JiaGaSongTang improves chronic cholestasis via enhancing FXR-mediated bile acid metabolism.

BACKGROUND: Bile acid (BA) enterohepatic circulation disorders are a main feature of chronic cholestatic diseases. Promoting BA metabolism is thus a potential method of improving enterohepatic circulation disorders, and treat enterohepatic inflammation, oxidative stress and fibrosis due to cholestasis. PURPOSE: To investigate the effect of JiaGaSongTang (JGST) and its blood-absorbed ingredient 6-gingerol on α-naphthylisothiocyanate (ANIT)-induced chronic cholestasis, as well as elucidate the underlying regulatory mechanism. METHODS: Chronic cholestasis was induced in mice via subcutaneous injection of ANIT (50 mg/kg) every other day for 14 d. Treatment groups were administered JGST orally daily. Damage to the liver and intestine was observed using histopathological techniques. Biochemical techniques were employed to assess total BA (TBA) levels in the serum, liver, and ileum samples. Liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze fecal BA components. Bioinformatic methods were adopted to screen the core targets and pathways. The blood-absorbed ingredients of JGST were scrutinized via LC-MS/MS. The effects of the major JGST ingredients on farnesoid X receptor (FXR) transactivation were validated using dual luciferase reporter genes. Lastly, the effects of the FXR inhibitor, DY268, on JGST and 6-gingerol pharmacodynamics were observed at the cellular and animal levels. RESULTS: JGST ameliorated pathological impairments in the liver and intestine, diminishing TBA levels in the serum, liver and gut. Fecal BA profiling revealed that JGST enhanced the excretion of toxic BA constituents, including deoxycholic acid. Bioinformatic analyses indicated that JGST engaged in anti-inflammatory mechanisms, attenuating collagen accumulation, and orchestrating BA metabolism via interactions with FXR and other pertinent targets. LC-MS/MS analysis identified six ingredients absorbed to the bloodstream, including 6-gingerol. Surface plasmon resonance (SPR) and dual luciferase reporter gene assays confirmed the abilities of 6-gingerol to bind to FXR and activate its transactivation. Ultimately, in both cellular and animal models, the therapeutic efficacy of JGST and 6-gingerol in chronic cholestasis was attenuated in the presence of FXR inhibitors. CONCLUSION: The findings, for the first time, demonstrated that 6-gingerol, a blood-absorbed ingredient of JGST, can activate FXR to affect BA metabolism, and thereby attenuate ANIT-induced liver and intestinal injury in chronic cholestasis mice model via inhibition of inflammation, oxidative stress, and liver fibrosis, in part in a FXR-dependent mechanism.

Liang-Ge-San: a classic traditional Chinese medicine formula, attenuates acute inflammation via targeting GSK3ß.

Sepsis is a serious life-threatening health disorder with high morbidity and mortality rates that burden the world, but there is still a lack of more effective and reliable drug treatment. Liang-Ge-San (LGS) has been shown to have anti-inflammatory effects and is a promising candidate for the treatment of sepsis. However, the anti-sepsis mechanism of LGS has still not been elucidated. In this study, a set of genes related to inflammatory chemotaxis pathways was downloaded from Encyclopedia of Genes and Genomes (KEGG) and integrated with sepsis patient information from the Gene Expression Omnibus (GEO) database to perform differential gene expression analysis. Glycogen synthase kinase-3ß (GSK-3ß) was found to be the feature gene after these important genes were examined using the three algorithms Random Forest, support vector machine recursive feature elimination (SVM-REF), and least absolute shrinkage and selection operator (LASSO), and then intersected with possible treatment targets of LGS found through the search. Upon evaluation, the receiver operating characteristic (ROC) curve of GSK-3ß indicated an important role in the pathogenesis of sepsis. Immune cell infiltration analysis suggested that GSK-3ß expression was associated with a variety of immune cells, including neutrophils and monocytes. Next, lipopolysaccharide (LPS)-induced zebrafish inflammation model and macrophage inflammation model was used to validate the mechanism of LGS. We found that LGS could protect zebrafish against a lethal challenge with LPS by down-regulating GSK-3ß mRNA expression in a dose-dependent manner, as indicated by a decreased neutrophils infiltration and reduction of inflammatory damage. The upregulated mRNA expression of GSK-3ß in LPS-induced stimulated RAW 264.7 cells also showed the same tendency of depression by LGS. Critically, LGS could induce M1 macrophage polarization to M2 through promoting GSK-3ß inactivation of phosphorylation. Taken together, we initially showed that anti-septic effects of LGS is related to the inhibition on GSK-3ß, both in vitro and in vivo.

Jiawei Yanghe Decoction suppresses breast cancer by regulating immune responses via JAK2/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Yanghe Decoction (JWYHD) is a widely used traditional Chinese medicine prescription in the clinical setting for the treatment of autoimmune diseases. Many studies showed that JWYHD has anti-tumor activities in cell and animal models. However, the anti-breast cancer effects of JWYHD and the underlying mechanisms of action remain unknown. AIM OF STUDY: This study aimed to determine the anti-breast cancer effect and reveal the underlying mechanisms of action in vivo, in vitro and in silico. MATERIALS AND METHODS: Orthotopic xenograft breast cancer mouse model and inflammatory zebrafish model were used to observe the anti-tumor effect and immune cell regulation of JWYHD. Moreover, the anti-inflammatory effect of JWYHD were evaluated by the expression of RAW 264.7 cells. JWYHD active ingredients were obtained by UPLC-MS/MS and potential targets were screened by network pharmacology. The therapeutic targets and signaling pathways predicted by computer were assessed by Western blot, real-time PCR (RT-PCR), immunohistochemistry (IHC) staining, and Enzyme-linked immunosorbent assays (ELISA) to explore the therapeutic mechanism of JWYHD against breast cancer. At last, Colivelin and Stattic were used to explore the effect of JWYHD on JAK2/STAT3 pathway. RESULTS: JWYHD significantly decreased the tumor growth in a dose-dependent manner in the orthotopic xenograft breast cancer mouse model. Flow cytometry and IHC results indicated that JWYHD decreased the expressions of M2 macrophages and Treg while increasing M1 macrophages. Meanwhile, ELISA and Western blot results showed a decrease in IL-1ß, IL-6, TNFα, PTGS2 and VEGFα in tumor tissue of JWYHD groups. The results were also verified in LPS-induced RAW264.7 cells and zebrafish inflammatory models. TUNEL assay and IHC results showed that JWYHD significantly induced apoptosis. Seventy-two major compounds in JWYHD were identified by UPLC-MS/MS and Network pharmacology. It was found that the significant binding affinity of JWYHD to TNFα, PTGS2, EGFR, STAT3, VEGFα and their expressions were inhibited by JWYHD. IHC and Western blot analysis showed that JWYHD could decrease the expression of JAK2/STAT3 pathway. Furthermore, Colivelin could reverse the decrease effect of JWYHD in vitro. CONCLUSION: JWYHD exerts a significant anti-tumor effect mainly by inhibiting inflammation, activating immune responses and inducing apoptosis via the JAK2/STAT3 signaling pathway. Our findings provide strong pharmacological evidence for the clinical application of JWYHD in the management of breast cancer.

Quassinoids from Eurycoma longifolia with antiviral activities by inhibiting dengue virus replication.

BACKGROUND: Dengue caused by dengue virus (DENV) spreads rapidly around the world. However, there are no worldwide licensed vaccines or specific antivirals to combat DENV infection. Quassinoids are the most characteristic components of Eurycoma longifolia, which have been reported to display a variety of biological activities. However, whether quassinoids exert anti-DENV activities remains unknown. PURPOSE: To test the quassinoids of E. longifolia for their activity against DENV and to clarify the potential mechanisms. METHODS: The quassinoids from E. longifolia were isolated by chromatography techniques, and their chemical structures were elucidated by spectroscopic analysis. The anti-DENV activities of quassinoids on baby hamster kidney cells BHK-21 were determined by lactate dehydrogenase (LDH) assay. The synthesis of progeny virus was measured by plaque assay. The expression levels of envelope protein (E) and non-structural protein 1 (NS1) were evaluated by qRT-PCR, Western blot and immunofluorescence assays. Molecular docking was used to screen the potential targets of the most active quassinoid against DENV-2, and surface plasmon resonance analysis was employed to confirm the direct binding between the most active quassinoid and potential target. RESULTS: Twenty-four quassinoids, including three new quassinoids (1 - 3), were isolated from the ethanol extract of E. longifolia. Quassinoids 4, 5, 9, 11, 12, 15, 16, 17, 19 and 20 significantly reduced the LDH release at the stages of viral binding and entry or intracellular replication. Among them, 19 (6α-hydroxyeurycomalactone, 6α-HEL) exhibited the best anti-DENV-2 activities with an EC50 value of 0.39 ± 0.02 µM. Further experiments suggested that 6α-HEL remarkably inhibited progeny virus synthesis and mRNA and protein expression levels of E and NS1 of DENV-2. Time-of-drug-addition assay suggested that 6α-HEL inhibited intracellular replication of DENV-2 at an early stage. Moreover, 6α-HEL was shown to interact with NS5-RdRp domain at a binding affinity of -8.15 kcal/mol. SPR assay further verified 6α-HEL bound to RdRp protein with an equilibrium dissociation constant of 1.49 × 10-7 M. CONCLUSION: Ten quassinoids from E. longifolia showed anti-DENV activities at processes of virus binding and entry or intracellular replication. The most active quassinoid 6α-HEL exerts the anti-DENV-2 activities at intracellular replication stage by directly targeting the NS5-RdRp protein. These results suggest that 6α-HEL could be a promising candidate for the treatment of DENV-2 infection.

[A study on acquired acid resistance of enamel and dentine irradiated by Er, Cr: YSGG laser in vitro].

OBJECTIVE: To evaluate the acid resistance of enamel and dentin irradiated by Er, Cr: YSGG laser to artificial caries formation in vitro. METHODS: Enamel and dentin samples were irradiated with Er, Cr: YSGG laser at 6 W or 4 W for 6 second, respectively. Every sample was immersed in 3 ml of lactic demineralization solution at pH 4.8 and incubated at 37 degrees C for 24 hours. The parts per million of calcium ion of each solution was measured by atomic absorption spectrophotometry, the quantities of calcium and phosphate on the surface of enamel and dentin sample were examined by SED-X and the ultrastructure was investigated by SEM. RESULTS: In the enamel and dentin samples, the dissolved Ca(2+) concentration in laser group were significantly decreased compared with that of the control group (P < 0.05). The quantities of Ca (weight%) in enamel and dentin were significantly higher in the laser group than in the control group (P < 0.05). The content of P was increased in dentin and enamel samples, but only the increase in enamel sample was significant (P < 0.05). In both samples, no significant changes on Ca/P weight ratio were found between the groups. The irradiated enamel and dentin surface lacked a smear layer and showed various patterns of microirregulation with a scaly appearance, the openings of dentinal tubules were clearly visible, but no melting or carbonization was observed. CONCLUSION: Er, Cr: YSGG laser irradiation is effective for increasing acid resistance of dental hard tissue and does not cause thermal side effect.

[Research on the effect of root canal seal achieved by irradiation of Er, Cr:YSGG laser].

PURPOSE: To evaluate the effect of root canal seal achieved by routine root canal preparation and irradiation of Er, Cr:YSGG laser in root canal therapy. METHODS: One hundred and five freshly single-rooted extracted human teeth were selected for the study, which were randomly divided into seven groups of fifteen each. The anatomic crowns were removed at the cementoenamel junction and the remaining roots were prepared endodontically with conventional methods. Then they were treated with Er, Cr:YSGG laser in different parameters and patterns. The effect of root canal seal achieved by Er, Cr:YSGG laser was evaluated with microleakage measurements. Newman-Keuls test and one-way analysis of variance were used to determine the statistical differences between various groups. A value of P<0.05 was considered to be significant. RESULTS: The amount of dye penetration from an apical stop with irradiation of Er, Cr:YSGG laser was significantly lower (P<0.05) except 3W laser without water flow, and in 1W Er, Cr:YSGG laser without water flow, the amount of apical dye penetration was significantly decreased (P<0.01). CONCLUSIONS: The utilizing of Er, Cr:YSGG laser in small parameters prior to root canal filling could promote the effect of apical seal. Based on our results, it appeared that the seal effect of 1W Er, Cr:YSGG laser without water flow was superior to the effects of others.