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.

iTRAQ-based quantitative proteomic analysis of the anti-apoptotic effect of hyperin, which is mediated by Mcl-1 and Bid, in H2O2-injured EA.hy926 cells.

Endothelial injury has been implicated in the pathogenesis of many cardiovascular diseases, including thrombotic disorders. Hyperin (quercetin-3-O-galactoside), a flavonoid compound and major bioactive component of the medicinal herb Apocynum venetum L., is commonly used to prevent endothelium dysfunction. However, its mode of action remains unclear. To the best of our knowledge, we have for the first time investigated the protective effect hyperin exerts against H2O2-induced injury in human endothelium-derived EA.hy926 cells using isobaric tags for relative and absolute quantitation (iTRAQ)­based quantitative proteomic analysis. The results showed that H2O2 exposure induced alterations in the expression of 250 proteins in the cells. We noted that the expression of 52 proteins associated with processes such as cell apoptosis, cell cycle and cytoskeleton organization, was restored by hyperin treatment. Of the proteins differentially regulated following H2O2 stress, the anti-apoptotic protein, myeloid cell leukemia-1 (Mcl-1), and the pro-apoptotic protein, BH3-interacting domain death agonist (Bid), exhibited marked changes in expression. Hyperin increased Mcl-1 expression and decreased that of Bid in a dose-dependent manner. In addition, flow cytometric analysis and western blot analysis of the apoptosis-related proteins, truncated BID (tBid), cleaved caspase-3, cleaved caspase-9, Fas, FasL and caspase-8, demonstrated that the rate of apoptosis and the pro-apoptotic protein levels were decreased by hyperin pre­treatment. In the present study we demonstrate that hyperin effectively prevents H2O2­induced cell injury by regulating the Mcl­1­ and Bid-mediated anti­apoptotic mechanism, suggesting that hyperin is a potential candidate for use in the treatment of thrombotic diseases.