Accurate network pharmacology and novel ingredients formula of herbal targeting estrogen signaling for psoriasis intervention.

ETHNOPHARMACOLOGICAL RELEVANCE: As a common chronic inflammatory skin disease, psoriasis is incompletely understood and brings a lot of distress to patients. The estrogen signaling pathway has been implicated in its pathogenesis, making it a potential therapeutic target. Si Cao Formula (SCF) has demonstrated promise in treating psoriasis clinically. However, its molecular mechanisms concerning psoriasis remain largely unexplored. AIM OF THE STUDY: To elucidate the underlying mechanisms of the action of SCF on psoriasis. MATERIALS AND METHODS: Active ingredients were identified by LC-MS/MS. After the treatment with SCF, the exploration of differentially expressed proteins (DEPs) were conducted using tandem mass tag (TMT)-based quantitative proteomics analysis. By GO/KEGG, WikiPathways and network pharmacology, core signaling pathway and protein targets were explored. Consequently, major signaling pathway and protein targets were validated by RT-qPCR, immunoblotting and immunofluorescence. Based on Lipinski's Rule of Five rules and molecular docking, 8 active compounds were identified that acted on the core targets. RESULTS: 41 compounds of SCF and 848 specific targets of these compounds were identified. There were 570 DEPs between IMQ (Imiquimod) and IMQ + SCF group, including 279 up-regulated and 304 down-regulated proteins. GO/KEGG, WikiPathways and network pharmacology revealed estrogen signaling pathway as the paramount pathways, through which SCF functioned on psoriasis. We further show novel ingredients formula of SCF contributes to estrogen signaling intervention, including liquiritin, parvisoflavone B, glycycoumarin, 8-prenylluteone, licochalcone A, licochalcone B, oxymatrine, and 13-Hydroxylupanine, where targeting MAP2K1, ILK, HDAC1 and PRKACA, respectively. Molecular docking proves that they have good binding properties. CONCLUSION: Our results provide an in-depth view of psoriasis pathogenesis and herbal intervention, which expands our understanding of the systemic pharmacology to reveal the multiple ingredients and multiple targets of SCF and focus on one pathway (estrogen signaling pathway) may be a novel therapeutic strategy for psoriasis treatment of herbal medicine.

TMT-based quantitative proteomics analysis of neuroprotective effects of Forsythoside A on the MPTP-induced Parkinson's disease mouse model.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characteristized by the presence of dyskinesia and the progressive loss of dopaminergic neurons. Although certain drugs can mitigate the symptoms of PD, they are unable to delay the disease progression, and their prolonged use may result in complications. Therefore, there exists an urgent necessity to identify potential agents that can effectively delay PD progression with fewer side effects. Recent research has unveiled that several traditional Chinese medicines (TCM) exhibit neuroprotective properties in various models pertinent to PD. Forsythoside A (FSA), the primary bioactive compound derived from TCM Lianqiao, has undergone extensive research in animal models of Alzheimer's disease and cerebral ischemia. However, the investigation into the impact of FSA on PD is limited in existing research. In this study, we aimed to evaluate the neuroprotective effects of FSA on MPTP-induced PD mouse model. FSA demonstrated significant improvements in the behavioral and neuropathological changes triggered by MPTP in mice. Furthermore, it exerted a suppressive effect on the activations of astrocyte and microglia. Meanwhile, Tandem mass tag (TMT)-based quantitative proteomics of striatal tissue and bioinformatics analysis were performed to elucidate the underlying mechanisms of FSA on PD mouse model. Proteomics demonstrated a total of 68 differentially expressed proteins (DEPs) were identified between HFSA and MPTP groups including 26 upregulated and 42 downregulated. Systematic bioinformatics analysis of the 68 DEPs illustrated that they were predominantly related to estrogen signaling pathway and calcium signaling pathway. The related DEPs (PLCß4, Grm2, HPAC and Cox4i1) expression levels were verified by Western blot. FSA effectively restored the altered expression of the four DEPs induced by MPTP. Summarily, FSA exerted remarkable neuroprotective effects in MPTP-induced mice. Further, our research may provide proteomics insights that contribute to the further exploration of FSA as a potential treatment for PD.

Alleviation of endothelial dysfunction of Pheretima guillemi (Michaelsen)-derived protein DPf3 in ponatinib-induced thrombotic zebrafish and mechanisms explored through ox-LDL-induced HUVECs and TMT-based proteomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombus generation is one of the leading causes of death in human, and vascular endothelial dysfunction is a major contributor to thrombosis. Pheretima guillemi (Michaelsen), a traditional medicinal animal known as "Dilong", has been utilized to cure thrombotic disorders for many years. DPf3, a group of functional proteins extracted from P. guillemi, has been characterized and identified to possess antithrombotic bioactivity via in vitro and ex vivo experiments. AIM OF THE STUDY: This study is aimed to investigate the vascular-protection activity and related mechanism of antithrombotic protein DPf3 purified from Pheretima guillelmi systematically. MATERIALS AND METHODS: The antithrombotic activity and vascular endothelium protection effect of DPf3 was explored in vivo using ponatinib-induced vascular endothelial injury zebrafish thrombus model. Then, (hi) ox-LDL-induced HUVECs was applied to investigate the protection mechanism of DPf3 against the injury of vascular endothelium. In addition, TMT-based proteomics analysis was used to study the biomarkers, biological processes and signal pathways involved in the antithrombotic and vascular protective effects of DPf3 holistically. RESULTS: DPf3 exerted robust in vivo antithrombosis and vascular endothelial protection ability. DPf3 was identified to prevent HUVECs from damage by reducing ROS production, and to reduce monocyte adhesion by decreasing the protein content of adhesion factor VCAM 1. DPf3 was also observed to weaken the migration ability of injured cells and inhibit abnormal angiogenesis. The mechanism of DPf3's antithrombotic and vascular protective activity was mainly related to the regulation of lipid metabolism, energy metabolism, complement and coagulation system, ECM receptor interaction, MAPK signal pathway, etc. CONCLUSIONS: This study demonstrates that DPf3 has strong antithrombotic and endothelial protective effects. The endothelial protective ability and related mechanisms of DPf3 provide a scientific reference for the traditional use of earthworms in the treatment of thrombosis.

Proteomic Analysis Based on TMT Regarding the Therapeutic Action of Rhizoma Drynariae on Rats in an Osteoporosis Model.

BACKGROUND: Primary osteoporosis has increasingly become one of the risk factors affecting human health, and the clinical effect and action mechanism of traditional Chinese medicine in the treatment of primary osteoporosis have been widely studied. Previous studies have confirmed that in traditional Chinese medicine (TCM), Drynaria rhizome has a role in improving bone density. In this study, a tandem mass tag (TMT)-based proteomic analysis was conducted to derive potential targets for Drynaria rhizome treatment in postmenopausal osteoporosis. METHODS: The model group (OVX) and experimental group (OVXDF) for menopausal osteoporosis were established using the universally acknowledged ovariectomy method, and the OVXDF group was given 0.48g/kg Rhizoma Drynariae solution by gavage for 12 weeks. After 12 weeks, femurs of rats selected for this study were examined with a bone mineral density (BMD) test, Micro-CT, ELISABiochemical testing, hematoxylin and eosin (HE) staining, and immunohistochemistry. A certain portion of the bone tissue was studied with a TMT-based proteomic analysis and functional and pathway enrichment analysis. Finally, key target genes were selected for Western blotting for validation. RESULTS: The comparison of the OVXDF and OVX groups indicated that Drynaria rhizome could improve bone density. In the TMT-based proteomic analysis, the comparison of these two groups revealed a total of 126 differentially expressed proteins (DEPs), of which 62 were upregulated and 64 were downregulated. Further, by comparing the differential genes between the OVXDF and OVX groups and between the OVX and SHAM groups, we concluded that the 27 differential genes were significantly changed in the rats selected for the osteoporosis model after Drynaria rhizome intragastric administration. The gene ontology (GO) enrichment analysis of DEPs showed that molecular function was mainly involved in biological processes, such as glucose metabolism, carbohydrate metabolism, immune responses, and aging. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEPs revealed that multiple differential genes were enriched in the estrogen and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Relationships with nitrogen metabolism, glycerophospholipid metabolism, secretion systems, and tumor diseases were also observed. Western blotting was consistent with the analysis. CONCLUSIONS: We used TMT-based proteomics to analyze the positive effects of TCM Drynaria rhizome, which can regulate related proteins through the unique roles of multiple mechanisms, targets, and pathways. This treatment approach can regulate oxidative stress, improve lipid metabolism, reduce the inflammatory response mechanism, and improve bone density. These benefits highlight the unique advantages of TCM in the treatment of primary osteoporosis.

TMT-Based Proteomics Reveal the Mechanism of Action of Amygdalin against Rheumatoid Arthritis in a Rat Model through Regulation of Complement and Coagulation Cascades.

The limitations of current medications for treating rheumatoid arthritis (RA) emphasize the urgent need for the development of new drugs. This study aimed to investigate the potential anti-RA mechanism of amygdalin using tandem mass tag (TMT)-based quantitative proteomics technology. First, the anti-RA activity of amygdalin was evaluated in a Complete Freund's adjuvant (CFA)-induced rat model. Then, the roles and importance of proteins in the extracted rat joint tissue were evaluated using TMT-based quantitative proteomics technology. A bioinformatics analysis was used to analyze differentially abundant proteins (DAPs). A proteomics analysis identified 297 DAPs in the amygdalin group compared with the model group, of which 53 upregulated proteins and 51 downregulated proteins showed opposite regulatory trends to the DAPs produced after modeling. According to enrichment analyses of the DAPs, the signaling pathways with a high correlation degree were determined to be the complement and coagulation cascades. Furthermore, western blotting and molecular docking were used to further validate the key node proteins, e.g., complement C1s subcomponent (C1s), component C3 (C3) and kininogen 1 (Kng1). These results suggest that amygdalin may be a promising agent for treating RA by regulating the complement and coagulation cascades.

Mouse Model of Fragile X Syndrome Analyzed by Quantitative Proteomics: A Comparison of Methods.

Multiple methods for quantitative proteomics are available for proteome profiling. It is unclear which methods are most useful in situations involving deep proteome profiling and the detection of subtle distortions in the proteome. Here, we compared the performance of seven different strategies in the analysis of a mouse model of Fragile X Syndrome, involving the knockout of the fmr1 gene that is the leading cause of autism spectrum disorder. Focusing on the cerebellum, we show that data-independent acquisition (DIA) and the tandem mass tag (TMT)-based real-time search method (RTS) generated the most informative profiles, generating 334 and 329 significantly altered proteins, respectively, although the latter still suffered from ratio compression. Label-free methods such as BoxCar and a conventional data-dependent acquisition were too noisy to generate a reliable profile, while TMT methods that do not invoke RTS showed a suppressed dynamic range. The TMT method using the TMTpro reagents together with complementary ion quantification (ProC) overcomes ratio compression, but current limitations in ion detection reduce sensitivity. Overall, both DIA and RTS uncovered known regulators of the syndrome and detected alterations in calcium signaling pathways that are consistent with calcium deregulation recently observed in imaging studies. Data are available via ProteomeXchange with the identifier PXD039885.

Pharmacological effects and mechanism of Kaihoujian Throat Spray (children's type) in the treatment of pediatric acute pharyngitis and tonsillitis.

Context: Kaihoujian Throat Spray children's type (KHJSC) is a Chinese medicine prescription for treating pediatric acute pharyngitis and tonsillitis (APT). However, its relevant mechanisms remain unclear. Objective: To investigate the pharmacological effects of KHJSC on APT in vitro and in vivo, and explore the possible mechanism and target proteins. Materials and methods: The antiviral and antibacterial effects in vitro were evaluated by IC50 and MICs. Thirty-six Japanese white rabbits were averagely divided into control group, model group, amoxicillin group and 3 dose groups of KHJSC (720, 540 and 360 µL/kg/d). The model rabbits were injected with ß-hemolytic Streptococcus solution into the tonsils for 2 consecutive days. KHJSC treatment started on the third day. The whole blood, serum, tonsil tissues and pharyngeal mucosa tissues were collected for routine blood tests, proteomic, ELISA and other tests on the sixth day. Results: The IC50 of KHJSC on HCoV-229E, influenza PR8 and Ad3 were 1.99, 1.99 and 4.49 mg/mL, respectively; MICs of MDR-PA, MRSA and ß-hemolytic Streptococcus were 350, 350, and 175 mg/mL. KHJSC markedly decreased the number of white blood cells, lymphocytes, neutrophils, and the level of IL-1ß, IL-5, IL-6, IL-18, TNF-α and MCP-1; increased the content of IL-2 and IFN-γ. Proteomic analysis and ELISA revealed that PI3K-Akt signaling pathway, NF-κB signaling pathway and Toll-like receptor signaling pathway were the potential mechanisms of KHJSC against APT. Discussion and conclusion: These results provided the reference and scientific basis for the application of KHJSC in clinic and further mechanisms study.

Quantitative TMT-based proteomics revealing host, dietary and microbial proteins in bovine faeces including barley serpin Z4, a prominent component in the head of beer.

There has been little information about the proteome of bovine faeces or about the contribution to the faecal proteome of proteins from the host, the feed or the intestinal microbiome. Here, the bovine faecal proteome and the origin of its component proteins was assessed, while also determining the effect of treating barley, the major carbohydrate in the feed, with either ammonia (ATB) or sodium propionate (PTB) preservative. Healthy continental crossbreed steers were allocated to two groups and fed on either of the barley-based diets. Five faecal samples from each group were collected on Day 81 of the trial and analysed by quantitative proteomics using nLC-ESI-MS/MS after tandem mass tag labelling. In total, 281 bovine proteins, 199 barley proteins, 176 bacterial proteins and 190 archaeal proteins were identified in the faeces. Mucosal pentraxin, albumin and digestive enzymes were among bovine proteins identified. Serpin Z4 a protease inhibitor was the most abundant barley protein identified which is also found in barley-based beer, while numerous microbial proteins were identified, many originating bacteria from Clostridium, while Methanobrevibacter was the dominant archaeal genus. Thirty-nine proteins were differentially abundant between groups, the majority being more abundant in the PTB group compared to the ATB group. SIGNIFICANCE: Proteomic examination of faeces is becoming a valuable means to assess the health of the gastro-intestinal tract in several species, but knowledge on the proteins present in bovine faeces is limited. This investigation aimed to characterise the proteome of bovine faecal extracts in order to evaluate the potential for investigations of the proteome as a means to assess the health, disease and welfare of cattle in the future. The investigation was able to identify proteins in bovine faeces that had been (i) produced by the individual cattle, (ii) present in the barley-based feed eaten by the cattle or (iii) produced by bacteria and other microbes in the rumen or intestines. Bovine proteins identified included mucosal pentraxin, serum albumin and a variety of digestive enzymes. Barley proteins found in the faeces included serpin Z4, a protease inhibitor that is also found in beer having survived the brewing process. Bacterial and archaeal proteins in the faecal extracts were related to several pathways related to the metabolism of carbohydrates. The recognition of the range of proteins that can be identified in bovine faeces raises the possibility that non-invasive sample collection of this material could provide a novel diagnostic approach to cattle health and welfare.

International Music Therapists' Perceptions and Experiences in Telehealth Music Therapy Provision.

The use of telehealth within music therapy practice has increased through necessity in recent years. To contribute to the evolving evidence base, this current study on Telehealth Music Therapy (TMT) was undertaken to investigate the telehealth provision experiences of music therapists internationally. Participants completed an anonymous online cross-sectional survey covering demographics, clinical practice, telehealth provision, and telehealth perceptions. Descriptive and inferential statistics, in combination with thematic analysis, were used to analyze the data. A total of 572 music therapists from 29 countries experienced in providing TMT took part in this study. The results showed that the overall number of clinical hours (TMT and in-person hours combined) declined due to the pandemic. Participants also reported reduced perceived success rates in utilizing both live and pre-recorded music in TMT sessions when compared to in-person sessions. Although many music therapists rose to the challenges posed by the pandemic by incorporating TMT delivery modes, there was no clear agreement on whether TMT has more benefits than drawbacks; however, reported benefits included increased client access and caregiver involvement. Furthermore, a correlation analysis revealed moderate-to-strong positive associations between respondents who perceived TMT to have more benefits than drawbacks, proficiency at administering assessments over telehealth, and perceived likelihood of using telehealth in the future. Regarding the influence of primary theoretical orientation and work setting, respondents who selected music psychotherapy as a primary theoretical orientation had more experience providing TMT prior to the pandemic while those primarily working in private practice were most inclined to continue TMT services post-pandemic. Benefits and drawbacks are discussed and future recommendations for TMT are provided.

Dendrobium mixture ameliorates type 2 diabetes mellitus with non-alcoholic fatty liver disease through PPAR gamma: An integrated study of bioinformatics analysis and experimental verification.

Dendrobium mixture (DM) is a patented Chinese herbal medicine indicated which has anti-inflammatory and improved glycolipid metabolism. However, its active ingredients, targets of action, and potential mechanisms are still uncertain. Here, we investigate the role of DM as a prospective modulator of protection against non-alcoholic fatty liver disease (NAFLD) induced by type 2 diabetes mellitus (T2DM) and illustrate the molecular mechanisms potentially involved. The network pharmacology and TMT-based quantitative protomics analysis were conducted to identify potential gene targets of the active ingredients in DM against NAFLD and T2DM. DM was administered to the mice of DM group for 4 weeks, and db/m mice (control group) and db/db mice (model group) were gavaged by normal saline. DM was also given to Sprague-Dawley (SD) rats, and the serum was subjected to the palmitic acid-induced HepG2 cells with abnormal lipid metabolism. The mechanism of DM protection against T2DM-NAFLD is to improve liver function and pathological morphology by promoting peroxisome proliferator-activated receptor γ (PPARγ) activation, lowering blood glucose, improving insulin resistance (IR), and reducing inflammatory factors. In db/db mice, DM reduced RBG, body weight, and serum lipids levels, and significantly alleviated histological damage of liver steatosis and inflammation. It upregulated the PPARγ corresponding to the prediction from the bioinformatics analysis. DM significantly reduced inflammation by activating PPARγ in both db/db mice and palmitic acid-induced HepG2 cells.

TMT-based quantitative proteomics analysis of the effects of Jiawei Danshen decoction myocardial ischemia-reperfusion injury.

BACKGROUND: Every year, approximately 17 million people worldwide die due to coronary heart disease, with China ranking second in terms of the death toll. Myocardial ischemia-reperfusion injury (MIRI) significantly influences cardiac function and prognosis in cardiac surgery patients. Jiawei Danshen Decoction (JWDSD) is a traditional Chinese herbal prescription that has been used clinically for many years in China to treat MIRI. The underlying molecular mechanisms, however, remain unknown. To investigate the proteomic changes in myocardial tissue of rats given JWDSD for MIRI therapy-based proteomics. METHODS: MIRI rat model was created by ligating/releasing the left anterior descending coronary artery. For seven days, the drugs were administered twice daily. The model was created following the last drug administration. JWDSD's efficacy in improving MIRI was evaluated using biochemical markers and cardiac histology. Tandem mass tag-based quantitative proteomics (TMT) technology was also used to detect proteins in the extracted heart tissue. To analyze differentially expressed proteins (DEPs), bioinformatics analysis, including gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways, were employed. Furthermore, western blotting confirmed the potential targets regulated by JWDSD. RESULTS: The histopathologic characteristics and biochemical data showed JWDSD's protective effects on MIRI rats. A total of 4549 proteins were identified with FDR (false discovery rate) ≤1%. Twenty overlapping were identified (162 DEPs and 45 DEPs in Model/Control or JWDSD/Model group, respectively). Of these DEPs, 16 were regulated by JWDSD. GO analysis provided a summary of the deregulated protein expression in the categories of biological process (BP), cell component (CC), and molecular function (MF). KEGG enrichment analysis revealed that the signaling pathways of neutrophil extracellular trap formation, RNA polymerase, serotonergic synapse, and linoleic acid metabolism are all closely related to JWDSD effects in MIRI rats. Furthermore, T-cell lymphoma invasion and metastasis 1 (TIAM1) was validated using western blotting, and the results were consistent with proteomics data. CONCLUSIONS: Our study suggests that JWDSD may exert therapeutic effects through multi-pathways regulation in MIRI treatment. This work may provide proteomics clues for continuing research on JWDSD in treating MIRI.

Potential Anti-Tumor Activity of Nardoguaianone L Isolated from Nardostachys jatamansi DC. in SW1990 Cells.

Natural products (NPs) were a rich source of diverse bioactive molecules. Most anti-tumor agents were built on natural scaffolds. Nardostachys jatamansi DC. was an important plant used to process the traditional Chinese herbal medicines "gansong". Pancreatic cancer was the fourth most common cause of cancer-related death in the world. Hence, there was an urgent need to develop novel agents for the treatment of pancreatic cancer. In this paper, nardoguaianone L (G-6) is isolated from N. jatamansi, which inhibited SW1990 cells colony formation and cell migration, and induced cell apoptosis. Furthermore, we analyzed the differential expression proteins after treatment with G-6 in SW1990 cells by using iTRAQ/TMT-based quantitative proteomics technology, and the results showed that G-6 regulated 143 proteins' differential expression by GO annotation, including biological process, cellular component, and molecular function. Meanwhile, KEGG enrichment found that with Human T-cell leukemia virus, one infection was the most highly enhanced pathway. Furthermore, the MET/PTEN/TGF-ß pathway was identified as a significant pathway that had important biological functions, including cell migration and motility by PPI network analysis in SW1990 cells. Taken together, our study found that G-6 is a potential anti-pancreatic cancer agent with regulation of MET/PTEN/TGF-ß pathway.

Tandem mass tag-based proteomics analysis reveals the multitarget mechanisms of Phyllanthus emblica against liver fibrosis.

Phyllanthus emblica (PE), a traditional multiethnic herbal medicine, is commonly applied to treat liver diseases. Our previous study demonstrated that aqueous extract of PE (AEPE) could alleviate carbon tetrachloride (CCl4)-induced liver fibrosis in vivo, but the underlying molecular mechanisms are still unclear. The present study was undertaken to clarify the multitarget mechanisms of PE in treating liver fibrosis by proteomics clues. A CCl4-induced liver fibrosis rat model was established. The anti-liver fibrosis effects of chemical fractions from AEPE were evaluated by serum biochemical indicators and pathological staining. Additionally, tandem mass tag (TMT) - based quantitative proteomics technology was used to detect the hepatic differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, gene ontology (GO) enrichment and protein-protein interaction (PPI) network were used to perform bioinformatics analysis of DEPs. Western blot analysis was used to verify the key potential targets regulated by the effective fraction of AEPE. The low-molecular-weight fraction of AEPE (LWPE) was determined to be the optimal anti-liver fibrosis active fraction, that could significantly improve ALT, AST, HA, Col IV, PCIII, LN, Hyp levels and reduce the pathological fibrotic lesion of liver tissue in model rats. A total of 195 DEPs were screened after LWPE intervention. GO analysis showed that the DEPs were related mostly to extracellular matrix organization, actin binding, and extracellular exosomes. KEGG pathway analysis showed that DEPs are mainly related to ECM-receptor interactions, focal adhesion and PI3K-Akt signaling pathway. Combined with the GO, KEGG and Western blot results, COL1A2, ITGAV, TLR2, ACE, and PDGFRB may be potential targets for PE treatment of liver fibrosis. In conclusion, LWPE exerts therapeutic effects through multiple pathways and multiple targets regulation in the treatment of liver fibrosis. This study may provide proteomics clues for the continuation of research on liver fibrosis treatment with PE.

Effects of Omega-3 and Antioxidant Cocktail Supplement on Prolonged Bed Rest: Results from Serum Proteome and Sphingolipids Analysis.

Physical inactivity or prolonged bed rest (BR) induces muscle deconditioning in old and young subjects and can increase the cardiovascular disease risk (CVD) with dysregulation of the lipemic profile. Nutritional interventions, combining molecules such as polyphenols, vitamins and essential fatty acids, can influence some metabolic features associated with physical inactivity and decrease the reactive oxidative and nitrosative stress (RONS). The aim of this study was to detect circulating molecules correlated with BR in serum of healthy male subjects enrolled in a 60-day BR protocol to evaluate a nutritional intervention with an antioxidant cocktail as a disuse countermeasure (Toulouse COCKTAIL study). The serum proteome, sphingolipidome and nitrosoproteome were analyzed adopting different mass spectrometry-based approaches. Results in placebo-treated BR subjects indicated a marked decrease of proteins associated with high-density lipoproteins (HDL) involved in lipemic homeostasis not found in the cocktail-treated BR group. Moreover, long-chain ceramides decreased while sphingomyelin increased in the BR cocktail-treated group. In placebo, the ratio of S-nitrosylated/total protein increased for apolipoprotein D and several proteins were over-nitrosylated. In cocktail-treated BR subjects, the majority of protein showed a pattern of under-nitrosylation, except for ceruloplasmin and hemopexin, which were over-nitrosylated. Collectively, data indicate a positive effect of the cocktail in preserving lipemic and RONS homeostasis in extended disuse conditions.

Serum proteomic analysis reveals possible mechanism underlying physiological hemostasis of swim bladder.

The hemostatic effect of isinglass (dried swim bladder) in traditional Chinese medicine is well known. But its mechanism of action remains unclear. Here, mice were gavaged with the dried swim bladder of the chu's croaker (Nibea coibor). The hemostatic effect of swim bladder was investigated, tandem mass tag (TMT)-based quantitative proteomics analysis was performed to screen differentially abundant proteins associated with hemostasis in mouse serum. Results indicated that isinglass significantly shorten bleeding time and promoted coagulation after acute trauma (cut out mouse tail). In total, 57 differentially expressed proteins were identified in the sera between control and swim bladder group, of which 31 were up-regulated and 26 were down-regulated in swim bladder group. KEGG pathway enrichment analysis further demonstrated that the Neutrophil extracellular trap formation pathway was significantly affected. Combined with RT-qPCR verification, our findings further suggested that five candidate proteins in the pathway may be involved in the onset of hemostasis after swim bladder gavage, indicating their important role during the hemostasis process promoting by swim bladder. SIGNIFICANCE: Serum proteomics after swim bladder gavage described differentially enriched proteins related to hemostasis, and enriched pathways were validated. This study revealed the possible pathways involved in the hemostatic effect of swim bladder, which may provide a new effector target for the development of new hemostatic drugs.

TMT-based comparative proteomics reveals the role of acyl-CoA oxidase 4 in enhancing the drought stress tolerance in common buckwheat (Fagopyrum esculentum).

Drought stress has been the main abiotic factor affecting the growth, development and production of common buckwheat (Fagopyrum esculentum). To explore the response mechanisms of regulating buckwheat drought stress on the post-transcriptional and translational levels, a comparative proteomic analysis was applied to monitor the short-term proteomic variations under the drought stress in the seedling stage. From which 593 differentially abundant proteins (DAPs) were identified using the TMT-based proteomics analysis. A number of DAPs were found to be intimately correlated with the styrene degradation, phenylpropanoid biosynthesis and stimulus response, within which. The acyl-CoA oxidase 4 (ACX4), a key regulator in plant abiotic stress response, was selected for further elucidation. Overexpression of the FeACX4 not only conferred drought and salt tolerance in the Arabidopsis, but also significantly increased the root length and fresh weight in the overexpression lines plant relative to the wild type (WT) plant, accompanied by the elevated activities of catalase (CAT) and lowered malonaldehyde (MDA) and H2O2 contents, therefore allowing plants to better adapt to adverse environments. Our results provided information in the exploring of the molecular regulation mechanism responding to drought tolerance in common buckwheat.

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV. METHODS: A total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated. RESULTS: MT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-ß mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS) showed synergistic anti-PRRSV effect. CONCLUSIONS: The component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-ß may be one of the targets of the component formula possessed anti-PRRSV activity.

Comparative proteomics reveals biochemical changes in Salvia miltiorrhiza Bunge during sweating processing.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge is a bulk medicinal material used in traditional Chinese medicine, that can cure cardiovascular diseases, neurasthenia, and other conditions. Sweating is a frequently used method of processing S. miltiorrhiza for medical applications. We previously demonstrated changes to the metabolic profile of linoleic acid, glyoxylate, and dicarboxylate after Sweating. However, this alteration has not been explained at the molecular level. MATERIALS AND METHODS: Fresh roots of Salvia miltiorrhiza Bunge were treated by the Sweating processing, and then the tandem mass tag technique was used to compare the proteome difference between Sweating S. miltiorrhiza and non-Sweating S. miltiorrhiza. RESULTS: We identified a total of 850 differentially expressed proteins after Sweating treatment in S. miltiorrhiza, including 529 upregulated proteins and 321 downregulated proteins. GO enrichment analysis indicated that these differentially expressed proteins are involved in external encapsulating structure, cell wall, oxidoreductase activity, ligase activity, and others. Further analysis showed that CYP450, the pathogenesis-related protein Bet v 1 family, and the peroxidase domain were the major protein domains. KEGG enrichment identified 18 pathways, of which phenylpropanoid biosynthesis is the most important one related to the metabolite profile and is the principal chemical component of S. miltiorrhiza. CONCLUSION: This study addressed potential molecular mechanisms in S. miltiorrhiza after Sweating, and the findings provide reasons for the changes in biochemical properties and metabolites changes which might cause pharmacological variation at the proteome level.

Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus.

Suid herpesvirus 1 (SuHV-1), known as pseudorabies virus (PRV), is one of the most devastating swine pathogens in China, particularly the sudden occurrence of PRV variants in 2011. The higher pathogenicity and cross-species transmission potential of the newly emerged variants caused not only colossal economic losses, but also threatened public health. To uncover the underlying pathogenesis of PRV variants, Tandem Mass Tag (TMT)-based proteomic analysis was performed to quantitatively screen the differentially expressed cellular proteins in PRV-infected Vero cells. A total of 7072 proteins were identified and 960 proteins were significantly regulated: specifically 89 upregulated and 871 downregulated. To make it more credible, the expression of XRCC5 and XRCC6 was verified by western blot and RT-qPCR, and the results dovetailed with the proteomic data. The differentially expressed proteins were involved in various biological processes and signaling pathways, such as chaperonin-containing T-complex, NIK/NF-κB signaling pathway, DNA damage response, and negative regulation of G2/M transition of mitotic cell cycle. Taken together, our data holistically outline the interactions between PRV and host cells, and our results may shed light on the pathogenesis of PRV variants and provide clues for pseudorabies prevention.

The effect of Ma-Xin-Gan-Shi decoction on asthma exacerbated by respiratory syncytial virus through regulating TRPV1 channel.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of bronchial asthma are increasing, and respiratory syncytial virus (RSV) is widely regarded as the common cause of clinical exacerbation of asthma. Ma-Xing-Gan-Shi decoction (MXGSD), a classic traditional Chinese medicine prescription, is well-known for treating respiratory diseases, while the mechanism of effecting on RSV-exacerbated asthma remains to be explored. AIM OF THE STUDY: In this study, we investigated the mechanism by which MXGSD exerts a protective effect on asthma exacerbated by RSV in vivo and in vitro. MATERIALS AND METHODS: MXGSD is composed of four Chinese medicine, including Ephedra intermedia Schrenk & C.A.Mey. (herbaceous stem, 27g), Prunus armeniaca L. (dry seed, 27g), Glycyrrhiza uralensis Fisch. (radix and rhizome, 18g), and Gypsum fibrosum (main component: CaSO4·2H2O, 54g). In the present study, the exacerbated asthmatic mice model with the treatment of OVA plus RSV was replicated, and accompanied by the TMT proteomic analysis and further experimental investigations. Then, the protective effect of MXGSD (13.2, 6.6, 3.3 g/kg/d, 7d) on the mice treated by OVA plus RSV, and the mechanism of regulating TRPV1 was explored. In addition, the intracellular Ca2+ concentration of 16HBE cells pretreated with MXGSD medicated serum was also tested after stimulation with the TRPV1 agonist capsaicin. RESULTS: The results suggested that MXGSD could reduce the levels of inflammation cells, airway hyperresponsiveness, and pathological damage of lung tissue. TMT quantitative proteomics analysis and further experimental exploration revealed that MXGSD could reduce the levels of IL-4, IL-13, PGE2, and SP in BAL and down-regulate the expression of TRPV1 mRNA and protein in lung tissue. Furthermore, 16HBE cells stimulated by capsaicin showed an increased intracellular Ca2+ concentration, while the pretreatment of MXGSD medicated serum could reduce it. CONCLUSION: MSGSD showed a protective effect on RSV-exacerbated asthma, which may be related to its regulation of TRPV1 expression and reduction of Th2 cytokines and neurogenic inflammatory mediators. It may provide an objective basis and reference for the clinical application of MXGSD.