[Research on mechanism of hypolipidemic effect of Massa Medicata Fermentata based on metabolomics].

This paper aims to study the therapeutic effect of Massa Medicata Fermentata on hyperlipidemia model rats and investigate its mechanism of hypolipidemic effect with the help of non-targeted metabolomics. The mixed hyperlipidemia model rats were constructed by giving high-fat chow. After successful modeling, the rats were divided into the model group, pravastatin sodium group(4.4 mg·kg~(-1)), lipotropic group(0.1 g·kg~(-1)), high-dose group(2.4 g·kg~(-1)), medium-dose group(1.2 g·kg~(-1)), and low-dose group(0.6 g·kg~(-1)) of Massa Medicata Fermentata, and they were administered for four weeks once daily. An equal volume of ultrapure water was given to the blank group and model group. Serum lipid level and liver hematoxylin-eosin(HE) staining were used as indicators to estimate the intervention effect of Massa Medicata Fermentata on mixed hyperlipidemia, and the changes in metabolites in plasma of mixed hyperlipidemia model rats were analyzed by non-targeted metabolomics. The mechanism of the hypolipidemic effect of Massa Medicata Fermentata was analyzed through metabolite pathway enrichment. The results showed that compared with the model group, the Massa Medicata Fermentata administration group, especially the high-dose group, could significantly reduce the content of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c)(P<0.05 or P<0.01), and liver HE staining revealed that the number of adipocytes in the high-dose group was reduced to some extent. The potential biomarkers obtained by non-targeted metabolomics screening included glycerol 3-phosphate, sphingomyelin, sphingosine 1-phosphate, and deoxyuridine, which were mainly involved in the sphingolipid metabolism process, glycerophospholipid metabolism process, glycerol ester metabolism pathway, and pyrimidine metabolism pathway, totaling four possible metabolic pathways related to lipid metabolism. This study provides a reference for an in-depth investigation of the hypolipidemic mechanism of Massa Medicata Fermentata, which is of great significance for further promoting the clinical application of Massa Medicata Fermentata and increasing the indications.

Xianglian Zhixie Tablet Antagonizes Dextran Sulfate Sodium-Induced Ulcerative Colitis by Attenuating Systemic Inflammation and Modulating Gut Microbiota.

Purpose: Xianglian Zhixie Tablet (XLZXT), a classical traditional Chinese medicine formulation, is commonly used to treat Ulcerative Colitis (UC) in China. However, the therapeutic mechanisms of XLZXT for UC have yet to be fully understood. This study aimed to investigate the curative benefits of XLZXT and its associated mechanisms for healing UC in mice. Methods: In the present study, the 1% dextran sulfate sodium (DSS) solution was used to establish the UC model in C57BL/6N mice. To investigate the therapeutic effects of XLZXT on DSS-induced UC mice, several parameters were measured, including DAI score, colon length, spleen index, pathological changes in colon tissue, and levels of inflammatory factors in plasma and colon tissue. By investigating the gut microbiota, assessing the levels of intestinal mucosal protein expression, and looking at the proteins involved in the TLR4/MyD88/NF-B p65 signaling pathway, the mechanisms of XLZXT impact on UC were investigated. Mouse feces were examined for patterns of gut microbiota expression using high-throughput sequencing of 16S rRNA. Results: XLZXT effectively alleviated UC symptoms and colon pathological damage in DSS-induced UC mice. It improved body weight loss, stool consistency, and hematochezia, while also repairing colon damage. Moreover, it down-regulated pro-inflammatory cytokines (such as TNF-α, IL-1ß, and IL-6), and up-regulated anti-inflammatory cytokines (such as IL-10). XLZXT also increased the expression of MUC-2, Occludin and ZO-1, while decreasing the expression of NF-κB, MyD88 and TLR4. Additionally, it regulated gut microbiota disorder by increasing the abundance of beneficial bacteria and reducing the adhesion of intestinal harmful bacteria. Conclusion: XLZXT demonstrated therapeutic effects on DSS-induced UC mice. The mechanisms may be associated with repairing the intestinal mucosal barrier, regulating the TLR4/MyD88/NF-κB p65 signaling pathway, and restoring the balance of gut microbiota.

[Inhibitory effect and molecular mechanism of sinomenine on human hepatocellular carcinoma HepG2 and SK-HEP-1 cells].

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.

[Morin induces autophagy and apoptosis in hepatocellular carcinoma cells through Akt/mTOR/STAT3 pathway].

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 µmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 µmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.

Clinical antitumor application and pharmacological mechanisms of Dahuang Zhechong Pill.

Cancer still has elevated morbidity and mortality, which undoubtedly impacts the life quality of affected individuals. Remarkable advances have been made in cancer therapy, although the toxicities of traditional therapies remain an obvious challenge. Dahuang Zhechong Pill (DHZCP), developed by Zhongjing Zhang in the Synopsis of the Golden Chamber, represents an effective anticancer traditional Chinese medicine (TCM). In this review, it was found that DHZCP is therapeutically utilized in liver, lung, gastric, pancreatic and other cancers in clinic. Pharmacological evidence showed that its anti-tumor mechanisms mainly involve induced cell cycle arrest, apoptosis and autophagy, as well as suppressed tumor cell proliferation, obstructed angiogenesis and metastasis, enhanced immunity, and reversal of multidrug resistance. The present review provides a solid basis for the clinical application of DHZCP and may promote the wide use of TCM in clinical antitumor application.

Network pharmacology and experimental verification to explore the anti-migraine mechanism of Yufeng Ningxin Tablet.

ETHNOPHARMACOLOGICAL RELEVANCE: Yufeng Ningxin Tablet (YNT) is a traditional Chinese medicine formula, that has been used clinically to treat migraine for many years. It is composed of one herb Pueraria lobata var. lobata (Willd.) Ohwi (Relevant Chinese name: Gegen). Previously, it has been recorded by traditional Chinese doctor that Gegen could be used as medicine to treat migraine. However, the underlying mechanism of action remains to be investigated. AIM OF THE STUDY: It was to explore the effect and mechanism of YNT on migraine based on network pharmacology and experimental verification. MATERIALS AND METHODS: First, with the network pharmacology, the effective chemical components and target genes of YNT were filtrated, the YNT-compound-migraine-targets network was constructed. The protein-protein interaction network (PPI) and literature reports were combined to identify potential targets of YNT in the treatment of migraine. Then, the representative compounds of YNT were characterized by LC-MS/MS and the major effect components were identified. Finally, the prediction results of network pharmacology were verified by animal and cell experiments. RESULTS: 7 bioactive components of YNT could act on 97 migraine potential targets. The 5 bioactive components could be characterized comprehensively of YNT. The key therapeutic targets and pathways were collected including 5-HT, CGRP, inflammation and nociceptive factors, and NF-κB signaling pathway. Animal experiments showed that YNT could increase the expression level of 5-HT and reduce the expression of CGRP, NF-κB, c-fos and IL-1ß. YNT could inhibit LPS-induced neuroinflammation by NF-κB in BV2 cells in vitro. Western blotting analysis results showed YNT inhibited the NF-κB and phospho-NF-κB levels. CONCLUSIONS: It is the first time to verify the consistency between the metabolic components of YNT by LC-MS/MS and the active components predicted by network pharmacology. Meanwhile, the potential mechanism of YNT in the treatment of migraine was studied by combining network pharmacology and in vitro and in vivo experiments.

Integrated Solid-Phase Extraction, Ultra-High-Performance Liquid Chromatography-Quadrupole-Orbitrap High-Resolution Mass Spectrometry, and Multidimensional Data-Mining Techniques to Unravel the Metabolic Network of Dehydrocostus Lactone in Rats.

Dehydrocostus lactone (DL) is among the representative ingredients of traditional Chinese medicine (TCM), with excellent anticancer, antibacterial, and anti-inflammatory activities. In this study, an advanced strategy based on ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was integrated to comprehensively explore the metabolic fate of DL in rats. First, prior to data collection, all biological samples (plasma, urine, and feces) were concentrated and purified using solid-phase extraction (SPE) pre-treatment technology. Then, during data collection, in the full-scan (FS) data-dependent acquisition mode, FS-ddMS2 was intelligently combined with FS-parent ion list (PIL)-dynamic exclusion (DE) means for targeted monitoring and deeper capture of more low-abundance ions of interest. After data acquisition, data-mining techniques such as high-resolution extracted ion chromatograms (HREICs), multiple mass defect filters (MMDFs), diagnostic product ions (DPIs), and neutral loss fragments (NLFs) were incorporated to extensively screen and profile all the metabolites in multiple dimensions. As a result, a total of 71 metabolites of DL (parent drug included) were positively or tentatively identified. The results suggested that DL in vivo mainly underwent hydration, hydroxylation, dihydrodiolation, sulfonation, methylation, dehydrogenation, dehydration, N-acetylcysteine conjugation, cysteine conjugation, glutathione conjugation, glycine conjugation, taurine conjugation, etc. With these inferences, we successfully mapped the "stepwise radiation" metabolic network of DL in rats, where several drug metabolism clusters (DMCs) were discovered. In conclusion, not only did we provide a refined strategy for inhibiting matrix effects and fully screening major-to-trace metabolites, but also give substantial data reference for mechanism investigation, in vivo distribution visualization, and safety evaluation of DL.

Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy.

Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values of 5.96 ± 0.61 µM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.

Uric Acid Produces an Inflammatory Response through Activation of NF-κB in the Hypothalamus: Implications for the Pathogenesis of Metabolic Disorders.

Epidemiological studies have shown that an elevated uric acid (UA) level predicts the development of metabolic syndrome and diabetes; however, there is no direct evidence of this, and the underlying mechanism remains unclear. Here, we showed that a high-UA diet triggered the expression of pro-inflammatory cytokines, activated the NF-κB pathway, and increased gliosis in the hypothalamus. Intracerebroventricular injection of UA induced hypothalamic inflammation and reactive gliosis, whereas these effects were markedly ameliorated by the inhibition of NF-κB. Moreover, magnetic resonance imaging confirmed that hyperuricemia in rodents and humans was associated with gliosis in the mediobasal hypothalamus. Importantly, the rats administered UA exhibited dyslipidemia and glucose intolerance, which were probably mediated by hypothalamic inflammation and hypothalamic neuroendocrine alterations. These results suggest that UA can cause hypothalamic inflammation via NF-κB signaling. Our findings provide a potential therapeutic strategy for UA-induced metabolic disorders.

[Bioactivity assay of bupleurum injection for inhibiting PGE2 release in vitro].

OBJECTIVE: To establish the in vitro model of PGE2 released by hypothalamic neurocytes under rrIL-1beta in vitro interference, and investigate the correlation of the PGE2 content and the effect of the drug effect concentration in the model under the effect of Bupleurum injection. METHOD: Hypothalamic neurocytes were cultured in vitro, and added with rrIL-1beta (40 microg x L(-1)) stimulation. Cell sap was collected at different time points. ELISA was adopted to determine the content of PGE2 in cell sap collected at different time points. Hypothalamic neurocytes were cultured in vitro, added with rrIL-1beta (40 microg x L(-1)) stimulation and then different concentrations of Bupleurum injection. The changes in the content of PGE2 in cell supernatant were detected by ELISA. An analysis was made on the linear relationship between the sample concentration and the inhibition rate of PGE2. RESULT: The rrIL-1 cells could stimulate in vitro cultured hypothalamic neurocytes to release PGE2 and reach the peak at 10 h. Bupleurum injection could significantly interfere the release of PGE2 in the in vitro model (P < 0.01, P < 0.05), with a certain linear relationship between the interference effect and the effect concentration of Bupleurum injection (r = 0.911, P < 0.01). CONCLUSION: The rrIL-1 cells could stimulate in vitro cultured hypothalamic neurocytes to release PGE2, with a good correlation between the inhibition and generation effects of PGE2 and the drug concentration.