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1.
Journal of Traditional Chinese Medicine ; (12): 103-112, 2024.
Article in Chinese | WPRIM | ID: wpr-1005118

ABSTRACT

ObjectiveTo develop traditional Chinese medicine (TCM) formulae for the treatment of nonsevere coronavirus disease 2019 (COVID-19) and to explore its anti-inflammatory mechanism. MethodsThe dysregulated signaling pathways were determined in macrophages from bronchoalveolar lavage fluid of COVID-19 patients and in lung epithelial cells infected with SARS-CoV-2 in vitro based on transcriptome analysis. A total of 102 TCM formulae for the clinical treatment of nonsevere COVID-19 were collected through literature. The pathway-reversing rates of these formulae in macrophages and lung epithelial cells were evaluated based on signature signaling pathways, and the basic formula was determined in conjunction with TCM theory. The commonly used Chinese materia medica for nonsevere COVID-19 were summarized from the 102 TCM formulae as abovementioned. And together with the screening results from the Pharmacopoeia of the People's Republic of China, a “Chinese materia medica pool” was esta-blished for the development of TCM formulae for COVID-19. The regulatory effects of each herb on signaling pathways were obtained based on targeted transcriptome analysis. Oriented at reversing dysregulated signaling pathways of COVID-19, the calculation was carried out, and the artificial intelligent methods for compositing formulae, that are exhaustive method and parallel computing, were used to obtain candidate compound formulas. Finally, with reference to professional experience, an innovative formula for the treatment of nonsevere COVID-19 was developed. The ethanol extract of the formula was evaluated for its anti-inflammatory effects by detecting the mRNA expression of interleukin 1b (Il1b), C-X-C motif chemokine ligand 2 (Cxcl2), C-X-C motif chemokine ligand 10 (Cxcl10), C-C motif chemokine ligand 2 (Ccl2), nitric oxide synthase 2 (Nos2), and prostaglandin-endoperoxide synthase 2 (Ptgs2) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in RAW264.7 cells treated with lipopolysaccharide (LPS). ResultsIn macrophages and lung epithelial cells, 34 dysregulated signaling pathways associated with COVID-19 were identified respectively. The effects of the 102 formulae for clinical treatment of nonsevere COVID-19 were evaluated based on the dysregulated signaling pathways and targeted transcriptome, and the result showed that Yinqiao Powder and Pingwei Powder (银翘散合平胃散, YQPWP) ranked first, reversing 91.18% of the dysregulated signaling pathways in macrophages and 100% of the dysregulated signaling pathways in lung epithelial cells. Additionally, YQPWP had the function of scattering wind and clearing heat, resolving toxins and removing dampness in accordance with the pathogenesis of wind-heat with dampness in COVID-19. It was selected as the basic formula, and was further modified and optimized to develop an innovative fomula Qiaobang Zhupi Yin (翘蒡术皮饮, QBZPY) based on expert experience and artificial intelligence in composing formulae. QBZPY can reverse all the dysregulated signaling pathways associated with COVID-19 in macrophages and lung epithelial cells, with the reversing rates of 100%. The chief medicinal of QBZPY, including Lianqiao (Fructus Forsythiae), Xixiancao (Herba Siegesbeckiae) and Niubangzi (Fructus Arctii), can down-regulate multiple signaling pathways related with virus infection, immune response, and epithelial damage. RT-qPCR results indicated that compared with the model group, the QBZPY group down-regulated the mRNA expression of Il1b, tumor necrosis factor (Tnf), Cxcl2, Cxcl10, Ccl2, Nos2 and Ptgs2 induced by LPS in RAW264.7 cells (P<0.05 or P<0.01). ConclusionBased on targeted transcriptome analysis, expert experience in TCM and artificial intelligence, QBZPY has been developed for the treatment of nonsevere COVID-19. The ethanol extract of QBZPY has been found to inhibit mRNA expression of several pro-inflammatory genes in a cellular inflammation model.

2.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 79-90, 2024.
Article in Chinese | WPRIM | ID: wpr-1003411

ABSTRACT

ObjectiveTo explore the mechanism of Bushen Huoxue enema in treating the rat model of kidney deficiency and blood stasis-thin endometrium (KDBS-TE) by transcriptome sequencing. MethodThe rat model of KDBS-TE was established by administration of tripterygium polyglycosides tablets combined with subcutaneous injection of adrenaline. The pathological changes of rat endometrium in each group were then observed. Three uterine tissue specimens from each of the blank group, model group, and Bushen Huoxue enema group were randomly selected for transcriptome sequencing. The differentially expressed circRNAs, lncRNAs, and miRNAs were screened, and the disease-related specific competitive endogenous RNA (ceRNA) regulatory network was constructed. Furthermore, the gene ontology (GO) functional annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed for the mRNAs in the network. ResultCompared with the blank group, the model group showed endometrial dysplasia, decreased endometrial thickness and endometrial/total uterine wall thickness ratio (P<0.01), and differential expression of 18 circRNAs, 410 lncRNAs, and 7 miRNAs. Compared with the model group, the enema and estradiol valerate groups showed improved endometrial morphology and increased endometrial thickness and ratio of endometrial to total uterine wall thickness (P<0.05). In addition, 21 circRNAs, 518 lncRNAs, and 17 miRNAs were differentially expressed in the enema group. The disease-related specific circRNA-miRNA-mRNA regulatory network composed of 629 nodes and 664 edges contained 2 circRNAs, 34 miRNAs, and 593 mRNAs. The lncRNA-miRNA-mRNA regulatory network composed of 180 nodes and 212 edges contained 5 lncRNAs, 10 miRNAs, and 164 mRNAs. The mNRAs were mainly enriched in Hippo signaling pathway, autophagy-animal, axon guidance, etc. ConclusionBushen Huoxue enema can treat KDBS-TE in rats by regulating specific circRNAs, lncRNAs, and miRNAs in the uterus and the ceRNA network.

3.
International Eye Science ; (12): 677-685, 2024.
Article in Chinese | WPRIM | ID: wpr-1016577

ABSTRACT

AIM:To identify transcriptional differences between the ocular surface ectoderm(OSE)and surface ectoderm(SE)using RNA-seq, and elucidate the OSE transcriptome landscape and the regulatory networks involved in its development.METHODS:OSE and SE cells were differentiated from human embryonic stem(hES)cells. Differentially expressed genes(DEGs)between OSE and SE were analyzed using RNA-seq. Based on the DEGs, we performed gene ontology(GO)analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis, and protein-protein interaction(PPI)network analysis. Transcription factors(TFs)and hub genes were screened. Subsequently, TF-gene and TF-miRNA regulatory networks were constructed using the NetworkAnalyst platform.RESULTS:A total of 4 182 DEGs were detected between OSE and SE cells, with 2 771 up-regulated and 1 411 down-regulated genes in OSE cells. GO-BP analysis revealed that up-regulated genes in OSE were enriched in the regulation of ion transmembrane transport, axon development, and modulation of chemical synaptic transmission. Down-regulated genes were primarily involved in nuclear division, chromosome segregation, and regulation of cell cycle phase transition. KEGG analysis indicated that up-regulated genes in OSE cells were enriched in signaling pathways such as cocaine addiction, axon guidance, and amphetamine addiction, while down-regulated genes were enriched in proteoglycans in cancer, ECM-receptor interaction, protein digestion and absorption, and cytokine-cytokine receptor interaction. Additionally, compared with SE, 204 TFs(including FOS, EGR1, POU5F1, SOX2, and PAX6)were up-regulated, and 80 TFs(including HAND2, HOXB6, HOXB5, HOXA5, and HOXB8)were down-regulated in OSE cells. Furthermore, we identified 6 up-regulated and 9 down-regulated hub genes in OSE cells, and constructed TF-gene and TF-miRNA regulatory networks based on these hub genes.CONCLUSIONS:The transcriptome characteristics of OSE and SE cells were elucidated through RNA-seq analysis. These findings may provide a novel insight for studies on the development and in vitro directed induction of OSE and corneal epithelial cells.

4.
Chinese Journal of Clinical Pharmacology and Therapeutics ; (12): 252-259, 2024.
Article in Chinese | WPRIM | ID: wpr-1014535

ABSTRACT

AIM:To explore the molecular mechanism of Tiaopi Chengqi decoction (TpCqD) improving hyperthermia and high-protein food-induced hyperphagia mice based on transcriptomics. METHODS:C57 mice were randomly divided into a control group, model group, low-dose TpCqD group, high-dose TpCqD group, and domperidone group. The general condition of the experimental mice was observed and the average food intake was counted, and the rate of gastric emptying and intestinal propulsion was determined for each group of mice. H&E staining was used to observe pathological changes in gastric tissue. PAS staining was used to observe glycogen changes in gastric tissue. Pepsin activity was determined by colorimetry. pH value of gastric contents was measured by acid-base titration. Transcriptome sequencing was used to analyze the differential genes in gastric tissue, a volcano map and a cluster heat map were made for the differential genes, and KEGG was used to analyze the signal pathway enrichment of the differential genes. RT-qPCR verified the differential genes obtained by screening. RESULTS:After treatment with TpCqD, the body weight and average food intake of mice with food accumulation increased (P<0.05), and the intestinal propulsion rate and gastric emptying speed of mice with food accumulation accelerated (P<0.05). TpCqD could protect gastric tissue structure and glycogen degradation, increase pepsin activity (P<0.05), and reduce gastric content pH (P<0.05). Transcriptome results showed that TpCqD could regulate the expression of Acox2 and cilp2, regulate fat digestion and absorption, protein digestion and absorption, and pancreatic secretion signals. RT-qPCR showed that compare with model group, TpCqD up-regulated Acox2 (P<0.05) and down-regulated the mRNA level of cilp2 (P<0.05). CONCLUSION:TpCqD ameliorated digestive dysfunction in mice with high-calorie and high-protein diets leading to food accumulation involving the regulation of the fat and sugar metabolism genes Acox2 and cilp2, and pancreatic secretory signaling.

5.
Chinese Pharmacological Bulletin ; (12): 114-158, 2024.
Article in Chinese | WPRIM | ID: wpr-1013615

ABSTRACT

Aim To investigate the effect of benzyl iso-thiocyanate (BITC) on the proliferation of mouse U14 cervical cancer cells and to explore the mechanism of cytotoxicity based on transcriptomic data analysis. Methods The effect of BITC on U14 cell activity was detected by MTT, nuclear morphological changes were observed by Hochest 33258 and fluorescent inverted microscope, cell cycle and apoptosis were determined by flow cytometry, and the transcriptome database of U14 cells before and after BITC (20 μmol · L

6.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 47-61, 2024.
Article in English | WPRIM | ID: wpr-1011011

ABSTRACT

Artemisia argyi (A. argyi), a plant with a longstanding history as a raw material for traditional medicine and functional diets in Asia, has been used traditionally to bathe and soak feet for its disinfectant and itch-relieving properties. Despite its widespread use, scientific evidence validating the antifungal efficacy of A. argyi water extract (AAWE) against dermatophytes, particularly Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum, remains limited. This study aimed to substantiate the scientific basis of the folkloric use of A. argyi by evaluating the antifungal effects and the underlying molecular mechanisms of its active subfraction against dermatophytes. The results indicated that AAWE exhibited excellent antifungal effects against the three aforementioned dermatophyte species. The subfraction AAWE6, isolated using D101 macroporous resin, emerged as the most potent subfraction. The minimum inhibitory concentrations (MICs) of AAWE6 against T. rubrum, M. gypseum, and T. mentagrophytes were 312.5, 312.5, and 625 μg·mL-1, respectively. Transmission electron microscopy (TEM) results and assays of enzymes linked to cell wall integrity and cell membrane function indicated that AAWE6 could penetrate the external protective barrier of T. rubrum, creating breaches ("small holes"), and disrupt the internal mitochondrial structure ("granary"). Furthermore, transcriptome data, quantitative real-time PCR (RT-qPCR), and biochemical assays corroborated the severe disruption of mitochondrial function, evidenced by inhibited tricarboxylic acid (TCA) cycle and energy metabolism. Additionally, chemical characterization and molecular docking analyses identified flavonoids, primarily eupatilin (131.16 ± 4.52 mg·g-1) and jaceosidin (4.17 ± 0.18 mg·g-1), as the active components of AAWE6. In conclusion, the subfraction AAWE6 from A. argyi exerts antifungal effects against dermatophytes by disrupting mitochondrial morphology and function. This research validates the traditional use of A. argyi and provides scientific support for its anti-dermatophytic applications, as recognized in the Chinese patent (No. ZL202111161301.9).


Subject(s)
Antifungal Agents/chemistry , Arthrodermataceae , Artemisia/chemistry , Molecular Docking Simulation , Mitochondria , Microbial Sensitivity Tests
7.
Article | IMSEAR | ID: sea-223533

ABSTRACT

Background & objectives: During the COVID-19 pandemic, the death rate was reportedly 5-8 fold lower in India which is densely populated as compared to less populated western countries. The aim of this study was to investigate whether dietary habits were associated with the variations in COVID-19 severity and deaths between western and Indian population at the nutrigenomics level. Methods: In this study nutrigenomics approach was applied. Blood transcriptome of severe COVID-19 patients from three western countries (showing high fatality) and two datasets from Indian patients were used. Gene set enrichment analyses were performed for pathways, metabolites, nutrients, etc., and compared for western and Indian samples to identify the food- and nutrient-related factors, which may be associated with COVID-19 severity. Data on the daily consumption of twelve key food componentsacross four countries were collected and a correlation between nutrigenomics analyses and per capita daily dietary intake was investigated. Results: Distinct dietary habits of Indians were observed, which may be associated with low death rate from COVID-19. Increased consumption of red meat, dairy products and processed foods by western populations may increase the severity and death rate by activating cytokine storm-related pathways, intussusceptive angiogenesis, hypercapnia and enhancing blood glucose levels due to high contents of sphingolipids, palmitic acid and byproducts such as CO2 and lipopolysaccharide (LPS). Palmitic acid also induces ACE2 expression and increases the infection rate. Coffee and alcohol that are highly consumed in western countries may increase the severity and death rates from COVID-19 by deregulating blood iron, zinc and triglyceride levels. The components of Indian diets maintain high iron and zinc concentrations in blood and rich fibre in their foods may prevent CO2 and LPS-mediated COVID-19 severity. Regular consumption of tea by Indians maintains high high-density lipoprotein (HDL) and low triglyceride in blood as catechins in tea act as natural atorvastatin. Importantly, regular consumption of turmeric in daily food by Indians maintains strong immunity and curcumin in turmeric may prevent pathways and mechanisms associated with SARS-CoV-2 infection and COVID-19 severity and lowered the death rate. Interpretation & conclusions: Our results suggest that Indian food components suppress cytokine storm and various other severity related pathways of COVID-19 and may have a role in lowering severity and death rates from COVID-19 in India as compared to western populations. However, large multi-centered case?control studies are required to support our current findings.

8.
Indian J Exp Biol ; 2023 Mar; 61(3): 214-223
Article | IMSEAR | ID: sea-222589

ABSTRACT

Karnal bunt of wheat is an important quarantine disease that interrupts India’s wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809,and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores.Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease.

9.
J. appl. oral sci ; 31: e20230214, 2023. tab, graf
Article in English | LILACS-Express | LILACS | ID: biblio-1521077

ABSTRACT

Abstract Objective This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. Methodology A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). Results Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. Conclusion Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

10.
Acta cir. bras ; 38: e380923, 2023. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1429538

ABSTRACT

Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.


Subject(s)
Oncogenes , N-Acetylglucosaminyltransferases/analysis , Cell Migration Assays , Transcriptome , Esophageal Squamous Cell Carcinoma , Esophageal Neoplasms/physiopathology
11.
Acta Pharmaceutica Sinica ; (12): 3130-3139, 2023.
Article in Chinese | WPRIM | ID: wpr-999062

ABSTRACT

Analyze the changes in gene expression profiles during the process of Panax ginseng seed dormancy release, and screen for differential genes, providing a basis for analyzing the mechanism of P. ginseng seed dormancy release. Comparative transcriptome analysis was conducted by using RNA-Seq sequencing technology in P. ginseng seeds stored at different low temperature. A total of 80.97 Gb of Raw reads and 80.19 Gb of Clean reads were obtained from the transcriptome. Principal component analysis and correlation analysis showed that there were significant differences in gene expression patterns at different developmental stages. Upset results showed that 46 248 unigenes were co-expressed in four stages, and 414, 445, 400 and 389 unigenes were specifically expressed in 0, 8,14 and 28 days, respectively. Gene Ontology functional annotation showed that the differentially expressed genes were mainly involved in nsaturated fatty acid biosynthetic process, nuclear body and oxidoreductase activity. Encyclopedia of Genes and Genomes metabolic pathway showed that differentially expressed genes were mainly involved in peroxisome, mitogen-activated protein kinase signaling pathway-plant, plant hormone signal transduction, ribosome, biosynthesis of unsaturated fatty acid, circadian rhythm-plant and other metabolic pathways. In the process of P. ginseng seed dormancy release, multiple biological processes, such as unsaturated fatty acid biosynthesis and plant hormone signal transduction, are required to coordinate regulation, which constitutes a complex dormancy release regulation network. Transcriptome analysis and differential gene screening of P. ginseng seeds at different sand storage time laid a foundation for the analysis of P. ginseng seed dormancy release mechanism and molecular breeding.

12.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 21-28, 2023.
Article in Chinese | WPRIM | ID: wpr-996806

ABSTRACT

ObjectiveTo investigate the effects of using the polysaccharides from two Chinese medicine compound prescriptions as the carbon source on the growth of Bacteroides fragilis and to decipher the mechanism from the perspective of differential expression of polysaccharide utilization loci (PULs) based on transcriptomics. MethodThe media with different carbon sources [20% polysaccharides of Lizhongtang, polysaccharides of Shenling Baizhusan, glucose, and brain heart infusion (BHI) Broth] were used for the anaerobic culture of B. fragile ATCC25285. The effects of different carbon sources on the growth of B. fragilis ATCC25285 were determined by continuous sampling and spectrophotometry. Then, transcriptome sequencing was performed for the cultures obtained with different carbon sources to study the mechanism of different carbon sources in regulating bacterial growth. ResultThe concentration of bacteria with the polysaccharide of Lizhongtang, polysaccharide of Shenling Baizhusan, BHI Broth, and glucose as the carbon sources peaked at 26, 32, 26, 38 h, respectively, and the bacteria in all the four groups achieved robust growth. Gene ontology (GO) enrichment indicated that the differentially expressed genes in the Lizhongtang polysaccharide group and Shenling Baizhusan polysaccharide group were concentrated in the transport and transmembrane transport of dicarboxylic acid. The Shenling Baizhusan polysaccharide and BHI Broth groups showed high expression of PUL 4 and 27, glycoside hydrolase 13 (GH13), and glycosyl transferases 5 (GT5). PUL9 was highly expressed in Shenling Baizhusan polysaccharide group, and PUL 17, 19, and 20, GH3, and GH144 in the BHI Broth group. PUL27 and GT5 were highly expressed in Shenling Baizhusan polysaccharide and glucose groups. PUL 4 and 9 and GH13 were only highly expressed in Shenling Baizhusan polysaccharide group, and PUL 2, 17, and 19 and GH2 in the glucose group. Both Lizhongtang polysaccharide group and BHI group highly expressed PUL 4, 17, 19, 20, and 27, GH3, and GH144. PUL 2, 8, 23, and 27, GH2, and GH57 were highly expressed in Lizhongtang polysaccharide group, while GH13 showed high expression in the BHI group. Both the glucose and Lizhongtang polysacharride groups showed high expression of PUL 4 and 27 and GH2. PUL 4, 8, 20, and 23, GH3, and GH144 were highly expressed in Lizhongtang polysaccharide group, while PUL30 was highly expressed in the glucose group. ConclusionThe in vitro experiments and transcriptome sequencing results confirmed that the expression of PULs and GH may provide benefits or costs to the adaptive growth of Bacteroides fragilis ATCC25285 cultured with different carbon sources, which may be one of the mechanisms by which polysaccharides from Chinese medicine compound prescriptions regulate the growth of B. fragilis ATCC25285. The findings can provide a reference for further research on the relationship between B. fragilis metabolic pathway and polysaccharides of Chinese medicine compound prescriptions.

13.
Chinese Journal of Biologicals ; (12): 1039-1046+1053, 2023.
Article in Chinese | WPRIM | ID: wpr-996592

ABSTRACT

@#ObjectiveTo establish models of Dengue virus type Ⅲ(DENV-3,DV-3)infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)]. After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1. 0 × 104. 64PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated. All of these differential LncRNAs had open reading frames(ORFs). Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IRES).ConclusionIn DENV-3 infection of THP-1 cells and ADE infection mediated by DENV-3,the expression of LncRNAs has changed significantly,and may regulate the process of infection through a variety of biological functions,which is helpful for a deeper understanding of the mechanism of ADE infection.

14.
Chinese Journal of Ocular Fundus Diseases ; (6): 153-162, 2023.
Article in Chinese | WPRIM | ID: wpr-995605

ABSTRACT

Objective:To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process.Methods:An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log 2 (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results:When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 ( t=13.530), Icos ( t=3.482), Chst3 ( t=6.949), Kynu ( t=5.399), interleukin (IL)-1β ( t=2.960), TNF-α ( t=5.800), IL-6 ( t=3.130), interferon-γ ( t=7.690) and IL-17 ( t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion:TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.

15.
Chinese Journal of Microbiology and Immunology ; (12): 93-101, 2023.
Article in Chinese | WPRIM | ID: wpr-995261

ABSTRACT

Objective:To compare gene expression profiles in normal human cervical epithelial cells (HcerEpic) before and after Chlamydia trachomatis ( Ct) infection. Methods:HcerEpic cells that were pretreated with DEAE-D were infected with Ct serotype E standard strain and then cultured for 44 h. Uninfected HcerEpic cells were used as the control group. Total RNA was extracted from the cells in each group and reverse transcribed to construct a cDNA library. Differences in gene expression profiles between the two groups were analyzed by high-throughput sequencing and the representative genes were selected for verification by qPCR. Results:A total of 23 997 genes were detected, including 125 differentially expressed genes. Among the 125 genes, 119 were up-regulated and six were down-regulated. GO analysis showed that the differentially expressed genes were enriched in several biological processes including defense response to virus, typeⅠinterferon signaling pathway and cellular responses to typeⅠinterferons. KEGG enrichment analysis showed the differentially expressed genes were mainly enriched in the pathways related to virus infections, such as influenza A virus, herpes simplex virus, EB virus and HPV, and NOD-like receptor pathway.Conclusions:There were significant differences in transcriptome profiles of HcerEpic cells before and after Ct infection. The differentially expressed genes were mainly enriched in the interferon pathway, which was closely related to the antiviral processes in cells. qPCR verified the differentially expressed genes and the genes closely related to the interferon pathway, such as ISG15, IFIT2, OASL and UBE2L6.

16.
International Journal of Biomedical Engineering ; (6): 212-220, 2023.
Article in Chinese | WPRIM | ID: wpr-989341

ABSTRACT

Objective:To analyze the patterns of intercellular communication in facioscapulohumeral muscular dystrophy (FSHD) by single-cell nuclear transcriptome sequencing.Methods:Bilateral asymmetrical lesions mouth orbicular muscle of two patients with FSHD and mouth orbicular muscle of two healthy patients were selected. Six samples were obtained, and were divided into control group, mild group and severe group. The normal orbicularis muscle sample was collected from 2 healthy individuals (the control group). The muscle samples in the mild group were from two patients with relatively normal muscle sides, and the samples in the severe group were from two patients with more severe muscle damage sides. Single-cell nuclear transcriptome sequencing was performed on all cells of the three groups. Reduced dimension clustering and cell definition were performed to identify differentially expressed genes and enrichment pathways. Intercellular communication patterns among major cell types and key signaling pathways were explored by cellular communication analysis.Results:Differential gene expression analysis of FSHD bilateral muscle samples identified 46 functionally differentially expressed genes associated with the disease in different cell types, related to apoptosis, oxidative stress, immune inflammation, and muscle function. Intercellular communication was generally increased in the severe group. Fibro-adipogenic progenitors (FAPs) and macrophages are important signaling sources in the abnormal muscle microenvironment of FSHD and are closely associated with disease progression. There are six unique signaling pathways in the mild group, including bone morphogenetic proteins (BMP), transforming growth factor-β (TGF-β), CXC motif chemokine ligand (CXCL), adhesion G protein-coupled receptor E5 (ADGRE5), interleukin-16 (IL-16), and wingless-type MMTV integration site family (WNT) signaling pathways. These signaling pathways are mainly involved in the interaction between macrophages, FAPs, and adipocytes and may be involved in the regulation of fat deposition and fibrosis changes in the diseased muscle.Conclusions:Single-cell nuclear transcriptome sequencing provides a relatively comprehensive pattern of intercellular communication between key cell types in FSHD, providing an appropriate reference for understanding the intercellular regulatory mechanisms of the FSHD muscle microenvironment.

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Journal of Leukemia & Lymphoma ; (12): 147-152, 2023.
Article in Chinese | WPRIM | ID: wpr-988965

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Objective:To investigate the differences among targeted capture high depth sequencing (Panel-seq), transcriptome sequencing (RNA-seq) and traditional detection methods in cytogenetic and molecular genetic typing of childhood B-cell acute lymphoblastic leukemia (B-ALL) and their significances.Methods:The clinical data of 152 newly diagnosed childhood B-ALL cases in Guangzhou Women and Children's Medical Center from September 2020 to December 2021 were retrospectively analyzed. Along with traditional cytogenetic and molecular detection methods including karyotyping, fluorescence in situ hybridization (FISH) and 43 kinds of fusion gene quantitative screening for traditional cells and molecular genetic detection, both Panel-seq and RNA-seq were also performed. Panel-seq covered more than 600 genes with common mutations in hematological tumors, from which fusion genes and gene mutations were both analyzed. RNA-seq was used to analyze fusion genes, gene mutations, gene expression, and copy number variation at the chromosome level. High hyperdiploid karyotype was estimated by using gene expression profile clustering and copy number variations. The cytogenetic typing results of all detection methods were also analyzed.Results:Among 152 patients, 93 cases were males and 59 cases were females, with the median age of 4.0 years (0.8-13.0 years). The median blast cell ratio was 0.855 (0.215-0.965). The traditional detection methods could identify 4 cases (2.6%) with BCR-ABL1, 2 cases (1.3%) with CRLF2 gene-related fusion, 27 cases (17.8%) with ETV6-RUNX1, 1 case (0.7%) with iAMP21, 5 cases (3.3%) with MLL rearrangement, 8 cases (5.3%) with TCF3-PBX1 and 22 cases (14.5%) with high hyperdiploid karyotype. Panel-seq could identify 4 cases (2.6%) with BCR-ABL1, 2 cases (1.3%) with CRLF2 gene-related fusions, 27 cases (17.8%) with ETV6-RUNX1, 3 cases (2.0%) with MEF2D gene-related fusions, 1 case (0.7%) with MEIS1-FOXO1, 5 cases (3.3%) with MLL rearrangement, 5 cases (3.3%) with PAX5 gene-related fusions, 8 cases (5.3%) with TCF3-PBX1 fusions, 4 cases (2.6%) with ZNF384 gene-related fusions, and 2 cases (1.3%) with IKZF1 N159Y mutations. Among 152 patients, 1 case with MLL rearrangement didn't receive RNA-seq detection because of sample quality; in other 151 B-ALL cases, 1 case (0.7%) with ACIN1-NUTM1, 4 cases (2.6%) with BCR-ABL1, 3 cases (2.0%) with CRLF2 gene-related fusions, 8 cases (5.3%) with DUX4 gene-related fusions, 27 cases (17.9%) with ETV6-RUNX1, 3 cases (2.0%) with MEF2D gene-related fusions, 1 case (0.7%) with MEIS1-FOXO1, 4 cases (2.6%) with MLL rearrangement, 5 cases (3.3%) with PAX5 gene-related fusions, 1 case (0.7%) with ZMIZ1-ABL1, 8 cases (5.3%) with TCF3-PBX1,4 cases (2.6%) with ZNF384 gene-related fusions, 61 cases (40.4%) with hyperdiploid karyotypes, and 2 cases (1.3%) with IKZF1 N159Y mutations were detected; RNA-seq had obvious advantage in detecting fusion gene and hyperdiploid karyotype. The cytogenetic and molecular genetic typing rates of traditional method, Panel-seq and RNA-seq were 45.4% (69/152), 40.1% (61/152) and 87.4% (132/151), respectively. The combination of the three could identify 89.5% (136/152) of childhood B-ALL patients.Conclusions:The combination of Panel-seq and RNA-seq can increase the detection rate of genetic abnormality in childhood B-ALL, which provides a more accurate molecular genetic classification for B-ALL and the basis for treatment guideline and prognosis judgement.

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Journal of Environmental and Occupational Medicine ; (12): 1005-1013, 2023.
Article in Chinese | WPRIM | ID: wpr-988742

ABSTRACT

Background Paraquat (PQ) is one of the most widely used herbicides in the world and a risk factor for Parkinson's disease (PD), but the mechanisms underlying PD are poorly understood. Single-cell RNA sequencing (scRNA-seq) technology can study cellular heterogeneity at genetic level, providing insights into the pathogenesis of PQ-induced PD. Objective To analyze the brain cell grouping of PQ-infected mice and the biological processes involved in the subpopulation of PD-like changes cells by scRNA-seq, and to provide clues for revealing potential mechanisms of PQ-induced PD-like changes in mouse brains. Methods Six male 6-week-old C57BL/6 mice were randomly divided into a control group and an experimental group, three mice in each group, and were intraperitoneally injected with 0 (saline) and 10.0 mg·kg−1 PD respectively, once every two days, for 10 consecutive injections for modeling. After infection, mouse brains were taken and scRNA-seq was performed. Cell segmentation was performed according to gene expression characteristics of different cell types, PD-related cell subsets were screened by bioinformatics tools, and gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), protein interaction network analysis, and transcription factor prediction were performed on their characteristic genes. Finally, GO and KEGG analyses were performed on the differential genes of PD-associated cell subsets between the PQ-treated group and the control group, and the biological processes in which these genes may participate were analyzed. Results The sequencing data met quality control standards, a total of 55779 cells were obtained, and all cell dimensionality reduction analysis results showed that they could be further divided into 37 clusters, including 5 major cell types. Based on the KEGG analysis of the top 20 characteristic genes of each subpopulation, the specifically expressed Cluster 33 subpopulation (dopaminergic neurons) was screened and found to be significantly associated with PD. The results of GO analysis showed that the biological function of this subpopulation mainly enriched neurotransmitter transport and regulation. The results of GSEA analysis showed that the tyrosine metabolic pathway and the ligand-receptor interaction pathway of neural activity in brain tissues were significantly enriched. The analysis of transcriptional regulatory networks showed that 39 transcription factors were expressed differently. The metabolic pathway of the dopamine neuronal subset, endocytosis, Ras-associated protein 1 (Rap1) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway were all affected by PQ exposure, according to further analysis of its effects on this subpopulation. The GO analysis showed that differential genes were involved in biological processes such as ion transport and synaptic assembly regulation, and were involved in the cellular component formation of cytoplasm and synapses. Conclusion This study has initially mapped the transcriptome of single cells in the mouse brain after PQ exposure, and screened out the specific expression of Cluster 33 subgroup (dopaminergic neurons), which is significantly correlated with PD, and its biological function changes may be one of the mechanisms of PD-like changes in the mouse brain induced by PQ.

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Journal of Sun Yat-sen University(Medical Sciences) ; (6): 758-767, 2023.
Article in Chinese | WPRIM | ID: wpr-988721

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objectiveTo explore the specific molecular mechanism of neuronal apoptosis induced by ATM inactivation. MethodsCGNs matured 7 days in vitro were cultured 8 h with 25 K, 5 K or 25 K medium containing ATM-specific inhibitors (Ku55933, 10 µmol/L; Ku60019, 15 µmol/L) for Hoechst stain and apoptosis analysis, or cultured for different lengths of time (2, 4, 8 h) to detect the protein expression levels of ATM, caspase-3 and cleaved caspase-3 by Western blotting. ATM and GADD45α specific siRNA was transfected into C6 cells and CGNs, and its interference efficiency was verified by q-PCR and Western blotting. CGNs matured for 5 days in vitro were transfected with ATM specific siRNA and pCMV-EGFP by calcium phosphate for 48 h, Hoechst staining and apoptosis analysis were performed. CGNs matured for 7 days in vitro were treated with 25 K medium containing ATM specific inhibitors for 8 h, transcriptome sequencing, differential expression gene identification and pathway enrichment analysis were performed. CGNs matured for 5 days in vitro were co-transfected with GADD45α specific siRNA and pCMV-EGFP by calcium phosphate for 48 h, then treated with 5 K or 25 K medium containing 15 µmol/L Ku6 for 8 h. Hoechst staining and apoptosis analysis were performed. ResultsCompared with the 25 K, CGNs nuclear pyknosis rate, cleaved Caspase-3 and ATM protein expression level were increased in the 5 K and ATM-specific inhibitor groups. The mRNA and protein expression levels of ATM and GADD45α were effectively reduced after transfection of ATM and GADD45α specific siRNA in C6 cells and CGNs. Compared with control, CGNs transfected with ATM specific siRNA showed a higher nuclear pyknosis rate. Totally 835 genes were identified to be up-regulated and 848 genes to be down-regulated in the Ku55933 treatment group; 454 genes were identified to be up-regulated and 314 genes to be down-regulated in the Ku6 treatment group; 274 genes were co-up regulated in the Ku5 and Ku60019 treatment groups, while 179 genes were co-down-regulated in the Ku5 and Ku6 treatment groups and the expression of ATM downstream target GADD45α was upregulated. The enrichment results showed that TNF signaling pathway, NF-κB signaling pathway and Apoptosis signaling pathway were significantly enriched. Compared with control, mRNA and protein expression levels of GADD45α were increased in inhibitor treatment and 5 K, while knocking down GADD45α resulted in a decrease in nuclear pyknosis rate in the Ku60019 and 5 K treatment group. ConclusionLoss of ATM activity induces GADD45α-dependent cerebellar granular neuronal apoptosis.

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Chinese Journal of Experimental Traditional Medical Formulae ; (24): 142-151, 2023.
Article in Chinese | WPRIM | ID: wpr-969609

ABSTRACT

ObjectiveTo investigate the effects of flavanomarein on the transcriptome of small intestinal organoids in insulin-resistant mice. MethodFirstly, small intestinal organoids of C57BL/6J and db/db mice were established. Ki-67 and E-cadherin expression was determined by immunofluorescence. Small intestinal organoids were divided into the following three groups: C57BL/6J mouse small intestinal organoids as the normal control group, db/db mouse small intestinal organoids as the model group (IR group), and db/db mouse small intestinal organoids treated with flavanomarein as the administration group (FM group). Western blot was used to detect the expression of glucagon-like peptide-1(GLP-1) protein on the small intestinal organoids of the three groups. Finally, transcriptome sequencing was performed on samples from the three groups. ResultOn the 6th day of small intestine organoids culture, a cyclic structure was formed around the lumen, and a small intestine organoids culture model was preliminarily established. Immunofluorescence detection showed that ki-67 and E-cadherin were expressed in small intestinal organoids. Western blot results showed that the expression of GLP-1 protein was increased by flavanomarein. In the results of differential expressed gene (DEG) screening, there were 1 862 DEGs in the IR group as compared with the normal control group, and 2 282 DEGs in the FM group as compared with the IR group. Through protein-protein interaction(PPI) network analysis of the DEGs of the two groups, 10 Hub genes, including Nr1i3, Cyp2c44, Ugt2b1, Gsta1, Gstm2, Ptgs1, Gstm4, Cyp2c38, Cyp4a32, and Gpx3, were obtained. These genes were highly expressed in the normal control group, and their expression was reduced in the IR group. After the intervention of flavanomarein, the expression of the above genes was reversed. ConclusionFlavanomarein may play its role in improving insulin resistance by reversing the expression levels of 10 Hub genes, including Nr1i3, Cyp2c44, Ugt2b1, Gsta1, Gstm2, Ptgs1, Gstm4, Cyp2c38, Cyp4a32, and Gpx3.

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