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The Zhuanggu Guanjie herbal formula has been a famous Chinese prescription for treating bone diseases since time immemorial. The anti-osteoarthritis (OA) properties of this botanical prescription are well documented in the Chinese Pharmacopoeia. However, the detailed mechanisms behind the phenomenon have not been elucidated. Hence, we aimed to investigate the anti-OA efficacy of the Zhuanggu Guanjie herbal formula and its underlying mechanism. The anti-OA properties of Zhuanggu Guanjie capsule (ZGC) were determined by the cytokine contents and inflammatory-related proteins, which were measured by RT-PCR, flow cytometry, Western blot, and laser confocal assay in ATDC5 cells. The levels of interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin synthesis E2 have been markedly reduced after being treated with ZGC for 48 h in a dose-dependent manner. Furthermore, ZGC prevented the translocation of NF-κB from the cytosol to the nucleus. On the other hand, we used the mono-iodoacetate (MIA)-induced OA model to confirm the in vivo efficacies of this herbal formula. Oral administration of ZGC attenuated MIA-induced OA damage through changes in histopathological and knee joint volumes. The serum matrix metalloproteinase-13 contents in the ZGC treatment group declined as compared to those in the MIA model group. Through our in vitro and in vivo studies, we confirmed the anti-OA efficacy of ZGC and uncovered its detailed mechanism, and this treatment shed light on OA pathophysiology.

Laoxianghuang, fermented from Citrus medica L. var. Sarcodactylis Swingle of the Rutaceae family, is a medicinal food. The volatiles of Laoxianghuang fermented in different years were obtained by solid-phase microextraction combined with gas chromatography–mass spectrometry (SPME-GC–MS). Meanwhile, the evolution of its component-flavor function during the fermentation process was explored in depth by combining chemometrics and performance analyses. To extract the volatile compounds from Laoxianghuang, the fiber coating, extraction time, and desorption temperature were optimized in terms of the number and area of peaks. A polydimethylsiloxane/divinylbenzene (PDMS/DVB) with a thickness of 65 μm fiber, extraction time of 30 min, and desorption temperature of 200 °C were shown to be the optimal conditions. There were 42, 44, 52, 53, 53, and 52 volatiles identified in the 3rd, 5th, 8th, 10th, 15th, and 20th years of fermentation of Laoxianghuang, respectively. The relative contents were 97.87%, 98.50%, 98.77%, 98.85%, 99.08%, and 98.36%, respectively. Terpenes (mainly limonene, γ-terpinene and cymene) displayed the highest relative content and were positively correlated with the year of fermentation, followed by alcohols (mainly α-terpineol, β-terpinenol, and γ-terpineol), ketones (mainly cyclohexanone, D(+)-carvone and β-ionone), aldehydes (2-furaldehyde, 5-methylfurfural, and 1-nonanal), phenols (thymol, chlorothymol, and eugenol), esters (bornyl formate, citronellyl acetate, and neryl acetate), and ethers (n-octyl ether and anethole). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed a closer relationship between the composition of Laoxianghuang with similar fermentation years of the same gradient (3rd-5th, 8th-10th, and 15th-20th). Partial least squares discriminant analysis (PLS-DA) VIP scores and PCA-biplot showed that α-terpineol, γ-terpinene, cymene, and limonene were the differential candidate biomarkers. Flavor analysis revealed that Laoxianghuang exhibited wood odor from the 3rd to the 10th year of fermentation, while herb odor appeared in the 15th and the 20th year. This study analyzed the changing pattern of the flavor and function of Laoxianghuang through the evolution of the composition, which provided a theoretical basis for further research on subsequent fermentation.

Toll‐like receptors (TLRs) are an important family of pattern‐recognition receptors that recognize a series of pathogen‐associated molecular patterns to trigger the host immune response. In the present study, we identified three TLR13 genes (i.e., LcLTR13a, LcTLR13b, and LcTLR13c) in American bullfrogs, Lithobates catesbeianus, and explored their constitutive and pathogen‐induced expression profile in different tissues. The open reading frames of LcLTR13a, LcTLR13b and LcTLR13c are 2811, 2844, and 2814 bp long, encoding 955, 947, and 933 amino acids, respectively. Phylogenetic analysis showed that three LcTLR13 genes grouping in different subclades indicate the gene duplication of bullfrog TLR13 as in teleost. The three LcTLR13 genes were expressed in all the tissues examined, with the liver and spleen expressing the higher levels. Moreover, the expression levels of LcLTR13a, LcTLR13b, and LcTLR13c were significantly induced in the liver and spleen after bullfrogs were challenged with bacterial (i.e., Citrobacter freundii, Aeromonas hydrophila, and Edwardsiella tarda) pathogens. These results indicate that LcTLR13 genes play an essential role in the bullfrog antibacterial innate immune response, providing new insights into immune mechanisms of bullfrogs. [ABSTRACT FROM AUTHOR]

In recent years, bullfrog (Lithobates catesbeianus) has become one of the most commercially important amphibians for aquaculture in China, and its annual production has grown rapidly. However, bacterial diseases caused by Citrobacter freundii infection have resulted in enormous economic losses. The present study used RNA sequencing technology to analyze the transcriptomic profile and differentially expressed genes (DEGs) in the bullfrog liver and spleen post C. freundii infection. De novo assembly resulted in the generation of 77,556 L. catesbeianus transcripts, and 49,421 of these genes were successfully annotated to the databases. Overall, 1487 DEGs were identified in the liver group, including 788 upregulated and 709 downregulated DEGs. A total of 799 DEGs, including 311 upregulated and 488 downregulated genes, were detected in the spleen groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significantly enriched pathways, including pathways involved in metabolism and immune responses. The processes in which the DEGs were enriched were those related to metabolic activities, including the lipid metabolism pathway, amino acid pathway, immune‐related pathway, and inflammatory responses. The results of this study provide insight into the response of bullfrogs against C. freundii at the transcriptome level and reveal the mechanism of the immune response against C. freundii. [ABSTRACT FROM AUTHOR]