Specific expression of FABP5 in type II alveolar epithelial cells in silicosis pulmonary fibrosis based on single-cell transcriptome sequencing / 环境与职业医学

Background Silicosis is caused by long-term inhalation of large amounts of free silica (SiO2) particles, and exploring its mechanism can provide new directions for the treatment of silicosis fibrosis. Objective To investigate the expression and role of fatty acid binding protein 5 (FABP5) in a silica-induced silicosis model. Methods In combination with the results of single-cell transcriptome sequencing, the expression pattern of FABP5 in mouse alveolar epithelial cells was explored by bioinformatic analysis, and the distribution pattern of fabp5 was detected by spatial transcriptomics. An in vivo model of silicosis was established by intratracheal injection with SiO2 into mice and four groups were set up: normal saline (NS) 7 d group, NS 56 d group, SiO2 7 d group, and SiO2 56 d group. An in vitro model of silicosis was established in SiO2-treated mouse lung epithelial cell line (MLE-12). At the whole animal level, the marker of epithelial cells (E-Cad) and the protein level of FABP5 were detected by tissue immunofluorescence assay; in vitro, the changes of fabp5 mRNA expression and protein level in MLE-12. Results The results of single-cell transcriptome sequencing and spatial transcriptome sequencing showed that the mRNA expression of fabp5 was upregulated in type II alveolar epithelial cells in the focal area of silicosis in mice, accompanied by elevated tissue immunofluorescent protein levels, and there was co-localization of E-CAD. Meanwhile, SiO2 stimulation induced a 1.58-fold increase in fabp5 mRNA expression and a 2-fold increase in protein levels in MLE-12 cells, with significant differences (P<0.05). Conclusion The protein level of FABP5 is increased in alveolar epithelial cells in a pulmonary fibrosis model, suggesting that FABP5 may be involved in the pathological process of epithelial cells in pulmonary fibrosis.

PPP2R3A promotes silicosis by regulating the expression of p53 / 中国药科大学学报

@#Silicosis, one of the most serious occupational diseases in the world, is a complex pathological process with multi-cell involvement and multi-factor regulation, and its pathogenesis has not been fully elucidated.Protein phosphatase 2A (PP2A) regulates tumor signaling pathways, cell development and cell cycle.The regulatory subunit B of PP2A binds to the core enzyme, resulting in tissue expression specificity and substrate specificity of the PP2A holoenzyme complex.Protein phosphatase 2A regulatory subunit B"α (PPP2R3A) is a subunit of PP2A regulatory subunit B", which is a regulator of cell proliferation.However, the role of PPP2R3A in pulmonary fibrosis is still unclear.In this study, the pulmonary fibrosis model was established by endotracheal infusion of silica (SiO2, 250 mg/kg).Human pulmonary fibroblast-adult cells (HFP-a) were stimulated with 5 ng/mL TGF-β1 to construct fibro-related cell models.The transcription level of Ppp2r3a was detected by qRT-PCR assay.Immunofluorescence and Western blot experiments were performed to detect protein levels.Cell viability was detected by CCK-8 assay.The cell migration ability was detected by scratch test.Experimental results showed that silica nodules and collagen deposition were obvious in the SiO2 group, and the expression of PPP2R3A in lung fibroblasts increased, which could affect cell viability and migration ability, and may promote the progression of pulmonary fibrosis by regulating the expression of p53 signaling pathways.This study provides a new idea for the prevention and treatment of pulmonary fibrosis.

Macrophage differential genes and their functions in silicosis based on single cell RNA sequencing and spatial transcriptome sequencing / 环境与职业医学

Background The rise of single cell RNA sequencing (scRNA-seq) and spatial transcriptome sequencing technologies has allowed for intensive study of lung diseases, but both have been poorly studied in silicosis. Objective To explore differentially expressed genes DEGs in silicosis macrophages by scRNA-seq combined with spatial transcriptome sequencing and analyze the potential diagnostic genes. Methods Male C57BL/6 mice (5-6 weeks old, 22-30 g) were randomly divided into 4 groups: normal saline (NS) group for 7 d, NS group for 56 d, SiO2 group for 7 d, and SiO2 group for 56 d, with 1 mouse in each group. A silicosis model was constructed by tracheal drip injection of SiO2 suspension (0.2 g·kg−1, 50 g·cm−2), and the control mice were given the same volume of NS. The right lung was removed for scRNA-seq and the left lung for spatial transcriptome sequencing on day 7 and day 56, respectively. Cell populations were captured using principal component analysis techniques and dimensionality reduction of uniform manifold approximation and projection. The Find Markers function in R language was applied to analyze the DEGs changes of macrophages in two groups of lung tissues, and the corresponding DEGs were subjected to Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes signaling pathway analysis, while STRING and CytoHubba plug-ins of Cytoscape software were applied to protein-protein interaction network analysis to screen out key (Hub) genes. Spatial transcriptome sequencing was used to explore the original location of Hub genes on lung tissue sections and their mapping in lung macrophages. Finally, the correlation of Hub gene expression levels in lung tissues of silicosis patients and mouse silicosis models was verified, the diagnostic efficacy of Hub gene using subject operating characteristic curves (ROC). In vitro experiments by applying cell viability assay were conducted to verify the changes in viability of mouse macrophages (RAW264.7) under SiO2 stimulation. Results The scRNA-seq revealed a total of 20 clusters captured and defined. The results of scRNA-seq and spatial transcriptome sequencing showed an increased number of macrophages in the lung tissue of the SiO2 group compared to the NS group and clustered in the focal areas. Among the 97 macrophage DEGs screened out, 75 were up-regulated genes, and mainly enriched in chemotaxis and migration of neutrophils, chemokine receptor binding, tumor necrosis factor signaling pathway, cytokine-cytokine receptor interaction pathway, and interleukin-17 signaling pathway; and 22 were down-regulated genes, and mainly enriched in late endosomes, peroxisome proliferator-activated receptors signaling pathway, and alcoholic liver disease signaling pathway. A total of 2 core modules and 3 Hub genes were screened out, including Ccl2, Ccl7, and Ptgs2. The scRNA-seq showed that they were expressed at elevated levels in the SiO2 group compared to the NS group and clustered in additional macrophages, and the spatial transcriptome sequencing showed that they clustered in inflammatory areas with nodular lesions. The CCL7 and PTGS2 expressions were increased in the lung tissue of SiO2 patients compared with the healthy subjects, and the areas under the working curve of the subjects were 0.850 and 0.786, respectively. The viability of RAW264.7 cells was enhanced under SiO2 stimulation at 3 h, 6 h, and 12 h compared to those without the stimulation (P<0.05). Conclusion Bioinformatics screening have identified 3 Hub genes (Ccl2, Ccl7, and Ptgs2)and 2 potential diagnostic genes (CCL7 and PTGS2) in the lung tissue of silicosis mice, which may be potential molecular markers of early-stage silicosis with implications for the development and prognosis of silicosis.

Variation analysis of genomic methylation induced by high temperature stress in Pinellia ternata / 中国中药杂志

Pinellia ternata belongs to the Araceae family and is a medicinal herb. The tuber is the medicinal organ with antitussive, antiemetic and anti-tumor activities. It is easy to encounter high temperature environment during the growth periods, leading to decrease of tuber production. At present, the mechanism of response to high temperature stress in P. ternata is still unknown. DNA methylation plays a vital role in plant protection against adversity stress as a way of epigenetic regulation. In this study, P. ternata was used as material for treatment of high temperature stress at 0 h, 6 h and 80 h, and methylation sensitive amplification polymorphism(MSAP) analysis was conducted on the changes of DNA methylation in its genome. The results showed that 20 pairs of MSAP primers were selected from 100 MSAP primers with multiple clear and uniform bands, and 353, 355 and 342 loci were amplified from materials of P. ternata treated in the high temperature stress 0 h, 6 h and 80 h, respectively. Cytosine methylation levels of CCGG context in the above materials were characterized as 60.91%, 44.79% and 44.74%, respectively. And the full methylation ratios were 16.71%, 22.25% and 29.24, respectively. It demonstrated that high temperature stress significantly induced the down-regulation of DNA methylation level and up-regulation of the full methylation rate in P. ternata genome. This study provides a preliminary theoretical reference for analyzing the mechanism of P. ternata responding to high temperature stress from the epigenetic perspective.

Antitumor efficacy of the recombinant Newcastle disease virus rNDV-IL15 on melanoma models / 药学学报

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.

Efficacy observation on Jin's three-needle therapy for allergic rhinitis of lung qi deficiency and cold syndrome / 中国针灸

<p><b>OBJECTIVE</b>To compare the clinical efficacy in the treatment of allergic rhinitis (AR) of lung qi deficiency and cold syndrome between Jin's three-needle therapy and western medication.</p><p><b>METHODS</b>Sixty-six patients were randomized into an acupuncture group and a western medication group, 33 cases in each one. In the acupuncture group, acupuncture was applied at three-nose points [Yingxiang (LI 20), Shangyingxiang (EX-HN 8) and Yintang (GV 29); Cuanzhu (BL 2) was added for frontal headache] and three-back points [Dazhu (BL 11), Fengmen (BL 12) and Feishu (BL 13)], once every day. Ten treatments made one session. Two sessions of treatment were required. In the western medication group, desloratadine oral suspension was prescribed, 5 mg each time, once a day, for 20 days. The scores of the symptoms and physical signs in AR patients as well as the clinical efficacy were observed between the two groups.</p><p><b>RESULTS</b>The total effective rate was 93.9% (31/33) in the acupuncture group, which was better than 72.7% (24/33) in the western medication group (P < 0.05). After treatment, the scores of AR symptoms and physical signs as well as the total score were all reduced compared with those before treatment in the two groups (all P < 0.01). The score of every item in the acupuncture group was lower than that in the western medication group after treatment (score of symptoms: 4.70 +/- 2.07 vs 6.55 +/- 2. 69, score of physical signs: 0.85 +/- 0.67 vs 1.45 +/- +0.62, total score: 5.36 +/- 2.70 vs 8.00 +/- 2.91, all P < 0.01).</p><p><b>CONCLUSION</b>Jin's three-needle therapy achieves superior efficacy on AR of lung-qi deficiency and cold syndrome, which is better than desloratadine oral suspension.</p>

The synergism and mechanism of action of rClone30-hDR5 in combination with TRAIL on HCC / 药学学报

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.

Preparation antibodies against recombinant bovine IFN-gamma and development of sandwich ELISA for bovine IFN-gamma detection / 生物工程学报

This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.

Infectious bovine rhinotracheitis viral gG expression and gG-ELISA development / 生物工程学报

Taking the genome DNA of Infectious Bovine Rhinotracheitis Virus (IBRV) as the template, the gG gene was amplified with PCR and cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing, the insert was subcloned into the expression vector pGEX-KG. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assay showed that this gene was expressed as both soluble form and inclusion body by the transformed E. coli BL21 strain (DE3). The fusion protein was purified and used as the coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit (IDEXX) was made in the detection of 380 cow serum samples. The results demonstrated an agreement of 92%. By using this novel gG-ELISA, 1248 cow serum samples were tested and the average positive rate of IBRV antibodies for imported cows is 21.7%, while the positive rate ranged greatly from 0.0%-41.5% for Hubei local Chinese Black and White Dairy Cows.