Research On PPAR Pathway In Regulating TGF-β/MMP On Inhibiting Fibrosis Through Collagen Metabolism In Frozen Shoulder

Background Frozen shoulder is a disease of unknown etiology which is more susceptible in patients with glucolipid metabolism disorder.Previous studies found that,comparing with the patients with rotator cuff injury,the expression of matrix metalloproteinase 9(MMP-9)declined and some of the genes in the Peroxisome Proliferator-Activated Receptor(PPAR)pathway expressed increasingly.While genetic variations associated with extracellular matrix components.Our previous studies also found that the stimulus of transforming growth factor-beta(TGF-β)can significantly inhibit the expression of PPAR-γ.That indicated that the expressions of TGF-β/MMP-9 influenced the extracellular matrix metabolism,and PPAR pathway involved.This study prepared to 1)make suppression and overexpression with TGF-β/MMP-9 in fibroblasts,to see if they can regulate the activation of PPAR-γ and the collagen metabolism.2)Use PPAR-γ in the MMP-9 knockout animals to see if the activation of PPAR-γ can inhibit fibrosis.The study reveals the PPAR pathway,activated by lack of collagen metabolism,inhibiting of TGF-β to make the MMP-9 in the dominant position and then promote the collagen metabolism,thus inhibiting the fibrosis of shoulder joint capsule.The study offers a new perspective on the pathogenesis and treatment of frozen shoulder.Methods 1.Differential gene analysis of shoulder joint capsule in patients with frozen shoulder1)Using the method of transcriptomics to detect the difference of the level of fibrosis of the shoulder joint capsule and the normal shoulder joint capsule tissue in frozen shoulder patients;2)Find the specific genes related to metabolism in the frozen shoulder differential gene.2.The expression of collagen / MMP-9 / TGF-β in human fibroblasts and its effect by PPAR-γ1)to detect the expression of TGF-β / MMP-9 in human fibroblasts,and to detect the expression of type I,type II,type III and type IV collagen in fibroblasts and their extracellular matrix.2)The expression of TGF-β in human fibroblasts was increased by lentivirus infection,the changes of collagen in extracellular matrix were detected,and the effect of TGF-β on the expression of collagen was studied.3)The expression of MMP-9 in human fibroblasts was increased by lentivirus infection,the changes of collagen in extracellular matrix were detected,and the effect of MMP-9 on the expression of collagen was studied.4)To detect the expression of PPAR-γ in human fibroblasts;5)The expression of TGF-β / MMP-9 in fibroblasts and extracellular matrix was detected by using agonist CDDO-IM and antagonist GW9662 to stimulate the expression of PPAR-γ in human fibroblasts.The expression of PPAR-Γ on TGF-β / MMP-9 expression;6)The expression of PPAR-γ in fibroblasts and outer matrix was detected by using agonist CDDO-IM and antagonist GW9662 to stimulate the expression of PPAR-γ in human fibroblasts.The effects of PPAR-γ on type I and type II,Type III,type IV collagen metabolism regulation;7)Human fibroblasts overexpressing TGF-β were treated with lentivirus infection using agonist CDDO-IM and antagonist GW9662 to study the effect of PPAR-γ in human fibroblasts on type I,type II,Type III,type IV collagen,whether the change of TGF-β expression changes,and then study whether PPAR-γ through the TGF-β play a role in the regulation of collagen;8)Human fibroblasts overexpressing MMP-9 were treated with lentivirus infection using agonist CDDO-IM and antagonist GW9662 to study the effect of PPAR-γ in human fibroblasts on type I,type II,Type III,type IV collagen,whether it is through the changes in MMP-9 expression changes,and then study whether PPAR-γ through MMP-9 play a role in the regulation of collagen;3.The interaction of PPAR / TGF-β / MMP-9 and its regulation in the metabolism of collagen were confirmed by animals experiments1)The use of the same age as the human age of 40-50 years of age in New Zealand rabbits,shoulder joint capsule injection of adenovirus / adeno-associated virus overexpressing TGF-β to cause shoulder capsule fibrosis;2)To investigate the retraction(mobility)of the maximum abduction angle of the shoulder joint in the anesthesia state of the TGF-β injection side and the non-injection side and to understand whether TGF-β caused the contracture of the shoulder joint capsule;3)To give New Zealand rabbits oral commercial PPAR-γ agonists,detection of TGF-β injection side and non-injection side of the maximum expansion angle of the retraction of the situation(activity),to understand the PPAR-γ agonist for the shoulder capsule;4)To detect the HE staining of the shoulder joint capsule and the difference between the different groups when the TGF-β injection side and the non-injection side and the commercial commercial PPAR-γ agonist;4)TGF-β / MMP-9 and type I,type II,III,IV collagen protein expression were detected by immunohistochemistry in TGF-β injection side and non-injection side,and oral commercial PPAR-γ agonist Type,in order to understand the PPAR-γ agonist in the body state for the shoulder capsule in the different protein;5)Using the Western-Blot method to detect TGF-β injection side and non-injection side,and oral commercial PPAR-γ agonist after New Zealand rabbit shoulder joint capsule to understand the TGF-β / MMP-9 and type I,type II,Type III,type IV collagen,so as to understand the effect of PPAR-γ agonists on the different proteins in the shoulder capsule.Results 1.The differentially expressed genes in patients with frozen shoulder are mainly manifested in the PPAR signaling pathway1)The results showed that TGF-β could significantly increase the expression of type I collagen in extracellular matrix in vitro culture of chondrocytes.In addition,this study also found that TGF-β stimulation Inhibit the expression of PPAR-γ in chondrocytes.2)Through the transcriptome analysis of the m RNA of the shoulder joint capsule in 6 patients with frozen shoulder and 3 rotator cuff injury,it was found that the frozen shoulder patients were compared with the articular capsule tissue m RNA in patients with rotator cuff injury The enrichment of the biological process shows that the differential gene is associated with the response.3)The results of molecular functional enrichment showed that the differential gene mainly expressed protein binding(especially in the combination of globin)and other molecular functions in patients with frozen shoulder and rotator cuff injury.4)Frozen shoulder in patients with joint capsule m RNA and rotator cuff injury in patients with contrast,signal pathway enrichment results show that the difference gene is mainly reflected in the PPAR signal pathway.5)According to the interaction between proteins,the expression of MMP-9 gene was lower in the joint capsule tissue of frozen shoulder patients,and the expression of multiple genes was increased in PPAR signal pathway.2.The expression of collagen / MMP-9 / TGF-β in human fibroblasts was affected by PPAR-γ1)Human fibroblasts infected with TGF-β and MMP-9 lentiviral vector 48 hours after the green fluorescent marker,while the control group did not light,no-load virus also see green fluorescent markers,suggesting that fibroblasts infected with TGF-β and MMP-9 success.2)In the HFF cells,GW9662 at 40 u / ul can be more obvious inhibition effect,CDDOIM at 20 u / ul can be more obvious overexpression effect.In HSF cells,GW9662 could obtain more obvious inhibitory effect at 40 u / ul,CDDO-IM could get more obvious overexpression at 20 u / ul,and the effect of GW9662 in HSF cells was more obvious.3)Western-blot results showed that compared with the control group,the following results were obtained:(1.1)PPAR-γ antagonist GW9662 can inhibit the expression of PPAR-γ in human fibroblasts.PPAR-γ agonist CDDO-IM can increase the expression of PPAR-γ in human fibroblasts;(1.2)The use of PPAR-γ antagonist GW9662 inhibited the expression of TGF-β and MMP-9 after human fibroblasts,the difference was not statistically significant;(1.3)The use of PPAR-γ agonist CDDO-IM to stimulate the expression of TGF-β and MMP-9 after human fibroblasts,the difference was statistically significant;(1.4)overexpression of TGF-β and MMP-9 could induce the expression of PPAR-γ in human fibroblasts.(2.1)PPAR-γ antagonist GW9662,can inhibit the expression of TGF-β in human fibroblasts;PPAR-γ agonist CDDO-IM changes the expression of TGF-β in human fibroblasts,the difference is not statistically significance;(2.2)PPAR-γ antagonist GW9662 can inhibit the expression of TGF-β induced by overexpression of TGF-β,but it can not inhibit the expression of TGF-β induced by overexpression of MMP-9.(2.3)overexpression of MMP-9 can also cause TGF-β expression in human fibroblasts increased;(3.1)PPAR-γ antagonist GW9662 and agonist CDDO-IM,affect the expression of MMP-9 in human fibroblasts,the difference was not statistically significant;(3.2)PPAR-γ antagonist GW9662 could inhibit the expression of MMP-9 induced by overexpression of MMP-9.(3.2)PPAR-γ agonist CDDO-IM can induce the expression of MMP-9 in human fibroblasts after overexpression of TGF-β and MMP-9.(4.1)PPAR-γ antagonist GW9662,only inhibit the expression of type I collagen in human fibroblasts;and PPAR-γ agonist CDDO-IM can only increase the expression of type I collagen;(4.2)overexpression of TGF-β can increase the expression of I,II,IV collagen in human fibroblasts;(4.3)PPAR-γ antagonist GW9662 can inhibit the expression of four kinds of collagen,I,II,III and IV,which are caused by overexpression of TGF-β in human fibroblasts.(4.4)overexpression of MMP-9 can increase the expression of type I collagen;(4.5)human fibroblasts overexpressing MMP-9,PPAR-γ antagonist GW9662 and agonist CDDO-IM can lead to increased expression of type I,II,IV collagen;but the role of type III collagen in contrast.4)Real-time quantitative PCR results showed that compared with the control group,the following results were obtained:(1.1)PPAR-γ agonist CDDO-IM can increase the transcription of PPAR-γ in human fibroblasts,and can inhibit the transcription of PPAR-γ in human fibroblasts.(1.2)The use of PPAR-γ antagonist GW9662 inhibited the expression of TGF-β and MMP-9 after human fibroblasts,the difference was not statistically significant;(1.3)The use of PPAR-γ agonist CDDO-IM to stimulate the expression of TGF-β and MMP-9 after human fibroblasts,the difference was statistically significant;(1.4)overexpression of TGF-β can cause PPAR-γ transcription in human fibroblasts increased;(1.5)overexpression of MMP-9 did not cause the transcriptional level of PPAR-γ in human fibroblasts increased;(2.1)PPAR-γ antagonist GW9662 can inhibit the transcription of TGF-β in human fibroblasts;PPAR-γ agonist CDDO-IM can increase the transcription of TGF-β in human fibroblasts;(2.2)PPAR-γ antagonist GW9662,can inhibit the overexpression of TGF-β induced TGF-β transcription increased;(2.3)overexpression of MMP-9 can induce the transcriptional level of TGF-β in human fibroblasts;and the transcriptional enhancement can be inhibited by PPAR-γ antagonist GW9662;(3.1)PPAR-γ antagonist GW9662 can inhibit the transcription of MMP-9 in human fibroblasts;PPAR-γ agonist CDDO-IM can increase the transcription of MMP-9 in human fibroblasts;(3.2)PPAR-γ antagonist GW9662,can inhibit overexpression of MMP-9 caused by MMP-9 transcription increased;(4.1)PPAR-γ antagonist GW9662,can inhibit the transcription of II,III,IV collagen in human fibroblasts;PPAR-γ agonist CDDO-IM can elevate III,type IV collagen in human fibroblasts Transcription in cells;(4.2)overexpression of TGF-β can increase the transcription of I,II,III,IV four kinds of collagen in human fibroblasts;(4.3)PPAR-γ antagonist GW9662,can inhibit the overexpression of TGF-β caused by I,II,III,IV four kinds of collagen in human fibroblasts in the transcription increased;(4.4)overexpression of MMP-9 can increase the transcription of type IV collagen;(4.5)human fibroblasts overexpressing MMP-9,PPAR-γ antagonist GW9662 and agonist CDDO-IM can lead to the transcription of I,II,IV collagen,but the role of type III collagen in contrast..3.TGF-β regulates the activation of PPAR-γ pathway and the regulation of collagen metabolism in vivo experiments1)4 weeks after surgery,to detect the different groups of experimental animals in the plasma rosiglitazone plasma concentration,plasma concentration within the effective range to determine the efficacy of oral drugs in experimental animals in vivo;2)At 4 weeks after operation,the results of pathological sections can be observed,compared with the control group and the simple group compared with the TGF-β overexpression group in the shoulder capsule collagen formation of the direction of the fiber bundle slightly disordered,and the collagen density increased And the TGF-β overexpression + group was higher than that in the TGF-β overexpression group.The collagen fibers in the synovial capsule of the TGF-β overexpression group were significantly higher than those in the TGF-β overexpression group.There is no significant difference in the arrangement of the bundles,and the collagen is still denser and aggregated into clusters,and strong acidity.3)The results of immunohistochemistry showed that:(1)Rosiglitazone was administered orally for 4 weeks.No PPAR-γ expression was observed in the shoulder capsule of the experimental animals.Rosiglitazone was orally administered for 4 weeks.No TGF-β expression in the shoulder joint capsule was inhibition;(2)The expression of TGF-β in the shoulder of the experimental animals was increased by overexpressing the adeno-associated virus vector in the shoulder of the experimental animals.(3)The expression of PPAR-γ in the shoulder capsule of the experimental animals was increased by overexpression of TGF-β in the experimental animals.(4)The expression of MMP-9 in the shoulder capsule of experimental animals was not increased after overexpression of TGF-β in the experimental animals.(5)The expression of type I and type III collagen in the shoulder joint capsule of experimental animals can be increased by overexpression of TGF-β in the experimental animals.(6)Rosiglitazone was administered orally for 4 weeks(no high expression of PPAR-γ).Compared with the control group,the expression of I,II,III and IV collagen in the shoulder joint capsule was not changed.There was also no change in the expression of type I and type III collagen induced by overexpression of TGF-β.4)Animal experiment WB results showed:(1)Rosiglitazone was administered orally for 4 weeks.There was no expression of PPAR-γ in the shoulder capsule of the experimental animals.Rosiglitazone was orally administered for 4 weeks.No TGF-β expression in the shoulder joint capsule To suppress(2)The expression of TGF-β in the shoulder of the experimental animals was increased by overexpressing the adeno-associated virus vector in the shoulder of the experimental animals.(3)The expression of PPAR-γ in the shoulder capsule of the experimental animals was increased by overexpression of TGF-β in the experimental animals.(4)The expression of type I and type III collagen in the shoulder joint capsule of experimental animals can be increased by overexpression of TGF-β in the experimental animals.(5)Rosiglitazone was administered orally for 4 weeks(no high expression of PPAR-γ).There was no change in the expression of I,II,III and IV collagen in the shoulder capsule of experimental animals.-β-induced expression of type I and type III collagen changes.