| 1 Background and objectiveUlcerative colitis (UC) is a chronic non-specific inflammatory disease of the rect um and colon and is a kind of inflammatory bowel disease (IBD).Which is often recu rrent, refractory protracted illness, has been recognized as one of the three most import ant risk factors for colon cancer. Its etiology and pathogenesis are complicated. There is no technique to affect a radical cure at present. Therefore, UC-related mechanisms o f pathogenesis and drug intervention research is very critical. Huangqin Tang (HQT) is a famous prescription from Shanghan Lun, It has been widely used to treat gastroi ntestinal diseases, such as diarrhea, abdominal spasms, fever, headache, vomiting, nau sea, extreme thirst, and subcardiac distention. Currently it is used in the treatment of ulcerative colitis. Chinese herbal formulae are complex mixtures of herbs consisting of multiple bioactive ingredients which bind to corresponding targets simultaneously, t ransiently, or weakly, and treat complex diseases in a systematic manner. On the basi s of previous research, this paper intends to focus on the regulation of TLR4/MyD88/NF-κB signaling pathway by HQT, and to identify the genes related to the develop ment and progression of UC disease by microarray detection, To construct the HQT to regulate gene expression profiling of related genes in ulcerative colitis and identif y key molecular targets. At the same time, combined with metabolomics research to i dentify potential molecular markers of UC.Ultimately bridging genomics and metabolo mics, from the perspective of multi-target and multi-channel to explain the treatment mechanism of HQT on UC, to explore the pathogenesis of UC and targeted therapy to provide molecular basis.2 Methods and results2.1 Effect of Huangqin Tang on the Function of Regulatory TLR4/MyD88 Signal Pathway in Rats with Ulcerative ColitisThe model of UC rats with cell immunoreactivity was made by using compound method(trinitrobenzene sulfonic acid and ethanol). Rats were randomly divided into control group,model group, SASP group and high dose, middle dose and low dose of Huangqin Tang group.After being treated for three days, Production of NO was detected by Griess assay, the expression levels of IL-4, IL-10,IL-17 and PGE2 were detected by ELISA. After five days of treatment, ICH method was undertaken to determine the positive expression of COX-2 and iNOS protein, and Western blotting was employed to evaluate expression of TLR4 and MyD88 protein in colon tissue. This study shows that the expression level of NO, IL-17,PGE2 in serum, TLR4, MyD88 protein of colon tissue and the positive expression of COX-2,iNOS was apparently higher than that of control group (P < 0.05). Moreover, the above indexes in high dose of Huangqin Tang groups were significantly ameliorated (P < 0.05).Thus, Huangqin Tang may exert the possible effect on the treatment of pathogenesis of UC by the inhibition of the relative activity of TLR4/MyD88/NF-κB Signaling Pathway and decrease the expression levels of NO, IL-17 and PGE2.2.2 Gene expression profiling identifies the potential targets of Huangqin-Tang in ulcerative colitisThe gene expression profiles of the control group,the model group and the HQT group were detected by genome-wide microarray , and the differential expression dat a were analyzed. We identify 569 various genes (199 upregulated and 370 downregul ated genes, | fold change (FC)|≥1.5, p-value<0.05) in the model group compared to the normal group. Then, according to the network pharmacological analysis of IPA da tabase and Cytoscape software, 126 genes with effective interaction were screened o ut. Among them,we filtered out "candidate targets"(ITGB1,FN1,CASP3,LDLR,B MP4, FABP1,ABCB1,FABP2, SLC51B) in terms of the "degree"≥10 or | fold cha nge (FC)| ≥4 in the network. Combined with disease and functional enrichment analy sis, 126 differences in genes may cause changes in disease and biological functionma inly concentrated in energy generation, lipid metabolism, cell development, growth an d proliferation, neurological diseases, tissue Damage and abnormalities, amino acid m etabolism and so on. Key target genes are closely related to immune response, infla mmation, apoptosis and metabolism, and these genes are revealed to be involved in multiple inflammatory-related pathways such as Agranulocyte Adhesion and Diapedesis,Integrin Signaling, NF-κB Activation by Viruses, FAK Signaling, PAK Signaling, ER K/MAPK Signaling, PI3K/AKT Signaling.2.3 Effect of Huangqin Tang on serum metabolic profile in rats with ulcerative colitis based on UPLC-MSUltra performance liquid chromatography tandem massspectrometry (UPLC-MS),principal component analysis(PCA) and partial least squares- discriminant analysis (PLS-DA)were employed to analyze the metabolic profile among normal group, the model group, HQT group. Serum and urine metabolomics pathways of enrichment analysis are: valine, leucine,isoleucine biosynthesis; alanine, aspartic acid and glutamic acid metabolism; peanuts Tetraacetic acid metabolism; arginine and proline metabolism; ketone synthesis and degradation; glycine, serine,threonine metabolism; tyrosine metabolism; tryptophan metabolism; linoleic acid metabolism. Potential biomarkers in the serum were screened based on the variable importance projection(VIP) value> 1, P<0. 05. As compared with the normal group, 16 potential biomarkers such as valine, tryptophan, lactic acid and urea were found and identified in the serum of model group rats. As compared with the model group,Parts of the biomarkers were regulated nearly to a normal state after HQT administration for 10 days. Metabolomic analysis revealed that the HQT has a certain therapeutic effect for UC rats,and the mechanism may be related to regulation of lipid metabolism , amino acid metabolism and energy metabolism.3 ConclusionIn this paper, through the study of TLR4/MyD88/NF-KB classical inflammatory pathway,it is clear that HQT plays an important anti-inflammatory effect in the treatment of ulcerative colitis. The gene chip obtained 126 HQT modally regulated UC differential genes and screened out 20 key target genes, which are mainly related to inflammatory response,metabolism, immune system.Based on the metabolomics of UPLC-MS, the specific biomarkers associated with the disease were screened. The treatment of UC by HQT may be related to the regulation to disorder of amino acid metabolism, energy metabolism and lipid metabolism in rats. At the same time, combined with gene and metabolic network analysis showed that the gene expression with fatty acid oxidation and transport function decreased,which may be the direct cause of fatty acid accumulation in metabolites, The accumulation of fatty acids may be an important factor in the development of UC inflammation; and cell migration and proliferation-related gene functions are activated, them may be the direct cause of further deterioration of inflammatory response. Our work provides a novel and powerful means to clarify the mechanisms of HQT acting on UC on the basis of multi-targets and multi-pathways. At the same time, it shows great significance to account for the theory of TCM in a holistic way. |
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