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Estrogen exerts essential role in liver metabolism, and its deficiency is frequently accompanied by a series of metabolic disorder diseases. To investigate the role of estrogen deficiency in fluorine ions (F–) induced liver injury, the ovariectomy (OVX) rat models were performed by surgically removing the ovaries, and the rats from OVX and non-OVX models were exposed to differential dose of F– (0, 25, 50 and 100 mg/L) in drinking water for 90 days. The liver morphological structure was evaluated by hematoxylin–eosin staining. Proliferation ability of hepatocytes was evaluated by 5-bromo-2-deoxyuridine (BrdU) assay. And distribution of lipid droplets in liver tissue was observed via oil red O staining. In addition, the liver function and lipid metabolism parameters in serum were detected by commercial kits. Results showed that F– induced hepatocytes morphological damage and inhibited the proliferation ability of hepatocytes; estrogen deficiency exacerbated these changes. The deposition of lipid droplets in the liver tissue was multiplicative with increased F– dose, especially after estrogen deficiency. In addition, F– exposure increased (P

C2C12 cells were cultured on medium containing fluoride (0, 1, and 2.5 mmol/L) for 48 h to investigate the effect of excessive fluoride on T helper 17 (Th17)-related cytokine expression profile in skeletal muscle cells, and the culture supernatant was collected and subjected for the detection of 18 cytokines via Th17 array. Results showed that compared with the control group, no differential expression proteins (DEPs) were found in the 1 mmol/L fluoride group; however, eight DEPs were upregulated in the 2.5 mmol/L fluoride group, including macrophage inflammatory protein-3α (MIP-3α), interleukin-21 (IL-21), IL-13, IL-17F, IL-28A, transforming growth factor type beta 1 (TGF-β1), IL-23, and IL-17A. In addition, five DEPs (MIP-3α, IL-13, IL-21, TGF-β1, and IL-17F) were upregulated in the 2.5 mmol/L fluoride group compared with the 1 mmol/L fluoride group. Gene ontology analysis revealed that the positive regulation of cytokine production, cytokine activity, receptor ligand activity, and cytokine receptor binding accounted for high percent of DEPs present. Kyoto Encyclopedia of Genes and Genomes analysis showed that these DEPs primarily involved 12 pathways enriched in the cytokine-cytokine receptor interaction and IL-17 signaling pathway after 2.5 mmol/L fluoride treatment. The results indicated that fluoride might induce cytotoxicity by disturbing Th17-related cytokine expression.

Ligand fishing with target biomolecule-immobilized magnetic beads (MBs) has been established and developed for nearly 10 years. Advantages of this technique, such as the ease of operation, associated with a diversity of automated online approaches, make it a valuable tool for affinity studies. However, transmembrane proteins have not been used as the target biomolecules in the assay, since they are usually not available in a purified and bioactive form. In addition, few publications have reported the use of this method for screening active compounds derived from natural products. In this work, for the first time, cellular membrane-coated MBs, which to a large extent maintain the activity of the transmembrane proteins, were used for the fishing assay. We demonstrated application of red blood cell membrane-coated MBs for fishing potential active components from a natural product (Angelica dahurica). The potential active compounds, such as imperatorin, bergapten, and pabulenol, were detected. The result correlated well with cell membrane chromatography (CMC) coupled with HPLC. Comparisons of the developed MBs fishing assay with the CMC method showed the noteworthy advantages of the fishing technique regarding the consumption of cellular membranes, buffers as well as length of operation time.

Target biomolecule-immobilized magnetic beads could be used as a powerful tool for screening active compounds present in natural products. Low damage rates of the target proteins, associated with the availability of diverse automated online approaches for analysis, make it a valuable tool for affinity studies. RAW264.7 cells (a kind of murine macrophage cell line) were used in this study. These cellular membranes were immobilized onto the surface of MBs and were used for screening the active compounds of Polygonatum sibiricum. Combining this technique with HPLC led to the identification of an active compound and its biological activity was confirmed. This is the first report establishing the use of RAW264.7 cellular membrane-coated magnetic bead fishing followed by HPLC analysis for screening active compounds from natural products.

Cell membrane chromatography is a useful tool for screening active compounds from natural products. As the reason of separation mechanism, traditional cell membrane chromatography could not be used for screening the active compounds absorbed through the cell membrane and influencing the cell signal transduction pathway. In this work, we establish a new method named cell extraction combined with off-line HPLC for screening the compounds penetrating the cell membrane. This is the first time 3 T3-L1 adipocyte culture has been combined with HPLC technology. Compared with other cell membrane chromatography methods, there is good resolution and no further analysis by other chromatographic steps is required. On co-incubating crude extracts of Coptis chinensis with cells and analyzing the compounds extracted by the cells, active compounds such as berberine were detected. Glucose consumption tests showed that berberine could increase glucose consumption by insulin-resistant 3 T3-L1 adipocytes. The levels of intracellular berberine correlated with its activity. The results indicate that the developed method could be an alternative method for screening active compounds from natural products.