Effects And Mechanisms Of Mesenchymal Stem Cells And Curcumin On Immune Disorders And Epithelial-Mesenchymal Transition In PBC

Background:Primary biliary cholangitis(PBC)is an autoimmune liver disease characterized by progressive cholestasis and non-suppurative,destructive inflammation within the medium and small bile ducts.The "upstream" immune injury and the "downstream" fibrosis are the two keys in the development and progression of PBC.Mesenchymal stem cells(MSCs)have a reduced immunogenicity and unique immunoregulatory function.Although several studies have shown that MSCs transplantation could alleviate the symptoms of refractory PBC patients,its specific mechanism has not been fully elucidated.Galectin-9(Gal-9)belongs to the galectin(Gal)superfamily,it is involved actively at various immune responses.Nevertheless,it remains largely unknown whether there is a close association between Gal-9 and PBC as well as MSCs.Biliary epithelial cells(BECs)are the primary "victim" in the pathogenesis of PBC.Epithelial-mesenchymal transition(EMT)is an important "bridge",which mediates the development of fibrosis in PBC.Curcumin has been demonstrated to possess good anti-fibrosis effects.However,it is still not clear whether curcumin could effectively inhibit EMT process in BECs under PBC microenvironment.Objectives:To investigate the correlation between immune disorders and regulatory molecular Gal-9 in PBC patients.The short-term immunoregulatory effects after umbilical cord-derived MSCs(UC-MSCs)transplantation were evaluated.Furthermore,UC-MSCs transplantation was conducted to treat PBC murine model in effort to explore the efficacy and immunomodulatory mechanisms of UC-MSCs.Additionally,the effects and mechanisms of curcumin on EMT in BECs under chronic induction were investigated.Methods:1)The alterations of helper T cells 1(Thl),helper T cells 17(Th17)and regulatory T cells(Treg)in peripheral blood from PBC patients were detected by flow cytometry.Immunohistochemistry was used to detect the expression of interferon-?(IFN-?).interleukin-17A(IL-17A)and forkhead transcription factor p3(Foxp3)in the liver tissues of PBC patients.Enzyme-linked immunosorbent assay(ELISA)was used to determine the expression of Gal-9 in the serum from PBC patients.Correlation analyses were carried out regarding the relationship between Gal-9 and clinical biochemical indexes as well as T cell subsets.The changes of different CD4+T cells,as well as serum Gal-9 expression were compared after UC-MSCs transplantation.2)UC-MSCs were injected intravenously into 2-octynoic acid coupled to bovine serum albumin(20A-BSA)induced PBC murine model.After finishing the treatment,all the mice were sacrificed.Histopathology of livers was observed by H&E staining and assessed by histological score.Serum levels of biomarkers and autoantibodies were measured by ELISA respectively.Luminex was utilized to determine the expression of inflammatory cytokines in the serum.The alterations of CD4+T cell subsets in the liver,spleen and peripheral blood were analyzed by flow cytometry.The mRNA expression of inflammatory factors in liver tissues were detected by reverse transcription-polymerase chain reaction(RT-PCR).In vitro assays,the expression of Gal-9 in UC-MSCs after IFN-y stimulation was measured by RT-PCR,western blot and ELISA.Certain signaling pathways were examined by western blot and RT-PCR.Splenic CD4+T cells from C57BL/6 mice(B6 mice)were sorted by magnetic activated cell sorting(MACS),and co-cultured with UC-MSCs in the presence or absence of Gal-9 inhibitor a-lactose.Flow cytometry was then used to determine the apoptosis and proliferation rates of CD4+T cells.Splenic naive T cells from B6 mice were isolated,then co-cultured with UC-MSCs in the presence or absence of a-lactose under Thl or Th17 cells differentiation condition.Four days later,the frequencies of Thl or Thl 7 cells were examined by flow cytometry.After the stimulation of IFN-?,BECs were co-cultured with UC-MSCs,parallel experiment was conducted using a-lactose.The expression of pro-inflammatory chemokines was measured by RT-PCR.3)To evaluate the role of curcumin on TGF-31 induced EMT in BECs,the morphological changes of BECs were observed.The expression of E-cadherin,Vimentin,?-smooth muscle actin(a-SMA)and N-cadherin was determined by immunofluorescence,western blot and RT-PCR.Several key transcription factors of EMT,such as Snail2,Twistl and ZEB1 was detected by RT-PCR,Further,the Smad and Hedgehog signaling pathway were determined by western blot.Results:1)Compared to the healthy controls,the frequencies and cell numbers of Thl and Th17 cells in the peripheral blood of PBC patients were significantly increased,while the frequencies and cell numbers of Treg cells were decreased.The expression of IFN-y and IL-17A in the liver tissues of PBC patients was much higher than control group.The serum levels of Gal-9 in PBC patients were significantly decreased,and Gal-9 was negatively correlated with alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and glutamyl transpeptidase(GGT).Meanwhile,there existed a negative correlation between Gal-9 and the frequencies of Thl and Th17 cells in patients with PBC.After 24 hours of UC-MSCs transplantation,the frequencies of Thl and Th17 cells were decreased significantly in the peripheral blood from PBC patients,while the frequencies of Treg cells remained unchanged.2)After UC-MSCs transplantation,the liver injury in PBC mice was relieved,and the serum levels of anti-PDC-E2 antibodies were decreased.More importantly,UC-MSCs transplantation significantly down-regulated the percentages of Thl and Th17 cells from different tissues in PBC mice,and effectively reduced the expression levels of pro-inflammatory cytokines in the liver and serum.Under the stimulation of IFN-y,UC-MSCs could be induced to secrete high levels of Gal-9.Moreover,the inhibitory effects of UC-MSCs on both the proliferation of CD4+T cells and the differentiation from naive T cells to Thl and Thl7 cells were closely related to Gal-9.The signal transducer and activator of transcription(STAT)and c-Jun N-terminal kinase(JNK)signaling pathways were involved in the production of Gal-9 in UC-MSCs.In addition,through Gal-9,UC-MSCs could reduce the expression of pro-inflammatory chemokines in BECs under IFN-y stimulation.3)After 48 hours of curcumin intervention,the EMT morphological changes of BECs could be improved.As demonstrated by immunofluorescence,western blot and RT-PCR,the expression of E-cadherin in BECs under induction was increased after curcumin treatment,whilst the expression of Vimentin,a-SMA and N-cadherin were decreased.Besides,curcumin significantly repressed the expression of several transcription factors that mediated EMT,such as Snail2,Twistl,and ZEB1.What's more,curcumin could inhibit the activation of Smad and Hedgehog signaling pathway via up regulating the expression of Smad7 and SUFU,down regulating the expression of several activation markers.Conclusions:Local and systemic immune disfunctions and abnormal Gal-9 expression were observed in PBC patients.UC-MSCs could secret Gal-9 to regulate the local and systemic immune disorders,thus effectively limiting the "upstream" immune injury in PBC.Curcumin could interfere with the EMT in BECs under chronic induction,so as to prevent the "downstream"fibrotic process in PBC.Collectively,our results suggest that a combination of therapies targeting different pathways in different stages may be beneficial to patients with PBC.