Integration of metabolomics and transcriptomics reveals that Da Chuanxiong Formula improves vascular cognitive impairment via ACSL4/GPX4 mediated ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuanxiong Formula (DCX) is a traditional herbal compound composed of Gastrodia elata Bl. and Ligusticum chuanxiong Hort, which could significantly enhance blood circulation and neuroprotection, showing promise in treating Vascular Cognitive Impairment (VCI). AIM OF STUDY: This study aims to elucidate the potential of DCX in treating VCI and its underlying mechanism. MATERIALS AND METHODS: Firstly, the cognitive behavior level, blood flow changes, and brain pathology changes were evaluated through techniques such as the Morris water maze, step-down, laser speckle, coagulation analysis, and pathological staining to appraise the DCX efficacy. Then, the DCX targeting pathways were decoded by merging metabolomics with transcriptomics. Finally, the levels of reactive oxygen species (ROS), Fe2+, and lipid peroxidation related to the targeting signaling pathways of DCX were detected by kit, and the expression levels of mRNAs or proteins related to ferroptosis were determined by qPCR or Western blot assays respectively. RESULTS: DCX improved cognitive abilities and cerebral perfusion significantly, and mitigated pathological damage in the hippocampal region of VCI model rats. Metabolomics revealed that DCX was able to call back 33 metabolites in plasma and 32 metabolites in brain samples, and the majority of the differential metabolites are phospholipid metabolites. Transcriptomic analysis revealed that DCX regulated a total of 3081 genes, with the ferroptosis pathway exhibiting the greatest impact. DCX inhibited ferroptosis of VCI rates by decreasing the levels of ferrous iron, ROS, and malondialdehyde (MDA) while increasing the level of superoxide dismutase (SOD) and glutathione (GSH) in VCI rats. Moreover, the mRNA and protein levels of ACSL4, LPCAT3, ALOX15, and GPX4, which are related to lipid metabolism in ferroptosis, were also regulated by DCX. CONCLUSION: Our research findings indicated that DCX could inhibit ferroptosis through the ACSL4/GPX4 signaling pathway, thereby exerting its therapeutic benefits on VCI.

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.

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.

[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.

Emodin induces liver injury by inhibiting the key enzymes of FADH/NADPH transport in rat liver.

Emodin is a natural anthraquinone derivative that occurs in many Chinese medicinal herbs. It might induce liver damage, but the mechanism is not clear. In this research, seven groups of Sprague-Dawley (SD) rats with three doses of emodin were used. The liver injury was examined by analyzing biochemical indexes and histopathology. Altered proteins between the control group (CG) and the liver injury group were determined by proteomic technology. The results showed that emodin causes liver injury in a time- and dose-dependent manner. In the high-dosage 1-week group (HG1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was downregulated, and the activity of malate dehydrogenase (MDH) was inhibited by emodin. These might cause the inhibition of FADH or NADH/NADPH transport from the cytoplasm to mitochondria. The WB results showed that the inhibition of FADH/NADPH transport induced a high activity of caspase-9 and caspase-3, and the expressions of cytochrome c (Cyt C), caspase-9 and caspase-3 were high in HG1, which might lead to mitochondrial apoptosis pathway activation. In addition, whatever the HG1 or low-dose group (LG), the effects of emodin on mitochondria were observed. Overall, for the first time, we showed that emodin inhibited proton transport and induced the activation of the mitochondrial apoptosis pathway, which might be the reason for liver injury.

Diversified bioactivities of four types of naturally occurring quinochalcones.

Quinochalcones, quinone-containing chalcones, belong to the flavonoid family and have attracted increasing popularity in Western countries in the last decade due to their pharmacological activities. This review describes four types of naturally occurring quinochalcones and summarizes their different pharmacological activities, including anti-cerebral ischemia, anti-tumor, and anti-infection activities. In addition, the pharmacological activities and relevant structure-activity relationships of synthetic quinochalcones are also reviewed.