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.

Jia-ga-song-tang protection against alcoholic liver and intestinal damage.

Gut-liver axis and cellular homeostasis play key roles in alcohol liver disease (ALD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a stress-sensitive guarantor of cellular homeostasis. We investigated whether the beneficial effects and underlying mechanisms of Jia-ga-song Tang (JGST) against ALD were associated with gut-liver axis and cellular homeostasis. A predictive network depicting the relationship between Jia-Ga-Song-Tang (JGST) and alcoholic liver disease (ALD) was designed by Network pharmacology. Next, 5% v/v Lieber-DeCarli alcohol liquid diet was used to establish the ALD. JGST protected the liver damage, repaired the intestines to alleviate the Two-hit on the liver, and balanced the cellular homeostasis. It was manifested in repairing the liver and intestinal pathological structure, reducing serum ALT, AST, and liver TG, TC, MDA, CAT, and increasing liver GSH, and intestine GSH-Px. JGST mainly inhibited the liver mRNA levels of HO-1, NQO1, GCLC, FASN, and PPARα and activated the intestinal mRNA levels of HO-1 and NQO1, while inhibiting the liver protein levels of HO-1, NQO1. Furthermore, LPS and LBP in the plasma and the expression of inflammatory factors such as IL-1ß, TNF-α, IL-6, TGFß1, CD14, and Myd88 were reduced after treatment to prove that JGST protects the liver from Two-hit. Ethanol was used to intervene in HepG2 and IEC-6 to establish an ALD cell model and treated by Germacrone, ML385, and TBHQ. repaired the intestinal barrier, and inhibited Nrf2 in IEC-6, but protect the HepG2 by activating Nrf2 to balance cellular homeostasis. Our results reinforce that JGST provides an effective protective method for alcoholic liver disease (ALD) by regulating Gut-liver axis and cellular homeostasis.

Intrahepatic cholestasis induced by α-naphthylisothiocyanate can cause gut-liver axis disorders.

Clinical studies have shown that Intrahepatic cholestasis is closely related to intestinal injury. The gut-liver axis theory suggests that the intestine and liver are closely related, and that bile acids are important mediators linking the intestine and liver. We compared two cholestasis models: a single injection model that received a single subcutaneous ANIT injection (75 mg/kg), and a multiple subcutaneous injection model that received an injection of ANIT (50 mg/kg) every other day for 2 weeks. We used Transmetil (ademetionine 1,4-butanedisulfonate) to relieve intrahepatic cholestasis in the multiple injection group. In the multiple injection group, we found increased hepatic bile duct hyperplasia, increased fibrosis of the liver, increased small intestine inflammation and oxidative damage, increased harmful bile acids, decreased bile acids transporter levels. After treatment with Transmetil, the liver and gut injuries were relieved. These results suggest that intrahepatic cholestasis can cause disorders of the gut-liver axis.

Hepatic dysfunction induced by intestinal dysbacteriosis mainly manifests as immunologic abnormity in mice.

Currently, the potential role of the alterations occurring in the liver immune system and intestinal flora in liver injury remains unknown. Our study aimed to explore the impacts of intestinal microbial barrier damage induced by ceftriaxone on liver immunity. We developed the BALB/c mice model by administering ceftriaxone. The intestinal microbial barrier damage was observed by 16S rRNA, and the pathological changes of intestines and livers were detected by H&E or transmission electron microscope. The activation of immunocytes were tested by Flow Cytometry; the expression of LPS, ALT, AST, IL-6 and TNF-α were detected by Limulus Test or ELISA. Compared to the control, the intestinal microbes significantly decreased in ceftriaxone group. Additionally, the weight of cecum contents increased, the intestinal wall became thinner and the villus in the small intestine and cecum were damaged. The expression of LPS and the ratio of liver lymphocytes were significantly increased. H&E results indicated the structures of liver arose the pathologic changes. Meanwhile, the content of serum ALT, AST, IL-6 and TNF-α increased. Collectively, our study indicates that the damages of gut microbial barrier induced by ceftriaxone prompted activation of immunocytes and release of inflammatory cytokines, which may lead to chronic inflammation in liver.

SanWeiGanJiang San relieves liver injury via Nrf2/Bach1.

ETHNOPHARMACOLOGICAL RELEVANCE: San Wei Gan jiang San (SWGJS) also called Jia Ga Song Tang, is widely used in ancient medicine for liver diseases. THE AIM OF THIS STUDY: To identify the blood components of SWGJS. To determine the hepatoprotective effect and the mechanism of SWGJS by observing its effect on different degrees of liver damage and gene knockdown cells. MATERIALS AND METHODS: SWGJS treated serum was analyzed by UPLC-MS to identify blood components. CCl4-induced chronic liver injury in rats was treated with SWGJS. The viscera index was calculated. Pathological changes of the liver were determined by HE staining and analysis of by following: GSH-Px and MDA in liver homogenate; ALT and AST in serum; mRNA expression of Nrf2, Bach1, and HO-1 by RT-PCR; Nrf2 and Bach1 in the nucleus and cytoplasm; HO-1 total expression by Western blot; silencing Nrf2 and Bach1 in human L-02 cells by siRNA; MDA, GSH-Px, GST, and GR in cell supernatants; and GSH/GSSG within the cell. RESULTS: We found that 6-gingerol was one of the blood components in the serum treated with SWGJS. In CCl4-induced chronic liver injury in rats, SWGJS repaired the liver structure in the early stages of liver damage as evidenced by reduced ALT and AST in the serum, increased GSH-Px activity and decreased MDA levels in the liver over time. SWGJS has excellent antioxidant and hepatoprotective effects and prevents disease progression. The mechanism of SWGJS is related to the dynamics promoting Nrf2 entry to the nucleus and Bach1 exit from the nucleus. In L-02 cells with silenced Nrf2, the antioxidant enzyme system was disordered, and the change in the cellular redox state was not conducive to antioxidative stress. However, in cells with silenced Bach1, the antioxidant enzyme system could be activated to promote cellular antioxidant stress. SWGJS had a combined effect on Nrf2 and Bach1 contributing to antioxidant properties and liver protection. SWGJS increased GSH-Px and HO-1, decreased MDA and increased the ratio of GSH/GSSG by upregulating the expression of Nrf2 to enhance its antioxidant effects. At the same time, SWGJS had a specific impact on decreasing Bach1. Its elevation of GST is due to the overall performance of increasing Nrf2 and decreasing Bach1. This mechanism of action embodies the characteristics of the multitarget impact of traditional medicine and the antioxidation effect of SWGJS. CONCLUSIONS: 6-Gingerol is one of the blood components of SWGJS. SWGJS can regulate antioxidant enzymes, protect against liver damage in different stages, and slow the progression of liver cell damage and liver disease by increasing Nrf2 and reducing Bach1 in the nucleus, dynamically regulating Nrf2/Bach1.

Modification effects of SanWei GanJiang Powder on liver and intestinal damage through reversing bile acid homeostasis.

BACKGROUND: Sanwei Ganjiang Powder (SWGJ), derived from traditional Chinese medicine (TCM), has long demonstrated its effectiveness in long-term liver damage therapy. Recent studies indicated that it can also regulate the intestinal tract, although the underlying molecular mechanisms of this remain mysterious. The aim of the study is to investigate the mechanisms of SWGJ against dysbacteriosis and carbon tetrachloride (CCl4)-induced gut-liver axis damage underlying bile acid enterohepatic circulation. METHODS: To observe the regulatory effects of SWGJ on Liver and Intestinal Damage, we explored two animal models. In model 1, sixty BALB/c mice were subjected to oral gavage with 12 g/kg of ceftriaxone sodium for 10d; during this time, SWGJ, bifendate and bifico were sequentially administered over 7d. In model 2, the model of chronic liver injury was induced by subcutaneous injection of 40% CCl4 oil solution twice per week for 8 weeks. From the 3rd week, SWGJ, bifendate and bifico were sequentially administered for 6 weeks. Intestinal flora (16S rDNA analysis), histology (H&E staining), tight connections (Immunohistochemistry, IHC), ultrastructure (Transmission electron microscopy, TEM), inflammatory cytokines and LPS (Enzyme-linked immunosorbent assay, ELISA) of the intestines were assessed, and liver function was also evaluated by methods including ALT, AST and H&E staining. The levels of protein associated with bile acid metabolism were assessed by western blot. RESULTS: In model 1, SWGJ significantly decreased the activity of inflammatory cytokines and LPS compared with the ceftriaxone sodium group. In addition, SWGJ improved symptoms of intestinal flora imbalance; further, ZO-1 and occludin in the cytoplasm of intestinal villus epithelial cells was increased, and the histopathology of the ileum was improved. Notably, the expression of ALT and AST was significant increased, and disordered hepatic lobule structures were clearly observed in liver histopathology in model group; SWGJ can significantly improve these changes. Furthermore, the levels of proteins related to bile acid synthesis, such as CYP7A1, were significantly upregulated in the SWGJ group compared with the model, and proteins related to excretion and reabsorption, such as NTCP, Mrp2 and BESP, were also upregulated. Importantly, SWGJ increased the nuclear expression of nuclear factor-E2-related factor-2 (Nrf2). Similar results appeared in model 2. CONCLUSION: This study suggests that SWGJ may elicit significant effects on the treatment of gut-liver axis damage, potential mechanisms at least partially involve bile acid enterohepatic, and increasing of the nuclear Nrf2 levels.

Systems pharmacology-based investigation of Sanwei Ganjiang Prescription: related mechanisms in liver injury.

Liver injury remains a significant global health problem and has a variety of causes, including oxidative stress (OS), inflammation, and apoptosis of liver cells. There is currently no curative therapy for this disorder. Sanwei Ganjiang Prescription (SWGJP), derived from traditional Chinese medicine (TCM), has shown its effectiveness in long-term liver damage therapy, although the underlying molecular mechanisms are still not fully understood. To explore the underlining mechanisms of action for SWGJP in liver injury from a holistic view, in the present study, a systems pharmacology approach was developed, which involved drug target identification and multilevel data integration analysis. Using a comprehensive systems approach, we identified 43 candidate compounds in SWGJP and 408 corresponding potential targets. We further deciphered the mechanisms of SWGJP in treating liver injury, including compound-target network analysis, target-function network analysis, and integrated pathways analysis. We deduced that SWGJP may protect hepatocytes through several functional modules involved in liver injury integrated-pathway, such as Nrf2-dependent anti-oxidative stress module. Notably, systems pharmacology provides an alternative way to investigate the complex action mode of TCM.

Antioxidant and anti­inflammatory effects of DHK­medicated serum on high glucose­induced injury in endothelial cells.

It has been shown that oxidative damage and inflammation caused by hyperglycemia in endothelial cells are key factors triggering diabetic vascular complications. The aim of the present study was to investigate the antioxidant and anti­inflammatory effects of Danhong Huayu Koufuye (DHK)­medicated serum on high glucose (HG)­induced injury in endothelial cells, and examine its underlying mechanisms. EA. hy926 cells were treated with normal glucose, HG, or HG with DHK­medicated serum. Cell viability was assessed using the MTT method. Apoptosis was detected using flow cytometry. Intracellular reactive oxygen species (ROS) levels were measured using the 2',7'­dichlorodihydrofluorescein method. Cell culture supernatant was collected for detecting the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA). The protein expression levels of intercellular adhesion molecule­1 (ICAM­1), nuclear factor­κB (NF­κB), hypoxia­inducible factor­1α (HIF­1α) and vascular endothelial growth factor (VEGF) were determined using western blot analysis. The results revealed that DHK­medicated serum accelerated the proliferation and inhibited the apoptosis of cells treated with HG (P<0.01) in a dose­dependent manner. Compared with the HG group, the high levels of ROS and MDA were significantly reduced by DHK­medicated serum (P<0.01). A 10% concentration of DHK­medicated serum increased the activities of SOD and GPx by 59.4 and 95.5%, respectively. The high protein expression levels of ICAM­1, NF­κB, VEGF and HIF­1α were significantly ameliorated by DHK­medicated serum (P<0.01, vs. HG group). These findings indicated that DHK­medicated serum protected EA. hy926 cells from HG­induced injury and apoptosis through antioxidation and anti­inflammatory effects.

Triterpenoids with Antiplatelet Aggregation Activity from the Roots of Ilex pubescens.

Two new triterpenes and five new triterpene saponins, named ilexpusons A-G (1-7), as well as eight known compounds were isolated from Ilex pubescens. The structures of the new compounds were established by a combination of chemical and spectroscopic methods, including HRESIMS, 1H-NMR, 13C-NMR, 1H-1H COSY, HSQC, HMBC, and NOESY. Additionally, the biological activity of compounds 1 - 15 against adenosine diphosphate-induced platelet aggregation in rabbit plasma was determined. Among the tested compounds, 1, 2, 5, 6, 8, 13, 14, and 15 exhibited significant inhibition of platelet aggregation in vitro.

Antiplatelet aggregation triterpene saponins from the barks of Ilex rotunda.

Four new triterpene saponins, rotundinosides A-D (1-4) and seven known triterpene saponins (5-11) were isolated from a methanol extract of the barks of Ilex rotunda Thunb. The new saponins were characterized as 3-O-ß-d-glucopyranosy1-(1→2)-ß-d-xylopyranosyl siaresinolic acid 28-O-ß-d-glucopyranoside (1), 3-O-[ß-d-glucopyranosy1-(1→2)-ß-d-xylopyranosyl]-3ß,19α-dihydroxyurs-12-en-28-oic-O-ß-d-glucopranosy1ester (2), 3-O-[α-l-rhamnopyranosyl-(1→2)-ß-d-glucopyranosy1-(1→2)-α-l-arabinopyranosyl]-3ß,19α-dihydroxyurs-12-en-28-oic-O-ß-d-glucopyranosy1 ester (3), and 3-O-α-l-rhamanopyranosyl-(1→2)-ß-d-glucopyranosy1-(1→2)-α-l-arabinopyranosyl ilexgenin B 28-O-ß-d-glucopyranosy1 ester (4), respectively. Their structures were established by extensive spectroscopic analysis, including HSQC, HMBC, (1)H-(1)H COSY, NOESY and acid hydrolysis, and also by the comparison of their spectroscopic data with those of related compounds. The known compounds 5-11 were all obtained from this species for the first time. The biological activity of compounds 1-11 against ADP induced platelet aggregation in rabbit plasma was determined. Among the tested compounds 1, 3, 5 and 10 exhibited strong inhibition of platelet aggregation in vitro, with IC50 values of 11.4±2.2, 10.4±1.3, 13.2±2.4, and 15.1±3.4µM, respectively.

[Pattern Recognition Analysis for Quality Control of Jia Ga Song Tang by GC-MS].

OBJECTIVE: To establish a scientific method for identitication and evaluation of the Tibetan prescription Jia Ga Song Tang. METHODS: Volatile oil was extracted by water steam distillation and analyzed by GC-MS. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to the samples for chemical fingerprint pattern recognition research. RESULTS: 16 samples according to hierarchical cluster analysis (HCA) and principal component analysis (PCA) were divided into two classes, and results from two recognition analysis methods had good consistency. CONCLUSION: GC-MS-pattern recognition method was a kind of scientific, accurate and effective method for the quality evaluation of Jia Ga Song Tang.

Role of Nrf2 activation and NF-κB inhibition in valproic acid induced hepatotoxicity and in diammonium glycyrrhizinate induced protection in mice.

Diammonium glycyrrhizinate (DG), an active compound extracted and purified from liquorices root, has been reported to exhibit antioxidant and anti-inflammatory properties. The aim of this study was to investigate the effect and underlying mechanisms of DG on the hepatotoxicity induced by valproic acid (VPA). DG at the dose of 60mg/kg was orally administered with VPA (100mg/kg) to mice once daily for 14 consecutive days. DG treatment attenuated VPA-induced liver dysfunction, structural damage, glutathione depletion and decrease in antioxidant enzymes in BALB/C mice. DG prevented VPA-induced depletion of cytosolic nuclear factor E2-related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2, which, in turn, up-regulated phase II/antioxidant enzyme activities. The effects of VPA and DG on Nrf2 expression in HepG2 cells were in consistent with that of mice. Furthermore, an increase in the nuclear levels of nuclear factor-kappaB (NF-κB) was observed in the livers of VPA-treated mice that coincided with induction of inflammatory cytokines. In contrast, DG inhibited NF-κB translocation and that subsequently decreased inflammatory cytokines. Taken together, these results demonstrate that DG attenuates VPA-induced liver injury through increasing the expression of Nrf2 mediated phase II/antioxidant enzymes and simultaneously decreasing the expression of inflammatory mediators.

Diterpenoid alkaloids and flavonoids from Delphinium trichophorum.

Five hetisane-type C20-diterpenoid alkaloids, trichodelphinines A-E, one delnudine-type C20-diterpenoid alkaloid, trichodelphinine F and three known flavonoids, quercetin, quercetin 3-O-ß-D-glucopyranoside, and quercetin 3-O-ß-D-glucopyranoside-7-O-α-L-arabinopyranoside, were isolated from whole plants of Delphinium trichophorum Franch. Their structures were elucidated on the basis of extensive spectroscopic analysis, including HSQC, HMBC, (1)H-(1)H COSY, NOESY and X-ray crystallographic analysis, and from chemical evidence. The cytotoxic activities of the diterpenoid alkaloids were evaluated using the MTT method, and the IC50 values of their cytotoxicity against A549 cancer cells ranged from 12.03 to 52.79 µM.

[Analysis of chemical constituents of volatile components from Jia Ga Song Tang by GC-MS].

OBJECTIVE: To analyze the chemical components of volatile components from Jia Ga Song Tang. METHODS: The volatile oils were extracted by water steam distillation. The chemical components of essential oil were analyzed by GC-MS and quantitatively determined by a normalization method. RESULTS: 103 components were separated and 87 components were identified in the volatile oil of Zingiberis Rhizoma. 58 components were separated and 38 components were identified in the volatile oil of Myristicae Semen. 49 components were separated and 38 components were identified in the volatile oil of Amomi Rotundus Fructus. 89 components were separated and 63 components were identified in the volatile oil of Jia Ga Song Tang. CONCLUSION: Eucalyptol, ß-phellandrene and other terpenes were the main compounds in the volatile oil of Jia Ga Song Tang. Changes in the kinds and content of volatile components can provide evidences for scientific and rational compatibility for Jia Ga Song Tang.

Two new abietane diterpenoids from the caulis and leaves of Callicarpa kochiana.

Previous studies revealed that diterpenoids from Callicarpa genus were mainly of clerodane-type and phyllocladane-type, and abietane-type diterpenoids were seldom reported. In this paper, we reported two new abietane diterpenoids, kochianic acid A (1) and kochianic acid B (2), together with two known abietane-type diterpenoids, pedunculatic acid B (3) and 7α, 15-dihydroxydehydroabietic acid (4), which were all isolated from Callicarpa kochiana. The structures of the new compounds 1 and 2 were elucidated on the basis of extensive spectroscopic analysis, including HSQC, HMBC, (1)H-(1)H COSY, ROESY and finally confirmed by single-crystal X-ray diffraction.

New sphingolipid psychotramide A-D from the stem of Psychotria sp.

Chemical study of the Psychotria sp. led to the isolation of a new type of ceramide named psychotramide (1). HR-FAB-MS analysis revealed that psychotramide consists of four new sphingolipids. Their structures were determined on the basis of the spectroscopic data and chemical methods to be psychotramide A (1-a), psychotramide B (1-b), psychotramide C (1-c) and psychotramide D (1-d).

New triterpene saponins from the root of Ilex pubescens.

Two new triterpene glycosides named ilexpublesnin A (1) and ilexpublesnin B (2) were isolated from the root of Ilex pubescens. Their structures were determined as 3-O-(ß-D-xylopyranosyl)-28-O-(ß-D-glucopyranosyl)-3ß, 19α-dihydroxyurs-23-oxo-12-en-28-oic acid (1) and 28-O-(ß-D-xylopyranosyl-(2→1)-ß-D-glucopyranosyl)-3ß, 19α-dihydroxyurs-23-oxo-12-en-28-oic acid (2) on the basis of chemical and spectroscopic methods.

Gerardianin A, a new abietane diterpenoid from Isodon lophanthoides var. gerardianus.

A new abietane diterpenoid, gerardianin A (1), along with a known compound 6,7-dehydroroyleanone (2), has been isolated from the aerial parts of Isodon lophanthoides var. gerardianus [Bentham] H. Hara. The structure of 1 was determined on the basis of spectroscopic methods and X-ray single-crystal diffraction analysis.