ABSTRACT
Objective:To explore the effect of chemical compound of aconitum alkaloid on the lipopolysaccharide （LPS）-induced inflammatory response of RAW264.7 macrophages and investigate its mechanism. Method:The chemical compounds of Aconitum Kusnezoffii Reichb were collected from TCMSP database with consideration of oral bioavailability （OB）≥30% and drug-likeness （DL）≥0.18. The potential targets of each chemical component were predicted with use of Pubchem database and Swiss Target Prediction database. Rheumatoid arthritis （RA） targets were collected from GeneCards database and selected by intersection screening. Gene ontology （GO） classification enrichment and Pathway enrichment analysis were carried out with use of DAVID database. Cytoscape was used to construct "Chemical Compound-Potential Targets-Pathway-Disease" network. Protein-protein interaction （PPI） network was constructed by using STRING database and Cytoscape software. RAW264.7 macrophages were stimulated by LPS to establish macrophage inflammation model <italic>in vitro</italic>. Western blot was used to detect the effects of chemical compounds on the expression of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and cyclooxygenase-2 （COX-2） in RAW264.7 cells induced by LPS， as well as on the expression of JAK kinase and nuclear transcription factor- kappa B （NF-<italic>κ</italic>B） signal pathway. Result:A total of 27 chemical compounds were obtained by searching TCMSP database and consulting literature （OB≥30%， DL≥0.18）. 12 chemical compounds were obtained after screening. 177 potential targets were obtained after database prediction and screening， and 97 targets were obtained as potential targets for the treatment of RA after intersection between 177 potential targets and 4 329 RA targets. A total of 32 biological processes （BP）， 5 cellular components （CC）， and 12 molecular functions （MF） were enriched by DAVID database. The construction of network topology map showed that different chemical compounds can act on the same target and the same chemical compound can also act on different targets in the treatment of RA. Aconitum alkaloid can be connected with the same pathway through different targets or with different pathways through the same target， indicating that different targets may have synergistic effect， which fully reflected the complex multi-compound， multi-targets and multi-pathways mechanism. Different concentrations of LPS in stimulation （0-200 μg·L<sup>-1</sup>） can significantly up-regulate the expression of COX-2 protein in RAW264.7 macrophages （<italic>P</italic><0.05）， indicating that the inflammatory model was successful. Compared with the normal group， the expression of TNF-<italic>α</italic> and COX-2 protein in the inflammatory model of RAW264.7 cells increased significantly（<italic>P</italic><0.05）， while the expression of TNF-<italic>α</italic> and COX-2 protein in bulleyaconitine A（BLA）， songorine， yunaconitine and karacoline groups decreased in varying degrees compared with the model group （<italic>P</italic><0.05）. Compared with the normal group， the expression of IRAK4， NF-<italic>κ</italic>B， JAK1 and STAT3 in the inflammatory model of RAW264.7 cells were significantly increased （<italic>P</italic><0.05）， while such levels in BulleyaconitineA， songorine， yunaconitine and Karacoline groups were significantly lower than those in the model group（<italic>P</italic><0.05）. Conclusion:Based on systematic pharmacology and <italic>in vitro</italic> experiments， the related targets and signal pathways were analyzed to provide new insights into the pathogenesis of RA， reveal the molecular mechanism of aconitum alkaloid in the treatment of RA， and provide new ideas for the application of Mongolian medicine in modern medicine.
ABSTRACT
In this study, the effect of benzo[α]pyrene (BaP) on chaperone-mediated autophagy (CMA) in a simulated hypoxia environment was observed and the relationship to heat shock protein 90 (HSP90) was clarified. With HSP90 inhibitor geldanamycin (GA) and HSP90α silenced, the mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), HSP90, heat shock cognate protein 70 (HSC70), and lysosomal associated protein 2A (LAMP-2A) of A549 cells on hypoxic environment by BaP were tested. Alkaline comet experiment, immunofluorescence γ-H2AX focus experiment, quantitative real-time PCR (qPCR), and Western blot analyses were used to clarify the relationship between the DNA damage of different concentrations of BaP in A549 cells and the mRNA and protein expression of CMA-related factors. The results show that hypoxia can promote the expression of mRNA and protein of CMA-related factors in A549 cells. This study found that BaP has an inhibitory effect on CMA under the hypoxic environment. The inhibition or silencing of HSP90 will enhance the inhibitory effect of BaP on CMA. In a normoxic environment, BaP causes DNA damage and promotes CMA.
ABSTRACT
Objective: To study the benzo(a)pyrene (B[a]P)-induced mRNA expression of aromatic hydrocarbon receptor (AHR) and cytochrome P4501A1 (CYP1A1) genes in rat liver. Methods: Rats were injected intraperitoneally with 5, 10 and 15 mg/kg of B[a]P. The total RNAs were extracted from rat livers by RNA purification kit, and the mRNA expression of AHR and CYP1A1 genes was determined by reverse transcription polymerase chain reaction (RT-PCR). β-actin was used as the internal control. The mRNA expression of both AHR and CYP1A1 genes was measured at indicated time points (24, 48 and 72 h) after B[a]P treatment at three different concentrations (5, 10 and 15 mg/kg). Results: The mRNA expression of AHR gene increased in a time-dependent manner at the concentration of 10 mg/kg but not at 5 and 15 mg/kg of B[a]P. The mRNA expression of CYP1A1 gene differed significantly at 48 h and 24 h in rat livers treated with 10 and 15 mg/kg dosage of B[a]P. The mRNA expression of AHR and CYP1A1 genes increased with B[a]P treatment in a concentration-dependent manner. The time-dependent increase in mRNA expression was shown by AHR but not by CYP1A1 gene with B[a]P (10 mg/kg) treatment. Conclusion: This study demonstrates that toxic B[a]P increases the mRNA expression of both AHR and CYP1A1 genes in vivo, suggesting that B[a]P may play a role in cancer genesis by this way.
ABSTRACT
Objective: To estimate the relative risk for lung cancer associated with genetic polymorphism of T6235C mutation in 3′ non-coding region (MspI) of cytochrome P450 1A1 (CYP1A1) and glutathione S-transferase M1(GSTM1) in the Mongolian population in Inner Mongolian Region of China. Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR methods were used to analyze blood samples obtained from 263 case subjects and 263 control subjects to determine their genotypes for CYP1A1 and GSTM1. Control subjects were matched with case subjects by ethnic background, age and gender. Results: The frequencies of the variant CYP1A1 genotypes (CYP1A1C) and GSTM1-null in lung cancer groups were higher than those in control groups (38.4% vs. 28.5% and 57.8% vs. 48.0%). The individuals who carried with CYP1A1C genotype had a significantly higher risk of lung cancer (OR=1.56, 95% CI=1.08 to 2.25, P=0.016) than those who carried with non-variation CYP1A1 genotype. The ones who carried with GSTM1-null genotype also had a significantly higher risk of lung cancer (OR=1.49, 95% CI=1.06 to 2.10, P=0.023) than those who carried with GSTM1-present genotype. When combination of polymorphisms of CYP1A1 and GSTM1 genotypes was analyzed, the risk of lung cancer for combination of CYP1A1C and GSTM1-null genotypes was increased significantly (OR=2.084, 95% CI=1.27 to 3.42, P=0.003). Susceptibility to lung cancer was related to smoking (OR=2.10, 95% CI=1.48 to 2.98, P=0.000). Considering smoking status, the risk of lung cancer for combination of smoking and CYP1A1C genotype was remarkably increased (OR=2.76, 95% CI=1.74 to 4.37, P=0.000). It was the same case with combination of smoking and GSTM1-null genotype (OR=4.38, 95% CI=2.35 to 8.15, P=0.000). Conclusion: The polymorphisms of CYP1A1C genotype and GSTM1-null are the risk factors of lung cancer in the Mongolian population in Inner Mongolia Region of China. Smoking is also related to susceptibility to lung cancer. There may be a synergetic interaction between CYP1A1C and GSTM1-null in the elevated susceptibility of lung cancer. Smoking may have a synergetic interaction with CYP1A1C and GSTM1-null in the elevated susceptibility of lung cancer.
ABSTRACT
<p><b>OBJECTIVE</b>To study the polymorphism of CYP1A1 gene Msp I site in the Mongolian and Han nationality populations of Inner Mongolia.</p><p><b>METHODS</b>The PCR-restriction fragment length polymorphism(PCR-RFLP) technique was used to analyze the genotypes of CYP1A1 gene Msp I site in 80 subjects of Mongolian nationality and 120 subjects of Han nationality among whom there is no blood relationship each other.</p><p><b>RESULTS</b>The genotype frequency of CYP1A1 gene Msp I site showed that the wild-type, heterozygote, homozygous variants were 35.0%, 48.7%, 16.3% and 33.3%, 52.5%, 14.2% respectively distributions of Mongolian nationality and Han nationality population, and the Chi-square tests showed that there was no significant difference between the two groups.</p><p><b>CONCLUSION</b>The genotype frequency distributions of CYP1A1 gene Msp I site did not exhibit the obvious difference between Mongolian nationality and Han nationality population of Inner Mongolia.</p>
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Binding Sites , Genetics , China , Cytochrome P-450 CYP1A1 , Genetics , Deoxyribonuclease HpaII , Metabolism , Gene Frequency , Genotype , Mongolia , Ethnology , Polymerase Chain Reaction , Polymorphism, Genetic , Genetics , Polymorphism, Restriction Fragment LengthABSTRACT
<p><b>OBJECTIVE</b>To investigate the influence of the smoke tar on the expression of aromatic hydrocarbon receptor (AHR) and the cytochrome P4501Al (CYP1A1) gene of mice lungs.</p><p><b>METHODS</b>The smoke tar of 5.29, 10.58 and 15.87 mg/kg was administered intraperitoneally in mice respectively. RNA of mice lungs was got with RNA kit. RT-PCR technique was used for determining AHR and CYP1A1 gene expression with beta-actin as control.</p><p><b>RESULTS</b>The AHR gene expression level was (0.554 +/- 0.023) for the mice intraperitoneally administered with 5.29 mg/kg smoke tar for 72 hours with the significant difference in gene expression level compared with the Tween-80 group (0.484 +/- 0.045) (P < 0.05). The AHR gene expression levels were (0.555 +/- 0.014), (0.606 +/- 0.051), and (0.566 +/- 0.014), (0.684 +/- 0.069) for the mice intraperitoneally administered with 10.58 and 15.87 mg/kg smoke tar for 48 hours and 72 hours respectively with the significant difference in gene expression level compared with the Tween-80 group (0.486 +/- 0.060, 0.484 +/- 0.045) (P < 0.05, P < 0.01). The CYP1Al gene expression levels were (1.535 +/- 0.021), (1.643 +/- 0.046) and (1.624 +/- 0.056), (1.739 +/- 0.038) respectively with the significant difference compared with the Tween-80 group (l.436 +/- 0.016, 1.404 +/- 0.036) (P < 0.01).</p><p><b>CONCLUSION</b>The smoke tar can regulate up the expression of AHR and CYP1A1 gene at a certain dosage and time. The regulation of the smoke tar for the expression of AHR was earlier than for that of CYP1A1.</p>