ABSTRACT
The study aims to investigate the mechanism of Fengshining fecal microbiota transplants in the intervention of rheumatoid arthritis by ultra-performance liquid chromatography-quadrupole/electrostatic field obitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). All animal welfare and experimental procedures followed the regulations of the Medical Ethics Committee of Shanxi University of Chinese medicine. The rats were randomly divided into normal group, model group, fecal microbiota transplantation group and Tripterygium wilfordii polyglycoside group, and the collagen induced arthritis (CIA) was established. The changes of body weight and metatarpodal lesions of rats were evaluated. The serum of rats in each group was analyzed by liquid chromatography-mass spectrometry and metagenomic technology for differential metabolites and microflora. The protein expression levels of Toll-like receptors (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor of kappa B (NF-κB p65) were detected by Western blot. A total of 13 different metabolites, including arachidonic acid, docosahexaenoic acid, 13S-hydroxyoctadecanodienoic acid and L-phenylalanine were screened from serum. Three metabolic pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and arachidonic acid metabolism were identified through pathway enrichment. Metagenomic analysis showed that the abundance of g_Bacteroides, g_Prevotella and p_Actinobacteria in CIA group was higher. The abundance of c_Clostridia, g_Akkermansia and s_Akkermansia_muciniphila in fecal microbiota transplantation group is higher. The hierarchical cluster heat map showed that Akkermansia was negatively correlated with L-phenylalanine; while positively correlated with docosahexaenoic acid. Prevotella was positively correlated with L-phenylalanine. Fecal microbiota transplantation group could significantly inhibit the expression of TLR4, MyD88 and p65 proteins in the synovium of rats (P < 0.01). The anti-rheumatoid arthritis effects of fecal microbiota transplantation group is closely related to the intervention of the metabolism of phenylalanine and arachidonic acid, through Akkermansia, Prevotella and other microorganisms, inhibition the TLR4/MyD88/NF-κB pathway.
ABSTRACT
Objective:To elucidate the potential molecular markers and drug-compound-target mechanism of Epimedii Folium intervention on breast cancer stem cells（BCSCs） through chip analysis combined with network pharmacology and experimental validation. Method:Relevant drug information was retrieved in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） to obtain the active components and potential targets of Epimedii Folium. "Breast Cancer Stem Cells" were searched in Gene Expression Omnibus（GEO）database，and GSE98239 chip data were obtained through analysis and screening. Then GEO2R online analysis tool was used to obtain the differential genes to draw differential gene heat map and volcano map. The differential gene network map of Epimedii Folium intervention for breast cancer stem cells was constructed by Cytoscape 3.8.0，and Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis of drug and disease genes were performed. Human breast cancer MDA-MB-231 cells were divided into 20%，40%，60% Epimedii Folium drug-containing serum group and control group. Cell counting kit-8（CCK-8），and Western blot were used to detect the effect of Epimedii Folium drug-containing serum intervention on cell activity and target protein expression in breast cancer cells. Result:Twenty-three active components including flavones，sterols，alkaloids and sesquiterpenoids were obtained from Epimedii Folium. It was found that Epimedii Folium interacted with B-cell lymphoma-2-like protein 1（BCL2L1），matrix metallopeptidase 2（MMP2），prostaglandin-endoperoxide synthase 2（PTGS2），vascular endothelial growth factor A（VEGFA），transforming growth factor beta receptor 1（TGFBR1） and other pivotal genes in breast cancer stem cells，participated in the induction of new angiogenesis and cell migration，enabled the continuous self-renewal of BCSCs，decreased apoptosis and cell migration，thus promoting the recurrence and metastasis of breast cancer. KEGG results showed that Epimedii Folium intervened in multiple differential expressed genes（DEGs）of transforming growth factor-<italic>β</italic>（TGF-<italic>β</italic>），vascular endothelial growth factor（VEGF），phosphoinositide 3kinase/protein kenase B（PI3K/Akt），mitogen-activated protein kinese（MAPK）and mammalian target of rapamycin（mTOR）subpathways in cancer signaling pathways to exert its efficacy in intervening breast cancer stem cells. Experiments showed that the survival rate of breast cancer cells was significantly reduced and the expression levels of TGFBR1 and Smad2 in breast cancer cells significantly decreased after the intervention of Epimedii Folium drug-containing serum（<italic>P</italic><0.01）. Conclusion:Several components in different concentrations of drug-containing serum of Epimedii Folium can synergistically act on target differentially expressed genes of breast cancer stem cells，and inhibit the proliferation of breast cancer cells by down-regulating the expression levels of TGFBR1，a key molecule in the TGF-<italic>β</italic> pathway，and Smad2，a downstream signal.
ABSTRACT
To investigate the potential molecular markers and drug-compound-target mechanism of Mahuang Shengma Decoction(MHSM) in the intervention of acute lung injury(ALI) by network pharmacology and experimental verification. Databases such as TCMSP, TCMIO, and STITCH were used to predict the possible targets of MHSM components and OMIM and Gene Cards were employed to obtain ALI targets. The common differentially expressed genes(DEGs) were therefore obtained. The network diagram of DEGs of MHSM intervention in ALI was constructed by Cytoscape 3. 8. 0, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses of target genes. The ALI model was induced by abdominal injection of lipopolysaccharide(LPS) in mice. Bronchoalveolar lavage fluid(BALF) was collected for the detection of inflammatory factors. Pathological sectioning and RT-PCR experiments were performed to verify the therapeutic efficacy of MHSM on ALI. A total of 494 common targets of MHSM and ALI were obtained. Among the top 20 key active compounds of MHSM, 14 from Ephedrae Herba were found to be reacted with pivotal genes of ALI [such as tumor necrosis factor(TNF), tumor protein 53(TP53), interleukin 6(IL6), Toll-like receptor 4(TLR4), and nuclear factor-κB(NF-κB)/p65(RELA)], causing an uncontrolled inflammatory response with activated cascade amplification. Pathway analysis revealed that the mechanism of MHSM in the treatment of ALI mainly involved AGE-RAGE, cancer pathways, PI3 K-AKT signaling pathway, and NF-κB signaling pathway. The findings demonstrated that MHSM could dwindle the content of s RAGE, IL-6, and TNF-α in the BALF of ALI mice, relieve the infiltration of inflammatory cells in the lungs, inhibit alveolar wall thickening, reduce the acute inflammation-induced pulmonary congestion and hemorrhage, and counteract transcriptional activities of Ager-RAGE and NF-κB p65. MHSM could also synergically act on the target DEGs of ALI and alleviate pulmonary pathological injury and inflammatory response, which might be achieved by inhibiting the expression of the key gene Ager-RAGE in RAGE/NF-κB signaling pathway and downstream signal NF-κB p65.
Subject(s)
Animals , Mice , Acute Lung Injury/genetics , Drugs, Chinese Herbal/pharmacology , Lipopolysaccharides , Lung/metabolism , NF-kappa B/metabolism , Network Pharmacology , Receptor for Advanced Glycation End Products/metabolism , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To study the effect of intracellular reactive oxygen species (ROS) levels on T cell activation and apoptosis of synovial cells in collagen induced arthritis (CIA) rats, and to explore the mechanism of Fengshining Capsule (FSN) in the treatment of rheumatoid arthritis (RA).</p><p><b>METHODS</b>Sixty rats were randomly divided into the normal control group, the CIA model group, the Tripterygium Poly-glycoside Tablet (TPT) group, the low dose FSN group (at the daily dose of 0.33 g/kg), the middle dose FSN group (at the daily dose of 0.66 g/kg), and the high dose FSN group (at the daily dose of 1.32 g/kg), 10 in each group. T lymphocyte subsets were detected by flow cytometry. The content of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in plasma of rats were detected by ELISA. Its expression of hydroxyl radicals was detected by ultraviolet spectrophotometry. Caspase-3 and Caspase-9 protein expressions were measured by Western blot.</p><p><b>RESULTS</b>Compared with the CIA model group, the levels of ROS were elevated in each dose FSN group (P < 0.01). The level of CD4+ / CD8 was significantly reduced in the middle dose FSN group (P < 0.01). The content of IFN-gamma was obviously lowered in each dose FSN group (P < 0.01), while that of IL-4 was obviously elevated in the high dose FSN group (P < 0.01). Meanwhile, the expression of Caspase-9 and Caspase-3 significantly increased in each dose FSN group (P < 0.05). Besides, the average gray scale of Caspase-9 was significantly higher in the low and middle FSN groups than in the TPT group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>The mechanism of FSN for regulating the immune hyperfunction and inhibiting the proliferation of synovial cells in CIA rats might be associated with up-regulating in vivo ROS levels.</p>