S-allyl Cysteine Enhances Testosterone Production in Mice and Mouse Testis-Derived I-10 Cells

Hypogonadism, associated with low levels of testosterone synthesis, has been implicated in several diseases. Recently, the quest for natural alternatives to prevent and treat hypogonadism has gained increasing research interest. To this end, the present study explored the effect of S-allyl cysteine (SAC), a characteristic organosulfur compound in aged-garlic extract, on testosterone production. SAC was administered at 50 mg/kg body weight intraperitoneally into 7-week-old BALB/c male mice in a single-dose experiment. Plasma levels of testosterone and luteinizing hormone (LH) and testis levels of proteins involved in steroidogenesis were measured by enzymatic immunoassay and Western blot, respectively. In addition, mouse testis-derived I-10 cells were also used to investigate the effect of SAC on steroidogenesis. In the animal experiment, SAC significantly elevated testosterone levels in both the plasma and the testis without changing the LH level in plasma and increased phosphorylated protein kinase A (p-PKA) levels. Similar results were also observed in I-10 cells. The findings demonstrating the increasing effect of SAC on p-PKA and mRNA levels of Cyp11a suggest that SAC increases the testosterone level by activating the PKA pathway and could be a potential target for hypogonadism therapeutics.

Quantitative analysis of TMT phosphorylation modification to investigate the effect of verbascoside on the expression of phosphorylated protein in AD cell model

Background—The active monomer Verbascum glycosides in Cistanche tubulosa has good development prospects in terms of neuroprotection and delaying neurodegenerative diseases, and it has become one of the research hot spots. To investigate the effect of verbascoside (OC1) on the expression of phosphorylated protein in the protective effect of AD cell model by TMT labeling and phosphorylation enrichment technique and high-resolution liquid chromatography-mass spectrometry quantitative proteomics research strategy. Methods—The normal control group, the model group Aβ1-42(10μmol/L) group and the OC1(10μg/ml) administration group were set. (1)protein extraction quality control. (2)TMT mark. (3)HPLC classification and modification enrichment. (4)Analysis of mass spectrometry by liquid chromatography-mass spectrometry. (5)Analysis of bioinformatics results. (6)Western Blotting was used to detect the expression levels of p-CaMKII(Thr286), p-Synapsin1(Ser603)/Synapsin1, Synaptophysin and Synaptotagmin-1 protein. Results—The study finally identified 9020 phosphorylation sites on 3227 proteins, of which 8635 sites of 3134 proteins contained quantitative information. Screening of differential sites follows the following criteria: 1.2 times the change threshold and CV value < 0.1. Based on the above data and standards, we performed a systematic bioinformatics analysis of proteins containing quantitative information sites. Western Blotting results showed that Verbascoside could promote the expression of p-CaMKII (Thr286), p-Synapsin (Ser603)/Synapsin, Synaptophysin and Synaptotagmin-1 protein. Conclusions—Verbascoside(OC1) can increase the expression of phosphorylated protein in AD cell model, which provides a basis for further study on the molecular mechanism of verbascoside promoting neurotransmitter release.

Extracellular vesicles from mast cells induce mesenchymal transition in airway epithelial cells

Background In the airways, mast cells are present in close vicinity to epithelial cells, and they can interact with each other via multiple factors, including extracellular vesicles (EVs). Mast cell-derived EVs have a large repertoire of cargos, including proteins and RNA, as well as surface DNA. In this study, we hypothesized that these EVs can induce epithelial to mesenchymal transition (EMT) in airway epithelial cells. Methods In this in-vitro study we systematically determined the effects of mast cell-derived EVs on epithelial A549 cells. We determined the changes that are induced by EVs on A549 cells at both the RNA and protein levels. Moreover, we also analyzed the rapid changes in phosphorylation events in EV-recipient A549 cells using a phosphorylated protein microarray. Some of the phosphorylation-associated events associated with EMT were validated using immunoblotting. Results Morphological and transcript analysis of epithelial A549 cells indicated that an EMT-like phenotype was induced by the EVs. Transcript analysis indicated the upregulation of genes involved in EMT, including TWIST1, MMP9, TGFB1, and BMP-7. This was accompanied by downregulation of proteins such as E-cadherin and upregulation of Slug-Snail and matrix metalloproteinases. Additionally, our phosphorylated-protein microarray analysis revealed proteins associated with the EMT cascade that were upregulated after EV treatment. We also found that transforming growth factor beta-1, a well-known EMT inducer, is associated with EVs and mediates the EMT cascade induced in the A549 cells. Conclusion Mast cell-derived EVs mediate the induction of EMT in epithelial cells, and our evidence suggests that this is triggered through the induction of protein phosphorylation cascades.

Quantitative analysis of TMT phosphorylation modification to investigate the effect of verbascoside on the expression of phosphorylated protein in AD cell model

Ethnopharmacological relevance— The active monomer Verbascum glycosides in Cistanche tubulosa has good development prospects in terms of neuroprotection and delaying neurodegenerative diseases, and it has become one of the research hot spots. Aim of the study— To investigate the effect of verbascoside (OC1) on the expression of phosphorylated protein in the protective effect of AD cell model by TMT labeling and phosphorylation enrichment technique and high-resolution liquid chromatography-mass spectrometry quantitative proteomics research strategy. Materials and Methods— The normal control group, the model group (Aβ 1-42 10μmol/L) group and the OC1 administration group (10μg/ml) group were set. (1)protein extraction quality control.(2)TMT mark.(3)HPLC classification and modification enrichment. (4)Analysis of mass spectrometry by liquid chromatography-mass spectrometry. (5)Analysis of bioinformatics results.(6)Western Blotting was used to detect the expression levels of p-CaMKII(Thr286), p-Synapsin (Ser9) / Synapsin, Synaptophysin and Synaptotagmin-1 protein. Results— The study finally identified 9020 phosphorylation sites on 3227 proteins, of which 8635 sites of 3134 proteins contained quantitative information. Screening of differential sites follows the following criteria: 1.2 times the change threshold and CV value < 0.1. Based on the above data and standards, we performed a systematic bioinformatics analysis of proteins containing quantitative information sites. Western Blotting results showed that Verbascoside could promote the expression of p-CaMKII (Thr286), p-Synapsin (Ser9) / Synapsin, Synaptophysin and Synaptotagmin-1 protein. Conclusions— Verbascoside (OC1) can increase the expression of phosphorylated protein in AD cell model, which provides a basis for further study on the molecular mechanism of verbascoside promoting neurotransmitter release.

Lyn inhibited BCR signal pathway mediates B-cell anergy induced by avian leukosis virus subgroup J

Background: Immune tolerance induced by retrovirus is a prerequisite for tumorigeness. We had reported that B-cell anergy was the main reason for immune tolerance induced by avian leukosis virus subgroup J (ALV-J). However, the molecular mechanism remains unclear. Results: Initially, we found that Lyn showed down-regulation in chick embryo fibroblasts (CEF) and up-regulation in B-cells infected by ALV-J. So, we speculated that tyrosine kinase Lyn plays a key role in B-cell anergy induced by ALV-J. Confocal laser scanning microscopy (CLSM) and co-immunoprecipitation (Co-IP) results demonstrated that ALV-J indirectly regulated the expression of Lyn. To further investigate the role and regulatory mechanism of Lyn in B-cell anergy induced by ALV-J, the expression levels of Lyn and Syk at different phosphorylation site, the Ca 2+ mobilization, and the expression levels of NF-κB p65 protein in vitro and vivo were detected in B-cells. The result showed that Ca 2+ mobilization was delayed and p65 expression level was decreased in B-cells after ALV-J infection. Consistently, the retrieve of Ca 2+ mobilization, expression levels of NF-κB p65 were found after RNA interference of Lyn. Subsequently, we demonstrated that the activation of phosphorylated Lyn protein at Tyr507 site played a critical role in B-cells anergy, which were verified by the fact of the significantly up-regulation of the expression levels of phosphorylated Syk protein at Tyr525/526 site when RNA interference for Lyn were performed in B-cells. Furthermore, immunohistochemical (IHC) staining results confirmed that the expression levels of Lyn phosphorylated protein at Tyr507 site in bursal cells were increased, while the expression levels of Syk phosphorylated protein at Tyr525/526 sites were decreased. Conclusions: These findings suggested that Lyn inhibited BCR signal pathway mediates B-cell anergy under ALV-J infection，which will provide a new insight for revealing the molecular mechanism of immune tolerance induced by ALV-J. Keywords: avian leukosis virus subgroup J , B-cell anergy, immune tolerance, Lyn, Syk