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To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.
Subject(s)
Flavonoids/metabolism , Fruit/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Lycium/metabolism , Metabolic Networks and Pathways , TranscriptomeABSTRACT
Objective:To investigate the effect of salvianolic acid C (SalC) on fibroblast-like synoviocytes and through the role of nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway.Methods:Rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLSs) were exposed to different concentrations of SalC (0.1 μmol/L, 1 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L) for 24-72 h and measured for viability, proliferation, migration and invasion by Cell counting kit 8 (CCK-8) assay, wound-healing and transwell assay. The levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-1 beta (IL-1β) and IL-6 were measured by enzyme linked immunosorbent assay (ELISA). Western blot was used to detect the expression of matrix metallopeptidase (MMP)-9, MMP-13, apoptosis-related proteins and Nrf2 mediated gene. Then we used ML385 to inhibit Nrf2 signaling pathway. RA-FLSs were measured for migration and invasion, and the expression of proteins related to apoptosis, inflammation and Nrf2 pathway.Results:Compared with the control group, SalC inhibited the cell migration significantly (0.1 μmol/L, 0.75±0.05, t=7.65, P<0.001; 5 μmol/L, 0.50±0.05, t=14.25, P<0.001; 10 μmol/L, 0.26±0.05, t=20.67, P<0.001) and invasion (0.1 μmol/L, 0.75±0.11, t=4.93, P<0.001; 5 μmol/L, 0.49±0.06, t=10.32, P<0.001; 10 μmol/L, 0.26±0.07 , t=14.96, P<0.001) of RA-FLs, reduced the levels of MMP-9 (0.1 μmol/L, 0.72±0.10, t=5.60, P<0.001; 5 μmol/L, 0.48±0.08, t=11.03, P<0.001; 10 μmol/L, 0.27±0.06, t=15.94, P<0.001) and MMP-13 (0.1 μmol/L, 0.77±0.06, t=8.66, P<0.001; 5 μmol/L, 0.58±0.06, t=11.03, P<0.001; 10 μmol/L, 0.32±0.13, t=14.22, P<0.001), and promoted apoptosis. SalC reduced the level of pro-inflammatory cytokines significantly ( P<0.001) and activated the expression of Nrf2 signaling pathway proteins Nrf2, CAT, NQO1, SOD1 and GSS ( P<0.001). After ML385 was used to interfere Nrf2, the levels of SalC on Nrf2 pathway proteins, such as Nrf2 (0.68±0.06, t=5.08, P<0.001), CAT (1.44±0.12, t=4.77, P<0.001), NQO1 (0.65±0.12, t=5.04, P<0.001), SOD1 (1.43±0.10, t=6.36, P<0.001) and GSS (1.42±0.10, t=7.60, P<0.001), as well as the levels of TNF-α [(260±22) pg/ml, t=13.75, P<0.001], IL-1β [(701±30) pg/ml, t=12.98, P<0.001], IL-6 [(180±10) pg/ml, t=16.38, P<0.001) were significantly reduced. In addition, ML385 inhibited the inhibition of SalC on cell migration and invasion (0.70±0.09, t=11.24, P<0.001; 0.64±0.04, t=8.03, P<0.001) and induction of apoptosis (24.4±1.8, t=23.02, P<0.001). Conclusion:SalC may inhibit cell activity and inflammatory response, promote apoptosis via the upregulation of Nrf2 signaling pathway. SalC may have therapeutic potential in RA. However, further investigation are needed in animal models and human.
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Objective To investigate the induction and regulatory mechanism of placental trophoblast cell autophagy in women with preeclampsia (PE).Methods Twenty gravidas with severe PE who underwent cesarean section in the Department of Obstetrics and Gynecology of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University from August 2016 to November 2016 were enrolled in PE group.An equal number of normotensive gravidas without proteinuria who also underwent cesarean section during the same period were randomly selected as control group.Placental tissue samples were collected from all gravidas.Ultrastructure of placental trophoblast cells and changes in autophagosome formation were observed by transmission electron microscope.Expressions ofmicrotubule associated protein 1 light chain 3B (MAP1LC3B,or LC3B) and Beclin 1 in placental tissue samples were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot.Activities of protein kinase B (PKB,also known as Akt)/mammalian target of rapamycin (mTOR) pathway in placental tissue samples were detected by Western blot.Two independent samples t-test or Mann-Whitney U test was used for statistical analysis.Results Sparse and disordered villi and many typical autophagosomes were observed in placental trophoblast cells from patients with severe PE.Significantly enhanced expression of LC3B at mRNA and protein levels and increased ratio of LC3-Ⅱ/LC3-Ⅰ were observed in the PE group as compared with the control group [3.37 (2.37-6.11) vs 0.62 (0.25-4.15),1.40±0.17 vs 1.00±0.13,1.57±0.25 vs 1.00±0.31,Zor t=--4.440,3.274 and 3.113,all P＜0.05].No significant difference in the expression ofBeclin 1 at mRNA or protein level in placental tissues was found between the two groups (both P＞0.05).Furthermore,Akt and mTOR phosphorylation in the PE group was significantly suppressed as compared with that in the control group (1.00±0.29 vs 0.64±0.21,1.00±0.32 vs 0.60±0.22,t=--3.672 and-2.895,both P＜0.05).However,the two groups showed no significant difference in the expression of Akt or mTOR protein (both P＞0.05).Conclusions Suppressed activity of Akt/mTOR pathway and enhanced induction of trophoblast cell autophagy are detected in placental tissues of patients with severe PE,indicating that excessive trophoblast cell autophagy,induced by decreased activity of Akt/mTOR pathway,may be the pathogenesis for PE.
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Objective To evaluate the value of 2012 classification criteria for early rheumatoid arthritis (ERA),2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria,and 1987 ACR classification criteria in the diagnosis of early rheumatoid arthritis (RA).Methods Patients who had at least one swollen and tender joint with disease duration no more than 2 years,and age more than 16 years were enrolled.The patients were diagnosed as RA or other non-RA by 2 experienced rheumatologists.The clinical and laboratory parameters were recorded.The sensitivity and specificity of three RA classification criteria were compared by McNemar test,The areas under the receiver operating characteristic curve (ROC) curve (AUC) of each RA classification criteria were analyzed using MedCalc software.Results Atotal of 310 patients were enrolled in this study,including 182ERA and 128 non-RA.The sensitivity(88.5％) of ERA criteria was much higher than that of the 1987 ACR criteria (45.6％,x2=75.013,P＜0.05),and not significantly different with the 2010 ACR/EULAR criteria (91.8％,X2=1.042,P＞0.05).The specificity of ERA criteria (91.4％) of 2010 ACR/EULAR criteria (87.5％,x2=1.8,P＞0.05) was similar to that of the 1987 ACR criteria (96.1％,x2=3.1,P＞0.05).The AUC of ERA criteria was 0.962 [95％CI(0.934,0.980)],which was slightly better than that of the 2010 ACR/EULAR criteria 0.959 [95％CI(0.931,0.978)],Z=0.380,P=0.7038,and much higher than that of the 1987 ACR criteria 0.885 [95％CI (0.845,0.919)],Z=4.517,P＜0.01.Conclusion Overall evaluation,the diagnostic value of ERA criteria is better than 1987 ACR and 2010 ACR/EULAR criteria in early rheumatoid arthritis.Compared to 2010 ACR/EULAR classification criteria,ERA criteria is more simple and practical.
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Objective To investigate the clinical significance of D-dimer in systemic lupus erythematosus (SLE). Methods The study group comprised 261 SLE patients who were admitted in ward from 2005 to 2008 in Peking University People's Hospital Collect the clinical data to investigate the clinical significance of D-dimer. Results ( 1 ) The D-dimer levels of 56 patients were increased due to coexist reduced renal function, infections, disseminated intravascular coagulation ( DIC), liver disorders, pregnancy and injury. With the exception of above patients, 142 ( 69. 3 ) patients were increased in total 205 patients. (2)The level of D-dimer was positively correlated with SLE disease activity index( SLEDAI ) score ( r =0. 598,P =0. 000), and was associated with anti-dsDNA antibody, ESR, C-reactive protein(CRP) and complement C3 and C4. (3)D-dimer level was associated with important organ involvement. (4)All patients with thrombosis had increased D-dimer, but patients without thrombosis had normal or increased D-dimer levels. Conclusion The level of D-dimer elevates in patients with active disease or important organ involvement , it can not identify thrombosis. All patients with thrombosis had increased D-dimer levels.