Screening anti-anaphylactoid components in Polygonum cuspidatum via cell membrane chromatography with LC-MS targeting Mas-related G protein-coupled receptor X2.

Mas-related G protein-coupled receptor X2 (MrgprX2) is acknowledged as a mast cell-specific receptor, playing a crucial role in orchestrating anaphylactoid responses through mast cell degranulation. It holds promise as a target for regulating allergic and inflammatory diseases mediated by mast cells. Polygonum cuspidatum (PC) has shown notable anti-anaphylactoid effects, while its pharmacologically active components remain unclear. In this study, we successfully utilized MrgprX2 high-expressing cell membrane chromatography (CMC), in conjunction with liquid chromatography-mass spectrometry (LC-MS), to identify active anti-anaphylactoid components in PC. Our study pinpointed polydatin, resveratrol, and emodin-8-O-ß-d-glucoside as potential anti-anaphylactoid compounds in PC. Their anti-anaphylactoid activities were evaluated through ß-aminohexosidase and histamine release assays, demonstrating a concentration-dependent inhibition for both ß-aminohexosidase and histamine release. This approach, integrating MrgprX2 high-expression CMC with LC-MS, proves effective in screening potential anti-anaphylactoid ingredients in natural herbal medicines. The findings from this study illuminated the anti-anaphylactoid properties of specific components in PC and provided an efficient method for the drug development of natural products.

Development of target-based cell membrane affinity ultrafiltration technology for a simplified approach to discovering potential bioactive compounds in natural products.

Target-based drug discovery technology based on cell membrane targets has gained significant traction and has been steadily advancing. However, current methods still face certain limitations that need to be addressed. One of the challenges is the laborious preparation process of screening materials, which can be time-consuming and resource-intensive. Additionally, there is a potential issue of non-specific adsorption caused by carrier materials, which can result in false-positive results and compromise the accuracy of the screening process. To address these challenges, this paper proposes a target-based cell membrane affinity ultrafiltration technology for active ingredient discovery in natural products. In this technique, the cell membranes of human lung adenocarcinoma epithelial cells (A549) with a high expression of epidermal growth factor receptor (EGFR) were incubated with candidate drugs and then transferred to an ultrafiltration tube. Through centrifugation, components that interacted with EGFR were retained in the ultrafiltration tube as "EGFR-ligand" complex, while the components that did not interact with EGFR were separated. After thorough washing and eluting, the components interacting with EGFR were dissociated and further identified using LC-MS, enabling the discovery of bioactive compounds. Moreover, the target-based cell membrane affinity ultrafiltration technology exhibited commendable binding capacity and selectivity. Ultimately, this technology successfully screened and identified two major components from the Curcumae Rhizoma-Sparganii Rhizoma (CS) herb pair extracts, which were further validated for their potential anti-tumor activity through pharmacological experiments. By eliminating the need for laborious preparation of screening materials and the potential non-specific adsorption caused by carriers, the development of target-based cell membrane affinity ultrafiltration technology provides a simplified approach and method for bioactive compounds discovery in natural sources.

Elemene induces cell apoptosis via inhibiting glutathione synthesis in lung adenocarcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Curcuma wenyujin Y.H. Chen & C. Ling, also known as Wen-E-Zhu, has been used for cancer treatment since ancient times, with roots dating back to the Song Dynasty. Elemene (EE), a sesquiterpene extract with potent anticancer properties, is extracted from Wen-E-Zhu, with ß-elemene (BE) being its main active compound, along with trace amounts of ß-caryophyllene (BC), γ-elemene and δ-elemene isomers. EE has demonstrated broad-spectrum anti-cancer effects and is commonly used in clinical treatments for various types of malignant cancers, including lung cancer. Studies have shown that EE can arrest the cell cycle, inhibit cancer cell proliferation, and induce apoptosis and autophagy. However, the exact mechanism of its anti-lung cancer activity remains unclear and requires further research and investigation. AIM OF THE STUDY: In this study, the possible mechanism of EE and its main active components, BE and BC, against lung adenocarcinoma was investigated by using A549 and PC9 cell lines. MATERIALS AND METHODS: The subcutaneous tumor model of nude mice was constructed to evaluate the efficacy of EE in vivo, then the in vitro half-inhibitory concentration (IC50) of EE and its main active components, BE and BC, on A549 and PC9 cells at different concentrations were determined by CCK-8. Flow cytometry was used to detect the apoptosis and cycle of A549 and PC9 cells treated with different concentrations of BE and BC for 24 h. Non-targeted metabolomics analysis was performed on A549 cells to explore potential target pathways, which were subsequently verified through kit detection and western blot analysis. RESULTS: Injection of EE in A549 tumor-bearing mice effectively suppressed cancer growth in vivo. The IC50 of EE and its main active components, BE and BC, was around 60 µg/mL. Flow cytometry analysis showed that BE and BC blocked the G2/M and S phases of lung adenocarcinoma cells and induced apoptosis, leading to a significant reduction in mitochondrial membrane potential (MMP). Results from non-targeted metabolomics analysis indicated that the glutathione metabolism pathway in A549 cells was altered after treatment with the active components. Kit detection revealed a decrease in glutathione (GSH) levels and an increase in the levels of oxidized glutathione (GSSG) and reactive oxygen (ROS). Supplementation of GSH reduced the inhibitory activity of the active components on lung cancer and also decreased the ROS content of cells. Analysis of glutathione synthesis-related proteins showed a decrease in the expression of glutaminase, cystine/glutamate reverse transporter (SLC7A11), and glutathione synthase (GS), while the expression of glutamate cysteine ligase modified subunit (GCLM) was increased. In the apoptosis-related pathway, Bax protein and cleaved caspase-9/caspase-9 ratio were up-regulated and Bcl-2 protein was down-regulated. CONCLUSIONS: EE, BE, and BC showed significant inhibitory effects on the growth of lung adenocarcinoma cells, and the mechanism of action was linked to the glutathione system. By down-regulating the expression of proteins related to GSH synthesis, EE and its main active components BE and BC disrupted the cellular redox system and thereby promoted cell apoptosis.

Bioactivity-guided discovery of quality control markers in rhizomes of Curcuma wenyujin based on spectrum-effect relationship against human lung cancer cells.

BACKGROUND: Due to the diversity of the ingredients, the complexity of the mechanism of action, the uncertainty of the effective ingredients, coupled with the multiple species and multiple growing areas, the quality control (QC) of Traditional Chinese Medicines (TCMs) is challenging. Discovering and identifying effective compounds from the complex extracts of TCMs and then establishing a scientific QC method is the key to the holistic QC of TCMs. PURPOSE: To develop an anti-lung-cancer-guided spectrum-effect relationship approach for the discovery of QC markers of the rhizome of Curcuma wenyujin (WEZ) and establish a bioactive compounds-based holistic QC method. METHODS: The chemical profiling of the volatile oil (WVO) from 42 batches of WEZ collected from different growing areas was performed by GC-MS. The anti-lung cancer activity of different WVO samples was determined by CCK-8 assay against human lung cancer cells (A549). The apoptosis and cell cycle analysis under different concentrations of WVO were detected by flow cytometry. SIMCA-P software was used to perform multivariate statistical analysis on the chemical composition of different WVO samples and to find the different components. Active compounds were screened using a PLSR model of the spectrum-effect relationship. Bioactive compounds-based fingerprint and quantification of the leading bioactive compounds were developed by GC-MS and GC-FID, respectively. RESULTS: Seventy-eight compounds were detected in WVO and 54 were successfully identified. The multivariate statistical analysis uncovered that WVO components and the anti-A549 activity of WVO at the concentration of 60 nl/ml differ greatly according to the origin of the plant. The WVO at the concentration of 60 nl/ml (IC50) increased A549 cells apoptosis significantly with late and early apoptosis of 15.61% and 7.80%, and the number of cells in the G2/M phase were also increased significantly under this concentration. The spectrum-effect relationship analysis revealed that 44 compounds were positively correlated with their activities, and the result was verified by A549 cell viability assay. Sixteen positively correlated compounds were further selected as QC markers according to their relative amount > 0.5% and anticancer activity. Finally, the 16 QC markers-based GC-MS fingerprint was established to holistically control the quality of WEZ, and a GC-FID method was developed for the quantification of leading bioactive compounds, ß-elemene and ß-caryophyllene. CONCLUSION: Based on an anti-lung-cancer-guided spectrum-effect relationship approach, the bioactive compounds-based holistic QC method was successfully developed for WEZ, which could provide a valuable reference for the QC of TCMs.

Anti-arthritis effect of berberine associated with regulating energy metabolism of macrophages through AMPK/ HIF-1α pathway.

Berberine (BBR) is the effective constituent of Cortex phellodendri and was characterized as an excellent anti-microbial agent with significant anti-inflammatory effects. Previously, we had demonstrated that BBR alleviated the inflammatory response in adjuvant-induced arthritis (AA) rats by regulating polarization of macrophages. However, the exact mechanics by which BBR regulates macrophage polarization remained unclear. Here, we showed that BBR treatment had little influence on total number of macrophages in joints of AA rats, but increased the proportion of M2 macrophages and decreased the proportion of M1 macrophages. Meanwhile, we found BBR up-regulated the expression of AMP-activated protein kinase phosphorylation (p-AMPK) and down-regulated the expression of Hypoxia inducible factor 1α (HIF-1α) in synovial macrophages of AA rats. In vitro, using LPS-stimulated peritoneal macrophages from normal rats, we also verified that pretreatment with BBR promoted transition from M1 to M2 by up-regulating the expression of p-AMPK and suppressing the expression of HIF-1α. Compound C (an AMPK inhibitor) could abrogate the inhibition of BBR on migration of macrophages. Glycolysis of M1 suppressed by BBR through decreasing lactate export, glucose consumption, and increasing intracellular ATP content, which was remarkably reversed by Compound C. These findings indicated that anti-arthritis effect of BBR is associated with regulating energy metabolism of macrophages through AMPK/HIF-1α pathway.

[Chloroquine enhances cisplatin-induced apoptosis of nasopharyngeal carcinoma cells by inhibiting autophagy via upregulating miR129].

OBJECTIVE: To investigate the role of miR129 in mediating the effect of chloroquine to enhance cisplatin- induced apoptosis in nasopharyngeal carcinoma cells (HNE1). METHODS: MTT assay was used to detect the viability of HNE1 cells treated with different concentrations of cisplatin. Colony formation of HNE1 cells treated with cisplatin and chloroquine, alone or in combination, was observed using crystal violet staining. BALB/C unde mice were inoculated with HNE1 cells and randomly divided into 4 groups with 6 mice in each group. The mice received intraperitoneal injections of cisplatin and chloroquine, alone or in combination once every 3 days for 4 consecutive weeks, and the tumor growth was observed in each group. The expression of miR129 in HNE1 cells treated with chloroquine, cisplatin, or both was detected with qPCR. The effects of miR129 suppression with a miR129 inhibitor on the expressions of autophagy related proteins p62, LC3B, Beclin1 and the drug-resistant related protein P-glycoprotein (P-gp) were examined using Western blotting in HNE1 cells treated with chloroquine, cisplatin, or both; the changes in cell apoptosis were detected Annexin V/PI double staining. RESULTS: Chloroquine combined with cisplatin significantly inhibited HNE1 cell proliferation in vitro and the growth of HNE1 cell-derived tumor in nude mice as compared with cisplatin alone (P < 0.01). In cultured HNE1 cells, inhibition of the expression of miR129 significantly promoted autophagy and up-regulated P-gp expression (P < 0.01); Chloroquine obviously inhibited cisplatin-induced autophagy and up-regulated the expression of miR129 in HNE1 cells (P < 0.01). Transfection of the cells with the miR129 inhibitor abolished the inhibitory effect of chloroquine on cisplatin-induced autophagy, and significantly increased the cell survival rate (P < 0.05) and lower the cell apoptotic rate (P < 0.01) after combined treatment with chloroquine and cisplatin. CONCLUSIONS: Chloroquine enhances the pro-apoptotic effect of cisplatin by up-regulating miR129 to inhibit autophagy and drug resistance in HNE1 cells.