Procine recombinant NK-lysin inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 and inhibiting oxidative phosphorylation and glycolysis: a proteomic analysis / 南方医科大学学报

OBJECTIVE@#To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NKlysin (prNK-lysin) against liver cancer cell metastasis.@*METHODS@#HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells. GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases. RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13 (GALNT13), transmembrane protein 51 (TMEM51) and FKBP prolyl isomerase 3 (FKBP3) in the cells, and the protein expression of FKBP3 was verified using Western blotting.@*RESULTS@#Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells, and 2753 compared with PBS-treated cells. Fifteen proteins were differentially expressed between PBS-treated and the control cells, and 1909 were differentially expressed in prNK- lysin group compared with both PBS and control groups. These differentially expressed proteins were involved mainly in the viral process, translational initiation and RNA binding and were enriched mainly in ribosome, protein process in endoplasmic reticulum, and RNA transport pathways. RT-qPCR showed that compared with the control group, prNK-lysin treatment significantly increased the mRNA expressions of GALNT13 (P < 0.05) and TMEM51 (P < 0.01) and lowered FKBP3 mRNA expression (P < 0.05). Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells (P < 0.001).@*CONCLUSION@#Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.

Preliminary study on differentially expressed proteins in a mouse model of secondary alveolar echinococcosis based on data independent acquisition proteomics / 中国血吸虫病防治杂志

Objective To identify the differentially expressed proteins in different liver tissues in the mouse model of alveolar echinococcosis using high-resolution mass spectrometry with data independent acquisition (DIA), and to identify the key proteins contributing to the pathogenesis of alveolar echinococcosis. Methods Protoscoleces were isolated from Microtus fuscus with alveolar echinococcosis and the experimental model of alveolar echinococcosis was established in female Kunming mice aged 6 to 8 weeks by infection with Echinococcus multilocularis protoscoleces. Mice were divided into the experimental and control groups, and animals in the experimental group was injected with approximately 3 000 protoscoleces, while mice in the control group were injected with the same volume of physiological saline. Mouse liver specimens were sampled from both groups one year post-infection and subjected to pathological examinations. In addition, the lesions (the lesion group) and peri-lesion specimens (the peri-lesion group) were sampled from the liver of mice in the experimental group and the normal liver specimens (the normal group) were sampled from mice in the control group for DIA proteomics analysis, and the differentially expressed proteins were subjected to bioinformatics analysis. Results A total of 1 020 differentially expressed proteins were identified between the lesion group and the normal group, including 671 up-regulated proteins and 349 down-regulated proteins, and 495 differentially expressed proteins were identified between the peri-lesion group and the normal group, including 327 up-regulated proteins and 168 down-regulated proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these differentially expressed proteins were involved in peroxisome, peroxisome proliferator-activated receptor (PPAR) and fatty acid degradation pathways, and the peroxisome and PPAR signaling pathways were found to correlate with liver injury. Several differentially expressed proteins that may contribute to the pathogenesis of alveolar echinococcosis were identified in these two pathways, including fatty acid binding protein 1 (Fabp1), Acyl-CoA synthetase long chain family member 1 (Acsl1), Acyl-CoA oxidase 1 (Acox1), Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (Ehhadh) and Acetyl-Coenzyme A acyltransferase 1B (Acaa1b), which were down-regulated in mice in the experimental group. Conclusion A large number of differentially expressed proteins are identified in the liver of the mouse model of alveolar echinococcosis, and Fabp1, Acsl1, Acox1, Ehhadh and Acaa1b may contribute to the pathogenesis of alveolar echinococcosis.

Characteristics of Atmospheric Fine Particulate Matter (PM ) Induced Differentially Expressed Proteins Determined by Proteomics and Bioinformatics Analyses / 生物医学与环境科学（英文）

Objective@#To screen the differentially expressed proteins (DEPs) in human bronchial epithelial cells (HBE) treated with atmospheric fine particulate matter (PM ).@*Methods@#HBE cells were treated with PM samples from Shenzhen and Taiyuan for 24 h. To detect overall protein expression, the Q Exactive mass spectrometer was used. Gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and Perseus software were used to screen DEPs.@*Results@#Overall, 67 DEPs were screened in the Shenzhen sample-treated group, of which 46 were upregulated and 21 were downregulated. In total, 252 DEPs were screened in the Taiyuan sample-treated group, of which 134 were upregulated and 118 were downregulated. KEGG analysis demonstrated that DEPs were mainly enriched in ubiquitin-mediated proteolysis and HIF-1 signal pathways in Shenzhen PM samples-treated group. The GO analysis demonstrated that Shenzhen sample-induced DEPs were mainly involved in the biological process for absorption of various metal ions and cell components. The Taiyuan PM -induced DEPs were mainly involved in biological processes of protein aggregation regulation and molecular function of oxidase activity. Additionally, three important DEPs, including ANXA2, DIABLO, and AIMP1, were screened.@*Conclusion@#Our findings provide a valuable basis for further evaluation of PM -associated carcinogenesis.

Effects and Mechanism of Qiwei Qinggan Powder on Hepatic Fibrosis Based on Proteomics / 中国药房

OBJECTIVE：To investigate the anti- hepatic fibrosis （HF）effects of Qiwei qinggan powder and explore its possible mechanism. METHODS ：Male Wistar rats were randomly divided into blank group ，HF model group ，Qiwei qinggan powder low-dose，medium-dose and high-dose groups [ 135，270，405 mg/（kg·d），by total amount of crude drugs] ，with 12 rats in each group. Except for blank group ，other groups were given 50％ CCl4-peanut oil solution intragastrically （2 mL/kg，twice a week ，for consecutive 8 weeks） to induce HF model. At same time ， blank group and model group were given constant volume of 0.5％ CMC-Na solution intragastrically ；administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 8 weeks. General situation of rats were observedand liver morphology was observed after last administration and hepatic indexes were detected. The contents of liverfunction indexes （ALT，AST，ALP，HYP）in serum and the expression of α-SMA in hepatic tissue were determined ， and HE and Masson staining were performed to observe the histopathology. Using the difference multiple of expression quantity as the index ，TMT technology was used to screen the differentially expressed protein in medicine group （combining the liver tissue samples of Qiwei qinggan powder groups ）and HF model group. Uniprot-GOA database and KAAS ，KEGG mapper online tools were used to analyze GO and KEGG pathway enrichment. RESULTS ：The rats in the blank group were in good health ；the liver was bright red and smooth ，the liver lobules were intact ，no degeneration and necrosis ，inflammatory cell infiltration or fibrous tissue proliferation was found. Compared with blank group ，the rats in HF model group had poor diet ，depressed spirit ，disordered and lusterless fur ；the liver was dark red or yellow with rough surface ，hard texture ，inflammatory cell infiltration ，fiber tissue destruction ，bridge connection and so on ；the hepatic index ，the contents of liver function indexes and the expression of α-SMA were increased significantly （P＜0.05）. Compared with HF model group ，above symptoms of rats were improved to different extent in different dose groups of Qiwei qinggan powder ；hepatic index in Qiwei qinggan powder low-dose group ，the content of ALP in high-dose group ，the contents of ALT，AST and HYP and the expression of α-SMA in different dose groups were decreased significantly （P＜0.05）. A total of 42 differentially expressed proteins related to HF were screened ，of which 15 were up-regulated and 27 were down-regulated in expression，including fatty acid binding protein 4（FABP4），cholesterol 7α-hydroxylase（CYP7A1）. The results of enrichment analysis showed that the differentially expressed proteins were mainly enriched in extracellular space ，blood particles and other cell parts，involving the molecular functions of oxidoreductase activity and fatty acid binding ，the biological processes of the regulation of heterotypic cell adhesion ，protein activation cascade ，as well as retinol metabolism ，arachidonic acid metabolism ，PPAR and other signal pathway. CONCLUSIONS ：Qiwei qinggan powder can reduce the hepatic index ，ALT，AST，ALP and HYP contents in serum ，down-regulate the expression of α-SMA，improve the degree of inflammation and fibrosis of liver tissue ，and have a certain protective effect on rats. The anti-HF mechanism of it involves multiple targets and signal pathways ，such as FABP 4， CYP7A1 and PPAR.

Screening for differentially-expressed proteins in ovary of primary dysmenorrheal mice with Xiangfu Siwu decoction administration using nano LC-LTQ-Orbitrap-MS/MS / 中国中药杂志

To study the mechanism of Xiangfu Siwu decoction in treating primary dysmenorrhea, differentially-expressed proteins in ovary of primary dysmenorrheal mice with Xiangfu Siwu decoction administration were screened based on proteome technology using nano LC-LTQ-Orbitrap-MS/MS. Estradiol benzoate and oxytocin were used to produce dysmenorrheal mice model. The model mice were orally administrated with Xiangfu Siwu decoction for 3 days, and 1 h after the last administration, the ovary samples were collected. After protein denaturation, reduction, alkylation, desalination and enzymatic hydrolysis, identification was carried out by nano LC-LTQ-Orbitrap-MS/MS technology. The obtained data was processed by using Thermo Proteome discoverer 1.4 software. The differentially-expressed proteins were screened and identified, and their biological information was also analyzed. The significant differentially-expressed protein was checked using Western blot technology in ovary samples. A total of 106 differentially-expressed proteins were identified during the normal, model and administration group. Most of them participate cellular processes. Adherens junction and focal adhesion pathways play a regulatory role in various cell signaling pathways. Protein ADRM1 was validated. Compared to the normal group, it was up-regulated expression in the model group. After administration, the expression of ADRM1 was down-regulated. Through the comparative analysis, a series of differentially-expressed proteins involved in primary dysmenorrheal mice with Xiangfu Siwu decoction administration were obtained. Protein ADRM1 may become a target for Xiangfu Siwu decoction.

Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis without Helicobacter pylori infection

Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.

Insights into the protein profiles of stress tolerant and mesophilic yeast by proteomics approach: a comparative study.

Proteins of the stress tolerant and mesophilic yeast were extracted using optimized protein extraction method and estimated by Bradford method. Immobilized pH gradient (IPG) strips were rehydrated with known concentrations of protein samples. Rehydrated IPG strips were run in isoelectric focusing (IEF) to separate the proteins on the basis of their pH gradient. 2DE gels were run, stained and image of the stress tolerant yeast was compared with the gel image of mesophilic yeast. The image analysis using the image master software resulted in the identification of differentially expressed spots in stress tolerant yeast. Among the differentially expressed spots, six were selected and characterized by MALDI-TOF as Enolase, Fructose bisphosphate aldolase, Alcohol dehydrogenase, 30KDa HSP, HSP70 and HSP90.

Effect of Acupuncture of "Taixi" (KI 3) on Protein Expression in the Renal Tissues of Healthy Rats / 针刺研究

Objective To observe the changes of renal protein expression after electroacupuncture (EA) of "Taixi" (KI 3) of the Kidney Meridian of Foot-Shaoyin in healthy rats so as to explore the relationship between KI 3 and the Kidney. Methods Twelve adult male Wistar rats were randomly divided into normal control group (n=6) and EA group (n=6). EA (2 Hz/100 Hz,2-4 mA) was applied to bilateral KI 3 for 20 min,once a day for 7 days. Under anesthesia,the rats were perfused transcardiacally with ice saline and their kidneys removed for extracting the total proteins and assaying the differentially expressed proteins with two-dimensional gel electrophoresis (2-DE),Image MasterTM 2 D Platinum Software and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) MALDI-TOF-MS,respectively. Results Image MasterTM 2 D Platinum Software analysis showed that after EA,9 protein points of the kidney tissues expressed differentially above 3-fold in comparison with control group were upregulated,2 of which were identified to be NAD-dependent isocitrate dehydrogenase and Quinone reductase. No downregulated differentially expressed proteins were found in the kidney tissues. Conclusion EA of "Taixi" (KI 3) can increase the expression of NAD-dependent isocitrate dehydrogenase and Quinone reductase in the kidney tissue,suggesting an increase of energy metabolism after EA and a close correlation between the KI 3 (Source-point) and the Kidney.