Polygoni Multiflori Radix interferes with bile acid metabolism homeostasis by inhibiting Fxr transcription, leading to cholestasis.

Objective: To explore the possible mechanisms of cholestasis induced by Polygoni Multiflori Radix (PM). Methods: Low and high doses of water extract of PM were given to mice by gavage for 8 weeks. The serum biochemical indexes of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyltransferase (GGT) alkaline phosphatase (ALP) and so on were detected in the second, fourth, sixth, and eighth weeks after administration. At the end of the eighth week of administration, the bile acid metabolic profiles of liver and bile were screened by high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS). Liver pathological changes were observed by hematoxylin and eosin staining. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA transcription of the target genes and Western blotting (WB) was used to the detect target protein expression. Results: Biochemical tests results showed the values of ALP and GGT were two and three times greater than the normal values respectively, and the value of R was less than 2. Histopathology also showed that PM caused lymphocyte infiltration, a small amount of hepatocyte necrosis and nuclear fragmentation in mouse liver. The proliferation of bile duct epithelial cells was observed in the high group. These results indicated that PM may lead to cholestatic liver injury. HPLC-QQQ-MS/MS analysis with the multivariate statistical analysis revealed significant alterations of individual bile acids in liver and gallbladder as compared to those of the control group. RT-qPCR showed that the transcription of Fxr, Shp, Bsep, Bacs, Mdr2, and Ugt1a1 were downregulated and that of Cyp7a1, Mrp3, and Cyp3a11 was significantly upregulated in the treatment group. WB demonstrated that PM also markedly downregulated the protein expression of FXR, BSEP, and MDR2, and upregulated CYP7A1. Conclusion: PM inhibited the expression of FXR, which reduced the expression of MDR2 and BSEP, leading to the obstruction of bile acids outflow, and increased the expression of CYP7A1, resulting in an increase of intrahepatic bile acid synthesis, which can lead to cholestasis.

Danhe granule ameliorates nonalcoholic steatohepatitis and fibrosis in rats by inhibiting ceramide de novo synthesis related to CerS6 and CerK.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhe granule (DHG) is used by Chinese doctors to treat blood stasis, phlegm and dampness. Its lipid-lowering ability has been investigated in our previous research. However, the anti-liver inflammatory and fibrotic effects and mechanism of action of DHG in non-alcoholic steatohepatitis (NASH) have not been explored. AIM OF THE STUDY: To evaluate the ameliorative effects of DHG on liver inflammation and fibrosis in a methionine/choline-deficient (MCD) diet-induced NASH rat model, and its underlying mechanism. MATERIALS AND METHODS: Sprague-Dawley rats were fed an MCD diet for two weeks and then treated with or without DHG by oral gavage for eight weeks. Their body weight and liver index were measured. The serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities as well as the liver triglyceride (TG) and free fatty acid (FFA) levels were tested using reagent kits. Inflammatory cytokines, including Tnf-α, Il-ß and Il-6, and fibrosis genes, including Acta2, Col1a1, Col1a2 and Tgf-ß were examined by real-time quantitative PCR (RT-qPCR). Hematoxylin-eosin (H&E), Oil Red O, Masson's and Sirius Red staining were used to observe liver changes. The plasma and liver ceramide levels were analyzed using HPLC-QQQ-MS/MS. The expression of serine palmitoyl-CoA transferase (Spt), ceramide synthase 6 (Cers6), dihydroceramide desaturase 1 (Des1), glucosylceramide synthase (Gcs), and ceramide kinase (Cerk) mRNA was assayed by RT-qPCR, while the protein expression of CerS6, DES1, GCS, CerK, and casein kinase 2α (CK2α) was tested by western blotting (WB). CerS6 degradation was evaluated using a cycloheximide (CHX) assay in vitro. RESULTS: The liver index decreased by 20% in DHG groups and the serum ALT and AST decreased by approximately 50% and 30%, respectively in the DHG-H group. The liver Oil Red O staining, TG, and FFA changes showed that DHG reduced hepatic lipid accumulation by approximately 30% in NASH rats. H&E, Masson's and Sirius Red staining and the mRNA levels of Tnf-α, Il-ß, Il-6, Acta2, Col1a1, Col1a2 and Tgf-ß revealed that DHG alleviated liver inflammation and fibrosis in NASH rats. The ceramide (Cer 16:0), and hexosylceramide (HexCer 16:0, HexCer 18:0, HexCer 22:0, HexCer 24:0 and HexCer 24:1) levels decreased by approximately 17-56% in the plasma of the DHG-M and H rats. The Cer 16:0 content in the liver decreased by 20%, 50%, and 70% with the DHG-L, M, and H treatments; additionally, the dhCer 16:0, Cer 18:0, HexCer 18:0, HexCer 20:0 Cer 22:0-1P, Cer 24:0-1p, Cer 24:1-1p, and Cer 26:1-1p levels decreased in the DHG groups. The mRNA and protein expression levels of DES1, GCS, Cerk, CerS6, and CHX assay indicated that DHG decreased the mRNA and protein expression levels of CerK and reduced CerS6 protein expression by promoting its degradation. Additionally, DHG attenuated the protein expression of CK2α which could increase CerS6 enzymatic activity by phosphorylating its C-terminal region. CONCLUSION: DHG ameliorated the levels of liver FFA and TG and inflammation and fibrosis in MCD-induced rats, which were associated with decreasing ceramide species in the plasma and liver by reducing the expression levels of CerS6 and CerK.

Shengyu Decoction treating vascular cognitive impairment by promoting AKT/HIF-1α/VEGF related cerebrovascular generation and ameliorating MAPK/NF-κB mediated neuroinflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Shengyu Decoction (SYD), a classical Chinese medicine formula, is good at nourishing blood, promoting blood circulation, and soothe the nerves. SYD can improve cognitive ability. This decoction is suitable for treating vascular cognitive impairment (VCI). however, its active ingredients and possible mechanism have not been investigated. AIM OF THE STUDY: This study was conducted to observe the effects of SYD on improving the cognitive abilities of rats with VCI, to explore its active ingredients and mechanism. MATERIALS AND METHODS: The rats with VCI model were established by bilateral common carotid artery occlusion (BCCAO), and the effects of SYD (5, 2.5 g/kg) on the cognitive abilities of VCI rats were evaluated using the Morris water maze (MWM) and neurological assessment. The pathological changes of hippocampal CA1 were observed by H &E and Nissl staining. The effect of SYD on cerebral blood flow (CBF) was evaluated by Laser Speckle Contrast Imager. The expression of CD31 in the cerebral cortex was measured by immunofluorescence (IF) to evaluate the number of cerebral micro vessels. The levels of IL-6, IL-1ß, and TNF-α in the hippocampus were determined using an ELISA kit, and the active components in the plasma and brain tissues of rats after SYD administration were analyzed using UPLC-Q-TOF-MS/MS. The interaction network of the compound-target pathway was established using the SWISS Target, GO, and DAVID databases. The expression of AKT/HIF-1α/VEGF and p38 MAPK signaling pathway in the brain tissues was determined using western blotting (WB). RESULTS: SYD (2.5, 5 g/kg) significantly improved the cognitive abilities of VCI rats in the MWM and neurological assessment. H&E and Nissl staining showed that SYD significantly ameliorated the pathological hippocampal CA1 area and increased the number of Nissl bodies. The Laser Speckle Contrast Imager showed that the cortical CBF of VCI rats in the SYD group was significantly increased, and the IF results showed that CD31 expression was significantly increased in the SYD group. The ELISA results showed that the contents of IL-6, IL-1ß, and TNF-α in SYD were significantly reduced. A total of 29 compounds were found in the plasma and brain tissues of the rats treated with SYD. Network pharmacology revealed 99 targets for the treatment of VCI. Pathway enrichment analysis showed that the HIF-1 and MAPK signaling pathways might be important for SYD to ameliorate VCI. WB showed that the expressions of AKT, HIF-1α, and VEGF in the brain tissues of rats were significantly increased; in addition, NF-κB and p38 MAPK were significantly reduced in the SYD group. CONCLUSION: SYD can improve the cognitive abilities of VCI rats. The mechanism of action of its active ingredients improves cognitive impairment by affecting the AKT/HIF-1α/VEGF and p38 MAPK/NF-κB signaling pathways, promoting cerebrovascular generation, and ameliorating neuroinflammation.

Discovery of Hepatotoxic Equivalent Markers and Mechanism of Polygonum multiflorum Thunb. by Metabolomics Coupled with Molecular Docking.

Polygonum multiflorum Thunb. (PMT), a commonly used Chinese herbal medicine for treating diseases such as poisoning and white hair, has attracted constant attention due to the frequent occurrence of liver injury incidents. To date, its hepatotoxic equivalent markers (HEMs) and potential hepatotoxic mechanisms are still unclear. In order to clarify the HEMs of PMT and further explore the potential mechanisms of hepatotoxicity, firstly, the chemical constituents in PMT extract were globally characterized, and the fingerprints of PMT extracts were established along with the detection of their hepatotoxicity in vivo. Then, the correlations between hepatotoxic features and component contents were modeled by chemometrics to screen HEMs of PMT, which were then further evaluated. Finally, the hepatotoxic mechanisms of PMT were investigated using liver metabolomics and molecular docking. The results show that the chemical combination of 2,3,5,4-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) and emodin-8-O-glucoside (EG) was discovered as the HEMs of PMT through pre-screening and verifying process. Liver metabolomics revealed that PMT caused liver injury by interfering with purine metabolism, which might be related to mitochondrial function disorder and oxidative injury via the up-regulations of xanthosine and xanthine, and the down-regulation of 5' nucleotidase (NT5E) and adenylate kinase 2 (AK2). This study not only found that the HEMs of PMT were TSG and EG, but also clarified that PMT might affect purine metabolism to induce liver injury, which contributed to our understanding of the underlying mechanisms of PMT hepatotoxicity.

Proteomics Unravels Emodin Causes Liver Oxidative Damage Elicited by Mitochondrial Dysfunction.

Emodin is one of the main active compounds in many Chinese traditional herbs. Due to its potential toxic effect on the liver, the possible injury mechanism needs to be explored. In the present study, we investigated liver injury mechanisms of emodin on rats by the technology of proteomics. Firstly, 4530 proteins were identified from the liver of rats treated with emodin by label free proteomics. Inside, 892 differential proteins were selected, presenting a downward trend. Bioinformatics analysis showed that proteins interfered with by emodin were mainly involved in oxidation-reduction biological processes and mitochondrial metabolic pathways, such as mitochondrial fatty acid ß-oxidation, citric acid cycle, and oxidative phosphorylation, which were further confirmed by western blot. The decrease in maximal respiration, ATP production, spare respiratory capacity, and coupling efficiency and increase in proton leakage were detected by seahorse XFe 24 analyzer, which confirmed the damage of mitochondrial function. The down-regulated expressions in antioxidant proteins were verified by western blot and a significant increase of ROS levels were detected in emodin group, which showed that emodin disrupted redox homeostasis in livers. Molecular docking revealed that the main targets of emodin might be acadvl and complex IV. Generally, emodin could induce oxidative stress in livers by directly targeting acadvl/complex IV and inhibiting fatty acid ß-oxidation, citric acid cycle, and oxidative phosphorylation taken place in mitochondria.

Investigation of the Lipid-Lowering Mechanisms and Active Ingredients of Danhe Granule on Hyperlipidemia Based on Systems Pharmacology.

OBJECTIVE: Investigate the active ingredients and underlying hypolipidemic mechanisms of Danhe granule (DHG). METHODS: The lipid-lowering effect of DHG was evaluated in hyperlipidemic hamsters induced by a high-fat diet. The ingredients absorbed into the blood after oral administration of DHG in hamsters were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). A systems pharmacology approach incorporating target prediction and network construction, gene ontology (GO) enrichment and pathway analysis was performed to predict the active compounds and map the compounds-targets-disease network. Real-time polymerase chain reaction (RT-PCR) and Western blot were utilized to analyze the mRNA and protein expression levels of predicted targets. RESULTS: DHG remarkably lowered the levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and arteriosclerosis index (AI), at the same time, elevated the levels of serum high-density lipoprotein cholesterol (HDL-c) and HDL-c/TC ratio in hyperlipidemic hamsters. Sixteen ingredients absorbed into blood after oral administration of DHG were identified as the possible components interacted with targets. Moreover, 65 potential targets were predicted after targets intersection and compounds-targets-disease network mapping. Then, compounds-targets-pathways network mapping revealed that six active compounds (emodin, naringenin, etc.) compounds could interact with 10 targets such as sterol regulatory element binding protein (SREBP) 1c, SREBP-2 and peroxisome proliferation-activated receptor (PPAR) α, regulate three lipid metabolism-related pathways including SREBP control of lipid synthesis pathway, PPAR signaling pathway and nuclear receptors in lipid metabolism and toxicity pathway, and further affect lipid metabolic processes including fatty acid biosynthesis, low-density lipoprotein receptor (LDLR)-mediated cholesterol uptake, bile acid biosynthesis, and cholesterol efflux. Experimental results indicated that DHG significantly increased SREBP-2, LDLR, PPARα, liver X receptor alpha (LXRα), cholesterol 7α-hydroxylase (CYP7A1), and ATP binding cassette subfamily A member 1 (ABCA1) mRNA and protein expressions while decreased SREBP-1c and fatty acid synthase (FAS) mRNA, and protein expressions. CONCLUSION: DHG possessed a good hypolipidemic effect that may be through affecting the mRNA and protein expressions of SREBP-1c, FAS, SREBP-2, LDLR, PPARα, LXRα, CYP7A1, and ABCA1, involving in fatty acid synthesis, LDLR-mediated cholesterol uptake, bile acid biosynthesis, and cholesterol efflux. This study further provided experimental evidence about its practical application for treating hyperlipidemia and its complications.

[Effects of emodin on lipid accumulation and inflammation in hepatocytes].

The aim of this study was to explore the effect of emodin on lipid accumulation and inflammation in hepatocytes. The cell morphology was observed by microscopy. LDH release was detected by the kit. Levels of intracellular lipid droplets were observed by oil red O staining. The contents of TC and TG in cells were detected by the kit. Western blot was used to determine protein expressions of FASN,SREBF2,APOB,IL-6 and p-NF-κB in hepatocytes. The results showed that the levels of L02 cell LDH were significantly increased after being treated with emodin,and the cells showed shrinkage,volume reduction,decrease in quantity with the increase of dose. Red lipid droplets were observed in L02 hepatocytes. Intracellular TC and TG contents of L02 cell increased in a concentrationdependent manner,with significant differences between medium and high-dose groups( P < 0. 05). Protein expressions of FASN,SREBF2,IL-6 and p-NF-κB were significantly higher than those of the control group,and the expression level of APOB was significantly lower than that of the control group( P<0. 05). In conclusion,emodin could induce lipid accumulation and inflammatory damage in hepatocytes in a dose-dependent manner,which in turn could damage liver cells. This process was related to the up-regulation of FASN,SREBF2,IL-6,p-NF-κB,as well as the down-regulation of the protein expression of APOB.

Analysis of pharmacological mechanisms and targets mining of Wuzi-Yanzong-Wan for treating non-obstructive oligoasthenospermia.

As a classical traditional Chinese medicine, Wuzi-Yanzong-Wan (WZYZW) has been widely applied for several centuries to treat non-obstructive oligoasthenozoospermia (NOA), although its pharmacological mechanisms remain largely unknown. In this study, both plasma and urine metabolomics profiling was first analysed to explore the therapeutic mechanisms of WZYZW in NOA rats induced by removal of the unilateral testicle. Then, 106 identified compounds comprising WZYZW (our previous work), for which putative targets were discovered using systems pharmacology, were systematically analysed via mRNA microarrays to validate their putative targets. Finally, metabolomics-tested WZYZW-regulated metabolites were connected with validated targets using Spearman correlation analysis to further confirm the targets from a biological perspective. The results suggested that WZYZW plays key roles in modulating the concentrations of 18 metabolites in the metabolism of amino acids, lipids and so on, normalizing the metabolic phenotype and regulating metabolic disorders. Moreover, 27 targets of WZYZW (23 compounds) against NOA were validated, and metabolomics-tested metabolites were also found to be significantly related to these identified targets, suggesting that these targets and compounds are worthy of further research. This work offers the first systematic investigation of the efficacy of WZYZW against NOA and illustrates a practicable approach for explaining the molecular mechanisms of multicomponent drugs.

Nux Vomica Exposure Triggered Liver Injury and Metabolic Disturbance in Zebrafish Larvae.

Zebrafish larvae were used to further understand the liver toxicity of nux vomica. The histopathology, protein expression, and gene expression were assessed to confirm apoptosis in the liver, and then, profiles of the metabolites in zebrafish were investigated by untargeted metabolomic assessment to understand the potential toxicity mechanism of nux vomica. Histopathological observations showed that nux vomica caused damage to liver cells. Western blot results indicated increased expression of activated caspase3, and the result of real-time polymerase chain reaction showed a significant increase in the expression level of caspase-3, caspase-8, and caspase-9 genes (p < 0.05) compared with the control group. The liver injury from nux vomica was linked to the downregulation of amino acid (e.g., proline and alanine) and fatty acid (e.g., palmitoleic acid) metabolism and upregulation of some other fatty acid (e.g., arachidic acid) and purine (e.g., xanthine and uric acid) metabolism. The hepatotoxicity of nux vomica resulted from metabolic pathway disturbances, including small molecules involved in energy, purine, lipids, and amino acid metabolism.