[Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment].

This study combined network pharmacology, molecular docking, and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC). Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF. The active components of NWXF were retrieved from SwissADME, and the candidate targets of these active components were retrieved from SwissTargetPrediction. Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC. Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms. Finally, molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0. The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9). Additionally, the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed. The effectiveness of the drug combination was evaluated based on the Q value. The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF. Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR), c-Jun N-terminal kinase(JNK), and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF. Western blot was employed to determine the protein levels of EGFR, p-EGFR, JNK, p-JNK, and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF. A total of 77 active components, 488 potential targets, and 49 key targets involved in the treatment of NSCLC with NWXF were predicted. The results of GO annotation showed that NWXF may treat NSCLC by regulating the biological processes such as cell proliferation, apoptosis, and protein phosphorylation. KEGG enrichment revealed that the key targets of NWXF in treating NSCLC were enriched in the mitogen-activated protein kinase(MAPK), phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), hypoxia-inducible factor-1(HIF-1), and microRNA-related signaling pathways. Molecular docking results showed that 91.9% of the docking scores were greater than 5, indicating the strong binding capability between main active components and key targets. The cell experiments demonstrated that NWXF combined with GEF synergistically inhibited the proliferation, promoted the apoptosis, decreased p-EGFR/EGFR and p-JNK/JNK values, down-regulated the mRNA levels of EGFR and JNK, and up-regulated the mRNA and protein levels of Bax in A549 and PC-9 cells. In conclusion, NWXF combined with GEF can regulate the EGFR/JNK pathway to promote the apoptosis of NSCLC cells, thus treating NSCLC.

Activated hepatic stellate cells directly induce pathogenic Th17 cells in chronic hepatitis B virus infection.

Th17 cells are involved in liver fibrosis by activating hepatic stellate cells (HSCs). We aimed to investigate whether HSCs are able to regulate the function of Th17 cells and to determine the relevant mechanism. Sixty-five patients diagnosed with chronic hepatitis B (CHB) were enrolled in this study. To determine the effect of HSCs on T cells, naïve CD4+T cells and Th17 cells were sorted from CHB patients and cultured with or without activated-HSCs, and cytokine expression and gene transcription were analyzed. In addition, the regulatory mechanism of HSCs was investigated. ELISA and qRT-PCR showed that Th17 cells from CHB patients were more pathogenic, on the basis of the expression of IL-17A, IL-23R, RORC, CCL20 and CCR6, and meanwhile, they could activate the primary HSCs. Co-culture experiments indicated that activated HSCs dramatically promoted proliferation of CD4+T cells in a time- and dose-dependent manner. In addition, they could induce naïve CD4+T cells to become Th17 cells which had a more pathogenic phenotype. Moreover, activated HSCs-mediated induction of Th17 cells might depend on the release of IL-1ß and IL-6 as well as on the COX-PGE2 pathway. Th17 cells cooperated with HSCs in a proinflammatory feedback loop might provide a better understanding of the pathogenic role of Th17 cells in the chronicity of HBV infection.

[Effect of estrogen receptor alpha gene polymorphism on the variations of T lymphocyte subsets and its related cell factors in female patients with primary cholestasis cirrhosis].

OBJECTIVE: To study the effects of estrogen receptor (ER) alpha gene polymorphism on the migration of T lymphocyte subsets and related cytokines in the female patients with primary biliary cirrhosis(PBC). METHODS: This study was conducted with sixty female PBC patients without treatment as the study group and fifty-two healthy people wtih sex and age met the requirements of the study as the control group. The polymorphism of restriction enzyme cutting site of Xba I and Pvu II in intron 1 of ERa gene was detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). CD4+, CD8+, CD4+CD25+ and CD4+CD28- T lymphocytes in peripheral blood were quantitatively detected by flow cytometry. RT-PCR method was used to detect the expression of TNFa, IL-2, IFNgamma, IL-4, IL-6 and IL-10 in peripheral mononuclear cells. RESULTS: The positive rate of Pp in ERa gene Pvu II enzyme gene subtypes of female PBC patients was significantly greater than that of the control group, and the positive rate of pp gene subtype was significantly smaller than that of the control group (X2 = 7.2880, P = 0.0261). The difference of Xba I genotype and allele frequency between the female PBC patients group and the control group was not of statistical significance (X2 = 6.5382, P = 0.5833). The proportion of CD4+ T in T lymphocytes of PBC patients was increased to 45.31%+/-5.26%, compared with 33.81%+/-3.87% in the control group; and the proportion of CD8+ T lymphocytes was decreased to 27.78%+/-1.43 % from 31.83%+/-1.73% in the control group. In comparison with the control group, the proportion of CD4+CD25+ T lymphocytes decreased significantly, while that of CD4+CD28- T lymphocytes rose significantly. The expression levels of TNFa, IL-2 and IFNgamma mRNA were 0.59+/-0.19, 0.71+/-0.29 and 0.67+/-0.21 respectively, which were significantly higher than that in the control group (0.22+/-0.13, 0.31+/-0.14, 0.27+/-0.13) (t = 6.93, 5.07, 7.01, P value less than 0.01); the expression level of IL-6 mRNA was increased to 0.45+/-0.21 from 0.34+/-0.16 in the control group (t = 1.84, P value less than 0.05); and the difference of the expression levels of IL-4 and IL-10 mRNA between two groups was not of statistical significance. CONCLUSION: Pp of gene Pvu II was a genetically susceptible genotype in female PBC patients, and the allele p was a susceptible gene. Th1 cell subsets and related cytokines were dominant in peripheral blood of PBC patients. As a background of genetic susceptibility, ERa gene polymorphism could affect the shift of T lymphocyte subsets and the expression of the related cytokines in PBC patients.