Study On The Effect And Mechanism Of Total Glucosides Of White Peony On Alleviating Scleroderma By Inhibiting Type I Interferon Respons

Systemic sclerosis(SSc),also called scleroderma,is an autoimmune disease that can result in localized or diffuse skin fibrosis,vasculopathy,and inflammation.Scleroderma occurs when the immune system loses its ability to maintain self-tolerance,producing an abnormal balance of hyperactive immune cells and cytokines.The current conventional treatment generally uses anti-inflammatory drugs and immunosuppressants,but these drugs only partially improve symptoms.Additionally,some drugs have many side effects,including infection,obesity,abnormal glucose metabolism,osteoporosis,and even ischemic necrosis.Therefore,it is necessary to further study the pathogenesis and corresponding treatment strategies of SSc.TypeⅠ interferons(IFNs),the members of cytokine family,are produced by innate immune cells and act on adaptive immune cells.Therefore,type Ⅰ IFNs play an important role in regulating immune responses to infections,cancers and autoimmune diseases.Clinical studies have found that there is a significant excess of type Ⅰ IFN in the blood and skin tissue of a large proportion of patients with SSc,and the abnormal expression of type Ⅰ IFN-induced genes is related to SSc pathogenesis,clinical manifestations and disease activity.Many inhibitors targeting type Ⅰ IFN signaling had shown positive results in clinical trials for autoimmune diseases.Our previous study found that Si-Ni-San(SNS),a Chinese herbal formula composed of Glycyrrhiza uralensis Fisch.,Bupleurum chinense DC.,Paeonia lactiflora Pall.,and Citrus aurantium L.,has inhibitory effects on type Ⅰ IFN responses.However,it remains to be further explored which monomer or mixture in SNS is the pharmacodynamic material basis for its activity.In addition,considering that type Ⅰ IFN is one of the important pathogenesis of autoimmune diseases,ingredient with type Ⅰ IFN inhibitory activity in SNS is promising to be developed as a therapeutic strategy for type Ⅰ IFN-related diseases.Aim of the study:We aim to identify bioactive ingredient with anti-type Ⅰ IFN activity from SNS and further elucidate its therapeutic effect against scleroderma and underlying mechanisms.Materials and methods:Firstly,we constructed Gaussia-luciferase(Gluc)reporter assay systems with IFNB1 as promoter to identify the component with anti-type Ⅰ IFN activity in SNS.RAW264.7 macrophages were treated with the Poly(I:C),lipopolysaccharide(LPS),DMXAA and IFNα2 to induce the upstream and downstream pathways of type Ⅰ IFN,respectively.Then,Real-time PCR(RT-PCR)and Western blot(WB)were utilized to detect type Ⅰ IFN signature genes and relative protein in RAW264.7 cells to explore whether the total glucosides of peony(TGP)affect type Ⅰ IFN signaling.We used bioinformatic analysis of gene expression matrix from skin biopsy samples of SSc patients to clarify the pathogenesis of SSc.To explore the protective effect of TGP on SSc,we used RT-PCR to detect the expression of fibrotic genes and type Ⅰ IFN-related genes and used H&E staining,Masson staining and immunohistochemistry analysis to measure the histopathological changes in a bleomycin(BLM)-induced experimental SSc model.In SSc mice model and immune cell model,macrophages,inflammatory cytokines,cytotoxic T lymphocytes(CTL)and cytotoxic granules were detected by RT-PCR and immunohistochemical staining to explore the mechanism of TGP in improving fibrosis in SSc by inhibiting type Ⅰ IFN.To further explore the effective monomer in TGP that can inhibit type Ⅰ IFN in vitro,high speed countercurrent chromatography was used to selectively collect and knock out the main active components of TGP.Results:In this study,by using Gluc reporter gene screening system,we found that Paeonia lactiflora Pall.in SNS had type Ⅰ IFN inhibitory effect,and further screening results showed that TGP was the pharmacological basis of Paeonia lactiflora Pall.for its inhibitory activity.In RAW264.7 cells,TGP selectively inhibited TLR3-mediated type Ⅰ IFN response and blocked Janus kinase(JAK)-signal transducer and activator of transcription(STAT)signaling.The results of bioinformatics analysis showed that type Ⅰ IFN-related genes were upregulated in the skin samples of SSc patients and were correlated with fibrotic genes.In the BLM-induced SSc mice model,H&E and Masson staining showed that TGP reduced dermal thickness and collagen deposition.RT-PCR results also showed that TGP reduced the mRNA levels of fibrosis-related genes Fibronectin(Fn),Collagen type Ⅰ α1(Col1a1),Actin alpha 2(Acta2),and Connective tissue growth factor(Ctgf)in SSc mice model.Additionally,TGP inhibited multiple targets in upstream and downstream of the type Ⅰ IFN signaling pathway in SSc model mice,especially genes related to fibrosis,such as Interferon regulatory factor 7(Irf7),C-X-C motif Chemokine Oligand 9(Cxcl9),and Cxcl10.Further data analysis showed that macrophages and CTLs were up-regulated in the skin of SSc patients,and were significantly correlated with type Ⅰ IFN and fibrosis.In BLM-induced SSc mice,TGP treatment reduced the increased M2 macrophages,the pro-inflammatory cytokines secreted by M1 macrophages,such as Interleukin-1β(Il-1β)and Il-6,and the pro-fibrotic cytokines secreted by M2 macrophages,such as Il-10 and Transforming growth factor-β(Tgf-β).In M2-polarized bone marrow-derived macrophages(BMDMs),TGP also inhibited the expression of Il-10 and Tgf-β.In addition,TGP inhibited BLM-induced CD4+T cells,CD8+T cells and cytotoxic effector molecules,including Granzyme B(GzmB)and Perforin.In RAW264.7 cells and A549-Gluc cells,the four fragments separated by high-speed countercurrent chromatography and the major active monomers of TGP(Benzoyl paeoniflorin(BP),Albiflorin(AF),Paeoniflorin(PAE),Oxidized paeoniflorin(OP))did not show type Ⅰ IFN inhibitory activity.Conclusions:In summary,the current study identified TGP as a natural inhibitor of typeⅠ IFN responses.In experimental scleroderma,TGP significantly improved skin fibrosis.Specifically,TGP abrogated the polarization of M2 macrophages and their profibrotic effects and reduced cytotoxic T lymphocytes and their cytotoxic granules by regulating type Ⅰ IFNinduced Cxcl9 and Cxcl10 expression.These findings not only shed novel lights into the immunoregulative effects of TGP but also provides convincing evidence to inspire the discovery of TGP-based therapies in the treatment of scleroderma and other autoimmune diseases associated with type Ⅰ IFN signatures.