Urolithin A attenuates RANKL-induced osteoclastogenesis by co-regulating the p38 MAPK and Nrf2 signaling pathway.

As a critical regulator of bone resorption. osteoclastogenesis is closely associated with osteoporosis (OP) and commonly induced by receptor activator of nuclear factor-κB ligand (RANKL), suggesting that suppression of inflammation may improve OP. Urolithin A (UroA), an active metabolite of ellagic acid, is known to exert anti-inflammatory and antioxidative effects. However, whether UroA attenuates osteoclastogenesis remains unclear. Using a lipopolysaccharide (LPS)-induced bone loss model, we evaluated the effects of UroA on inflammatory osteoclastogenesis in mice and explored the potential mechanism from RANKL-related signaling pathway. UroA significantly improved LPS-induced bone loss and rescued the imbalance in bone microarchitecture parameters. Hematoxylin&eosin (H&E) and tartrate resistant acid phosphatase (TRAP) staining of femurs showed that UroA suppressed LPS-induced osteoclastogenesis accompanied by the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling. In RANKL-triggered mouse bone marrow-derived macrophages (BMDMs), UroA inhibited the formation of osteoclasts and Fibrous actin rings (F-actin rings), and decreased TRAP activity. Moreover, UroA significantly decreased mRNA and protein expression of major inflammatory cytokines in LPS-challenged RAW264.7 cells by decreasing the phosphorylation of NF-κB p65, c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase1/2 (Erk1/2), and p38. Furthermore, UroA may activate the Nrf2 signaling pathway by increasing mRNA and protein expression of antioxidant proteins. We conclude that UroA attenuated RANKL-induced osteoclastogenesis by suppressing the p38 mitogen-activated protein kinase (MAPK) pathway and inducing Nrf2 nuclear translocation. Thus, supplementation with UroA may help alleviate inflammation-induced bone loss and bone resorption.

[Multi-index components of Scutellariae Barbatae Herba according to UHPLC-QTRAP-MS coupled with chemometrics].

The present study developed an ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap composite mass spectrometry(UHPLC-QTRAP-MS) to simultaneously determine the content of potential active components in Scutellariae Barbatae Herba and also to provide a reference approach for screening out the differential quality control components among different batches of Scutellariae Barbatae Herba. Chromatographic separations were conducted on a Thermo Acclaim~(TM) RSLC 120 C_(18) column(3.0 mm×100 mm, 2.2 µm) in a gradient program. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile, and the column temperature was maintained at 40 ℃. The flow rate was 0.4 mL·min~(-1) and the injection volume was 2 µL. The targeted compounds were monitored in the multiple reaction monitoring(MRM) mode. The acquired data were processed by hierarchical cluster analysis(HCA) and partial least square discriminant analysis(PLS-DA). Sixteen compounds all showed good linear relationship within the corresponding linear ranges and the R~2 values were all higher than 0.993 2. The RSDs of precision, repeatability, and stability were less than or equal to 3.7%. Mean recovery rates were in the range of 95.67% and 104.8% with RSDs≤3.2%. According to HCA and PLS-DA, all samples were clustered into four categories. Scutellarin, acteoside, scutellarein, and scutebarbatine X(VIP>1) were considered as differential chemical markers in the four categories. In conclusion, the developed method can be used for the simulta-neous determination of the multiple components and quality control of Scutellariae Barbatae Herba.

Selective enrichment of microbiota-derived tryptophan metabolites in mouse faeces based on molecularly imprinted solid-phase extraction for HPLC analysis.

Gut microbiota-derived tryptophan (TRP) metabolites, especially the indole derivatives, function as critical modulators of intestinal immune function and integrity via modulating the aryl hydrocarbon receptor pathway. Selective enrichment of these indole metabolites in biological samples is important for quantitative determination by routine methods such as HPLC. Here, we report a molecularly imprinted polymer (MIP) for solid-phase extraction (SPE) of TRP-derived indole metabolites from faeces of mice. The MIP was synthesized by surface polymerization by using indole-3-acetic acid (IAA) and acrylamide after cross-linking by ethylene glycol dimethacrylate (EGDMA) and initialed by 2',2-azobisisobutyronitrile (AIBN). The MIPs were then characterized by transmission electron microscope (TEM) and fourier transform infrared spectroscopy (FTIR), and the adsorption capacity, selectivity and reusability of MIPs were systematically evaluated. Results showed that IAA-MIPs showed a uniformly distributed nanoshell layer with a thin shell thickness of 26 nm. The IAA-MIPs could selectively adsorb IAA, indole-3-propionic acid (IPA), and indole-3-lactic acid (ILA) in a mixed solution that also contains TRP and tyrosine. The adsorption capacity of IAA-MIPs only slightly decreased with the increase of recycling use. As purification material for SPE, the IAA-imprinted polymers (IAA-MIPs) were successfully applied to extract IAA, IPA, and ILA from normal and colitis mice for HPLC determination. Collectively, these surface molecularly imprinted polymers could find extensive use to selective enrichment of microbiota-derived indole metabolites in biological samples.