Gut microbiota-dependent modulation of pre-metastatic niches by Jianpi Yangzheng decoction in the prevention of lung metastasis of gastric cancer.

AIM OF THE STUDY: To evaluate the in vitro and in vivo anti-metastasis efficacy of Jianpi Yangzheng (JPYZ) decoction against gastric cancer (GC) and its potential mechanisms. MATERIALS AND METHODS: The distant metastasis of GC cells administered via tail vein injection was assessed using the pre-metastatic niche (PMN) model. 16S rRNA sequencing and GC-MS/MS were applied to determine the component of the gut microbiota and content of short-chain fatty acids (SCFAs) in feces of mice, respectively. The proportion of myeloid-derived suppressor cells (MDSCs) in the lung was evaluated by flow cytometry and immunofluorescence. Serum or tissue levels of inflammation factors including IL-6, IL-10 and TGF-ß were determined by ELISA or Western blot respectively. RESULTS: Injecting GC cells into the tail vein of mice led to the development of lung metastases and also resulted in alterations in the composition of gut microbiota and the levels of SCFAs produced. Nevertheless, JPYZ treatment robustly impeded the effect of GC cells administration. Mechanically, JPYZ treatment not only prevented the alteration in gut microbiota structure, but also restored the SCFAs content induced by GC cells administration. Specifically, JPYZ treatment recovered the relative abundance of genera Moryella, Helicobacter, Lachnoclostridium, Streptococcus, Tuzzerella, GCA-900066575, uncultured_Lachnospiraceae, Rikenellaceae_RC9_gut_group and uncultured_bacterium_Muribaculaceae to near the normal control levels. In addition, JPYZ abrogated MDSCs accumulation in the lung tissue and blocked inflammation factors overproduction in the serum and lung tissues, which subsequently impede the formation of the immunosuppressive microenvironment. Correlation analysis revealed that the prevalence of Rikenellaceae in the model group exhibited a positive correlation with MDSCs proportion and inflammation factor levels. Conversely, the scarcity of Muribaculaceae in the model group showed a negative correlation with these parameters. This suggests that JPYZ might exert an influence on the gut microbiota and their metabolites, such as SCFAs, potentially regulating the formation of the PMN and consequently impacting the outcome of GC metastasis. CONCLUSION: These findings suggest that GC cells facilitate metastasis by altering the gut microbiota composition, affecting the production of SCFAs, and recruiting MDSCs to create a pro-inflammatory pre-metastatic niche. JPYZ decoction counteracts this process by reshaping the gut microbiota structure, enhancing SCFA production, and inhibiting the formation of the pre-metastatic microenvironment, thereby exerting an anti-metastatic effect.

Nobiletin targets SREBP1/ACLY to induce autophagy-dependent cell death of gastric cancer cells through PI3K/Akt/mTOR signaling pathway.

BACKGROUND: Autophagy could sense metabolic conditions and safeguard cells against nutrient deprivation, ultimately supporting the survival of cancer cells. Nobiletin (NOB) is a kind of bioactive component of the traditional Chinese medicine Citri Reticulatae Pericarpium and has been proven to induce GC cell death by reducing de novo fatty acid synthesis in our previous study. Nevertheless, the precise mechanisms by which NOB induces cell death in GC cells still need further elucidation. OBJECTIVES: To examine the mechanism by which NOB inhibits gastric cancer progression through the regulation of autophagy under the condition of lipid metabolism inhibition. METHODS/ STUDY DESIGN: Proliferation was detected by the CCK-8 assay. RNA sequencing (RNA-seq) was used to examine signaling pathway changes. Electron microscopy and mRFP-GFP-LC3 lentiviral transfection were performed to observe autophagy in vitro. Western blot, plasmid transfection, immunofluorescence staining, and CUT & Tag-qPCR techniques were utilized to explore the mechanisms by which NOB affects GC cells. Molecular docking and molecular dynamics simulations were conducted to predict the binding mode of NOB and SREBP1. CETSA was adopted to verify the predicted of binding model. A patient-derived xenograft (PDX) model was employed to verify the therapeutic efficacy of NOB in vivo. RESULTS: We conducted functional studies and discovered that NOB inhibited the protective effect of autophagy via the PI3K/Akt/mTOR axis in GC cells. Based on previous research, we found that the overexpression of ACLY abrogated the NOB-induced autophagy-dependent cell death. In silico analysis predicted the formation of a stable complex between NOB and SREBP1. In vitro assays confirmed that NOB treatment increased the thermal stability of SREBP1 at the same temperature conditions. Moreover, CUT&TAG-qPCR analysis revealed that NOB could inhibit SREBP1 binding to the ACLY promoter. In the PDX model, NOB suppressed tumor growth, causing SREBP1 nuclear translocation inhibition, PI3K/Akt/mTOR inactivation, and autophagy-dependent cell death. CONCLUSION: NOB demonstrated the ability to directly bind to SREBP1, inhibiting its nuclear translocation and binding to the ACLY promoter, thereby inducing autophagy-dependent cell death via PI3K/Akt/mTOR pathway.

Preparative Separation of Phenolic Compounds from Chimonanthus praecox Flowers by High-Speed Counter-Current Chromatography Using a Stepwise Elution Mode.

High-speed counter-current chromatography (HSCCC) has been successfully used for the separation of eight compounds from Chimonanthus praecox flowers. Firstly, the crude extract of Chimonanthus praecox flowers was dissolved in a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-H2O (5:5:3:7, v/v) and divided into two parts: the upper phase (part I) and the lower phase (part II). Then, HSCCC was applied to separate the phenolic acids from part I and part II, respectively. Considering the broad polarity range of target compounds in part I, a stepwise elution mode was established. Two optimal solvent systems of petroleum ether-ethyl acetate-methanol-H2O-formic acid (FA) (5:5:3:7:0.02, 5:5:4.3:5.7:0.02, v/v) were employed in this separation. Five phenylpropanoids and two flavonoids were successfully separated from 280 mg of part I, including 8.7 mg of 3,4-dihydroxy benzoic acid (a, 95.3% purity), 10.9 mg of protocatechualdehyde (b, 96.8% purity), 11.3 mg of p-coumaric acid (c, 98.9% purity), 12.2 mg of p-hydroxybenzaldehyde (d, 95.9% purity), 24.7 mg of quercetin (e, 97.3% purity), 33.8 mg of kaempferol (f, 96.8% purity), and 24.6 mg of 4-hydroxylcinnamic aldehyde (g, 98.0% purity). From 300 mg of part II, 65.7 mg of rutin (h, 98.2% purity), 7.5 mg of 3,4-dihydroxy benzoic acid (a, 77.4% purity), and 4.7 mg of protocatechualdehyde (b, 81.6% purity) were obtained using the solvent system EtOAc-n-butanol (n-BuOH)-FA-H2O (4:1:0.5:5, v/v). The structures of the eight pure compounds were confirmed by electrospray ionization-mass spectrometry (ESI-MS), ¹H-NMR and (13)C-NMR. To the best of our knowledge, compounds a-d and f were the first separated and reported from the Chimonanthus praecox flower extract.