of anti-inflammatory mediators.

In vitro anti-inflammatory effects of different solution fractions of ethanol extract from Melilotus suaveolens Ledeb.

BACKGROUND: Melilotus suaveolens Ledeb (M. suaveolens Ledeb) has long been used as a folk medicine in inflammation-related therapy. This study was undertaken to determine the anti-inflammatory effect of the plant. METHODS: Petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, aqueous fraction were obtained from ethanol extract of M. suaveolens Ledeb and evaluated by high performance liquid chromatography (HPLC). While dexamethasone (DM) was used as a positive control, the effects of different solution fractions of ethanol extract on tumor necrosis factor alpha (TNF-alpha) mRNA, cyclooxygenase 2 (COX-2) mRNA, COX-2 and nuclear factor kappaB (NF-kappaB) of LPS-stimulated RAW 264.7 cells were studied by real-time PCR, Western blot analysis and immunocytochemical assay, respectively. RESULTS: Coumarin was one of the main ingredients in different solution fractions of ethanol extract except the aqueous fraction with no inflammatory effect. The petroleum ether fraction, ethyl acetate fraction and n-butanol fraction of ethanol extract could inhibit the production of TNF-alpha mRNA, COX-2 mRNA and NF-kappaB to some extent. CONCLUSIONS: Different solution fractions of ethanol extract from M. suaveolens Ledeb had similar anti-inflammatory effect as did dexamethasone except the aqueous fraction. Coumarin was likely to be essential to the anti-inflammatory effect, and other ingredients might attribute to their different anti-inflammatory effects from the HPLC fingerprint.

Inner anti-inflammatory mechanisms of petroleum ether extract from Melilotus suaveolens Ledeb.

UNLABELLED: Melilotus suaveolens Ledeb is a species of traditional medical plant for treating inflammation-related disease. This study is to explore the inner anti-inflammatory mechanism on petroleum ether extract from Melilotus suaveolens Ledeb. MATERIALS AND METHODS: Inflammatory cellular model was founded by intervention of lipopolysaccharide (LPS) on RAW264.7 cell line. Secretion of TNF-alpha, IL-1beta, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-alpha, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, activation of NF-kappaB and ingredients in the extract were assayed. RESULTS: The extract could not only reduce production of pro-inflammatory mediators by blocking NF-kappaB activation but promote release of anti-inflammatory mediator HO-1 significantly. The only active ingredient in the extract was coumarin and the concentration of coumarin in each 1 g extract was 0.27822 mg. CONCLUSION: Compared to Dexamethasone, the extract not only has similar effects on antagonizing pro-inflammatory mediators and cytokines but has effects on promoting production of anti-inflammatory mediators.