The Research On The Mechanisms Of Preventive Effect On The SSc By Using The Formula Of Bufei Qingyu Under The Method Of Xuanfu

Bac kground:Scleroderma is a connective tissue disorder characterized mainly by the thickening and hardening of the skin.There are two primary types of scleroderma:localized and systemic(also called systemic sclerosis,SSc).Currently,there is still no curative therapy resistant to this complex disorder.Chinese medicine,as supplement of modern medicine,need to be exploited.Bufei Qingyu Granules(BQG),which is composed of Astragalus mongholicus(Huangqi),Salvia miltiorrhiza(Danshen),Angelica sinensis(Danggui)and other seven Chinese herbs,has been developed and extensively used by Jiangsu Province Hospital of TCM(Nanjing,China)for the treatment of SSc and a portion of pulmonary fibrosis,demonstrating to be highly beneficial in clinical practice.In a preliminary evidence,we previously found the utility of alleviating sclerotic skin with appropriate dosage of BQG in Bleomycin(BLM)-induced mice model.In recent years,some advanced technology and methods have boomed in TCM-based research fields,such as transcriptomics and bioinformatics analyses,and ultra-performance liquid chromatography(UPLC)coupled with quadrupole-time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)analysis,etc.It is possible to provide important lines for understanding of genes' regulations and the mechanisms behind them.Objective:In order to research the preventive effect and its mechanisms of Bufei Qingyu Granule on SSc mouse model.Methods:1)Under the suitable conditions,UHPLC-Q-TOF/MS analysis was conducted to study the representative components,which are the material basis of BQG.2)The mice were randomly divided into normal group,BLM model group,BLM + BQG group.The scleroderma mouse models were estabilished by continuous subcutaneous injection of bleomycin solution into the back skin of mice for 4 weeks.After administrating BQG,the different therapeutic effects of BQG were observed,thus the overall health of the mice was evaluated and the index of each organ was calculated.In addition,the protective role of BQG on a mouse scleroderma model were evaluated as follows:histopathological analysis of skin tissue,quantitative determination of hydroxyproline content by alkaline hydrolysis,enzyme-linked immunosorbent assay(ELISA)method for the determination of chemokine(CXC motif)ligand 2(Chemokine(CXC motif)ligand 2,Cxcl2),immunohistochemistry(IHC)determination of a-Smooth muscle actin(a-SMA).3)The total RNA was extracted by the Trizol method in the skin tissues of all the groups.The RNA samples were enriched for mRNA,and then the mRNA library was constructed and tested in Hiseq X Ten(Illurmina).Data analysis was followed,mainly including:the assessment of sequencing raw data quality and quality control,RNA sequencing evaluation,gene expression statistics,screening differential expression genes,Venn diagram,visualization analysis of differentially expressed gene(volcano map),analysis of protein interaction network,differential genes heat map,Unigene annotation(GO annotation,KEGG annotation),GO enrichment analysis,KEGG enrichment analysis.Three predicted target genes were selected for validation by Quantitative real-time PCR.Then differential genes were analyzed by protein-protein interaction(PPI)network.After that corresponding signal pathways were screened,the associated genes on the signaling pathway were also verified by Quantitative real-time PCR.In addition,the relevant proteins were confirmed by immunoblotting.Results:1)Typical chemical components and its contents in BQG were identified and ordered by peak as follows:Amygdalin,Paeonol,Schisandrol A,Dihydrotanshinone I,Cryptotanshinone,Schizandrin A,Tanshinone IIA,Schizandrin B,Danshensu,acteoside,Ferulic acid,Lobetyolin,Platycodin D,Astragaloside IV.2)Compared with the normal group,although the model group mice did not perform exactly the same at different time points,the overall trend showed impotence,reduced food intake,decreased body size and hardened skin.Spleen index and hydroxyproline content increased significantly.The model group showed typical manifestation in pathology.The level of Cxcl2 was also significantly increased.Thus,the number of vascular smooth muscle positive cells rised.Compared with the model group,the mice in the BQG group had milder skin sclerosis,significantly lower spleen index and hydroxyproline content.The thickness of the dermis was significantly reduced.The level of Cxcl2 was lower than the model group,thus the number of vascular smooth muscle positive cells was decreased.3)The differential expression genes were screened by transcriptomics analysis,and the target genes were verified by Quantitative real-time PCR.The fold change of synaptosome associated protein 25(Snap25)and eukaryotic translation initiation factor 3,subunit J2(Eif3j2)was consistent with transcriptome analysis data,to some extent,indicating that transcriptomic data was credible.The signal pathway was chose by KEGG pathway analysis,finding that the Notch signaling pathway was in the top five pathways for enrichment of significantly differential expression genes.The related genes Notch-1 and Jagged-2 were confirmed at not only in gene level by Quantitative real-time PCR but also in protein level by immunoblotting.We further found that BQG could significantly reduce the mRNA and protein expression levels of Notch-1 and Jagged-2.Conclusion:There are representatively four types of chemical constituents in BQG.The saponins in BQG mainly originated from Huangqi,Dangshen,Taoren,Jiegeng and Lingxiaohua;phenolic acids were mainly from Daripi and Danggui;Salvia miltiorrhiza components were chiefly derived from Danshen;Wuweizi had the highest content of lignans compounds.BQG has protective effect on bleomycin-induced SSc mouse model,which may be partly due to partial involvement of Cxcl2 or neutrophil involvement,vascular correlation and Notch signaling.