A systematic study of Erzhu Erchen decoction against damp-heat internalized type 2 diabetes based on data mining and experimental verification

Background:Erzhu Erchen decoction(EZECD),which is based on Erchen decoction and enhanced with Atractylodes lancea and Atractylodes macrocephala,is widely used for the treatment of dampness and heat(The clinical manifestations of Western medicine include thirst,inability to drink more,diarrhea,yellow urine,red tongue,et al.)internalized disease.Nevertheless,the mechanism of EZECD on damp-heat internalized Type 2 diabetes(T2D)remains unknown.We employed data mining,pharmacology databases and experimental verification to study how EZECD treats damp-heat internalized T2D.Methods:The main compounds or genes of EZECD and damp-heat internalized T2D were obtained from the pharmacology databases.Succeeding,the overlapped targets of EZECD and damp-heat internalized T2D were performed by the Gene Ontology,kyoto encyclopedia of genes and genomes analysis.And the compound-disease targets-pathway network were constructed to obtain the hub compound.Moreover,the hub genes and core related pathways were mined with weighted gene co-expression network analysis based on Gene Expression Omnibus database,the capability of hub compound and genes was valid in AutoDock 1.5.7.Furthermore,and violin plot and gene set enrichment analysis were performed to explore the role of hub genes in damp-heat internalized T2D.Finally,the interactions of hub compound and genes were explored using Comparative Toxicogenomics Database and quantitative polymerase chain reaction.Results:First,herb-compounds-genes-disease network illustrated that the hub compound of EZECD for damp-heat internalized T2D could be quercetin.Consistently,the hub genes were CASP8,CCL2,and AHR according to weighted gene co-expression network analysis.Molecular docking showed that quercetin could bind with the hub genes.Further,gene set enrichment analysis and Gene Ontology represented that CASP8,or CCL2,is negatively involved in insulin secretion response to the TNF or lipopolysaccharide process,and AHR or CCL2 positively regulated lipid and atherosclerosis,and/or including NOD-like receptor signaling pathway,and TNF signaling pathway.Ultimately,the quantitative polymerase chain reaction and western blotting analysis showed that quercetin could down-regulated the mRNA and protein experssion of CASP8,CCL2,and AHR.It was consistent with the results in Comparative Toxicogenomics Database databases.Conclusion:These results demonstrated quercetin could inhibit the expression of CASP8,CCL2,AHR in damp-heat internalized T2D,which improves insulin secretion and inhibits lipid and atherosclerosis,as well as/or including NOD-like receptor signaling pathway,and TNF signaling pathway,suggesting that EZECD may be more effective to treat damp-heat internalized T2D.

2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-Glucoside modulates CHEK2 and CCND1 alternative splicing to inhibit MCF-7 cells proliferation

Background:In our previous study,we observed a synergistic effect of 2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-glucoside combined with adriamycin to induce apoptosis in MCF-7 breast cancer cells.However,the underlying mechanisms of epigenetic modifications,such as alternative splicing,have not been explored.In this study,we aimed to investigate the mechanism by which THSG inhibits MCF-7 cell proliferation using full-length transcriptome sequencing.Methods:First,cell viability was examined using the methyl thiazolyl tetrazolium method and full-length transcriptome sequencing was performed to identify genes and pathways.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify the principal pathways and targets of THSG.Flow cytometry analysis of cell cycle distribution was performed.Meanwhile,the analysis of alternative splicing and domains of the key proteins was conducted.Quantitative polymerase chain reaction and western blotting were performed for verification.Results:THSG showed significant cytotoxic activity in MCF-7 cells.Full-length transcriptome sequencing revealed differential alternative splicing with 173 upregulated and 263 downregulated genes.Further analysis identified distinct differential expression of genes(CHEK2-211 and CCND1-201)involved in the cell cycle in the THSG-treated group.Subsequently,alternative splicing types of CHEK2(mutually exclusive exon)and CCND1(intron retention).We found that THSG downregulated mRNA expression,as confirmed by quantitative polymerase chain reaction analysis.Interestingly,protein structural analysis revealed that THSG treatment led to the generation of CHK2-211,which was the result of a mutation in the amino acid residues(GLU-150,ASN-151)of the CHEK2 domain(VAL-150,GLY-151).and CyclinD1-201 were obtained when an amino acid(ASP-267)in the domain was lost in CyclinD1.Moreover,molecular docking analysis demonstrated that the domains of key proteins could bind THSG more effectively,with no difference in affinity.Western blotting confirmed that THSG inhibited the expression of CHK2 and CyclinD1.Conclusion:THSG modulated the alternative splicing of CHEK2 and CCND1 by inducing G0/G1 cell cycle arrest,consequently suppressing MCF-7 cell proliferation.