The Mechanism Of CTHRC1 Regulating Frozen Shoulder Through TGF-β/Smad Signal Pathway

Objective:Frozen shoulder(FS)is a common shoulder disorder,but the pathogenesis of FS is currently unknown.The aim of this study was to explore the mechanism of Collagen Triple Helix Repeat Containing-1(CTHRC1)on the pathogenesis of FS.Methods:Chapter 1:Based on bioinformatics analysis,to explore the potential regulatory relationship in the pathogenesis of FS and to determine the gene expression of CTHRC1 in FS patients during high-throughput sequencing.The main methods are as follows:(1)Using R packages,we first screened the differential genes between FS and control(CON)groups to clarify the expression of CTHRC1 between the two groups.(2)The screened differential genes were enriched for pathway and function to explore the pathways and biological processes associated with FS pathogenesis.(3)The protein-protein interaction(PPI)network of differential genes was constructed by STRING database,and the core interaction network and core genes in PPI were screened by algorithm to understand the interaction of genes in FS disease.(4)We used the Gene MANIA database to predict the core gene-related interactions genes and explore the molecules that may affect the development of FS.Chapter 2 : To determine the difference in CTHRC1 expression between patients in the FS and CON groups by tissue level and to validate the results of bioinformatics analysis.Here’s how:(1)The histomorphological differences of synovium between FS groups and CON groups were observed by using HematoxylinEosinstaining(HE)staining.(2)Molecular biology techniques such as real-time fluorescence quantitative PCR(q RT-PCR),Western Blot(WB)and Immunohistochemistry(IHC)were used to detect the difference of CTHRC1 expression between FS and CON groups.Chapter 3 : The effect of CTHRC1 on fibrotic phenotype and TGF-β/Smad signal of primary Fibroblast-Like Synoviocyte(FLS)were elucidated by cell experiment in vitro,and to clarify the molecular mechanism of CTHRC1 regulating FS.The main methods were as follows:(1)After extracting the primary FLS of patients in FS group,the expression levels of Collagen I(COL I),Collagen III(COL III),Fibronectin1(FN1)and α-Smooth Muscle Actin(α-SMA)in FLS transfected with CTHRC1 over expression or knockdown lentivirus were detected by q RT-PCR and WB to explore the effect of CTHRC1 on fibrotic phenotype of FLS.(2)In addition,the expression level of p-Smad2/3 in each group was detected by molecular biology technique to explore the effect of CTHRC1 on TGF-β/Smad signal pathway in FLS.(3)By regulating the activation and inhibitor of TGF-β/Smad signal pathway and conducting the rescue experiment,we explored whether CTHRC1 affected FLS fibrosis phenotype through the TGF-β/Smad signal pathway.Chapter 4 :The effect of CTHRC1 on the developmental process of FS was elucidated by animal experiments in vivo.SD rats were divided into sham + NS group,sham + LV-NC group,FS + LV-NC,FS + LV-CTHRC1 group,FS + shCTHRC1 group.After 8 weeks of modeling,shoulder joint specimens were obtained for the following tests:(1)The joint mobility of rats was detected in each group.(2)The synovial tissue was observed with HE staining.(3)The expression level of COL I was detected with IHC and immunofluorescence.(4)The collagen fiber deposition was observed with Sirius scarlet staining.Results:1.The results of bioinformatics analysis showed that CTHRC1,TGF-β signal pathway and the fibrotic phenotypes of Extracellular Matrix(ECM)were enriched,and CTHRC1 was highly expressed in synovium of FS patients.2.There were differences in the expression of CTHRC1 and COL I between CON and FS group,and the expression of CTHRC1 and COL I increased in synovium of FS patients.3.Overexpression of CTHRC1 increased the expression levels of COL I,COL III,FN1,α-SMA and p-Smad2/3 in primary FLS,whereas knockdown of CTHRC1 significantly downregulated the expression levels of these molecules.4.Rescue experiments showed that the regulation level of CTHRC1 on downstream molecules in primary FLS could be influenced by pathway agonists or inhibitors.5.After the establishment of FS model,joint mobility was significantly restricted and synovial hyperplasia was significantly aggravated.After transfection of CTHRC1-associated lentivirus,it was found that the overexpression of CTHRC1 could aggravate the limitation of joint and synovial hyperplasia in rats,while knockdown of CTHRC1 could improve joint mobility and synovial hyperplasia in FS model rats.6.After the establishment of FS model,the expression of COL I and the deposition of collagen fibers in synovium of rats significantly up-regulated.Transfection with CTHRC1-related lentivirus revealed that CTHRC1 overexpression aggravated the deposition of COL I and collagen fibers in the synovium,while CTHRC1 knockdown significantly improved the deposition of COL I and collagen fibers.Conclusion:1.We explored the influence of protein interactions,biological processes and related pathways on the pathogenesis of FS at the theoretical level through bioinformatics analysis,which provided new perspectives and ideas for future basic research on FS.Secondly,according to the results of bioinformatics analysis,it is proved that CTHRC1,TGF-β signal pathway and the fibrosis phenotype of ECM play an important role in the pathogenesis of FS,which provides a theoretical basis for the following chapters.2.CTHRC1,which is highly expressed in the synovial membrane of FS patients,is a key molecule in the pathogenesis of FS and may be a potential target for the treatment of FS.3.CTHRC1 can affect the fibrotic phenotype of FLS by regulating TGF-β/smad signal pathway,and then participate in the progression of FS.4.CTHRC1 has a regulatory effect on the formation of FS in rats.