The Mechanism Of Curcumin On ERK1/2 Signaling Way And The Synthesis Of CTGF In Pulmonary Fibrosis

Background and ObjectiveThe pathogenesis of pulmonary fibrosis is complicated, it is characterized with myofibroblast hyperplasia and deposition of extracellular matrix.Study unraveling the complexities of the cytokine and growth factor networks that underpin pulmonary fibrosis has always been an important thing in our understanding of these events. In particular, transforming growth factor-β(TGF-β)appears to be a pivotal contributor to pulmonary fibrosis. Many experiments demonstrated that the pathogenesis is associated with disorders of all kinds of cytokines or growth factors.Connective tissue growth factor(CTGF),which is a downstream cytokine of transforming growth factor-β(TGF-β), may be a critical cytokine in the development of bleomycin-induced pulmonary fibrosis . CTGF is a 38 kDa cysteine-rich peptide, which belongs to the CCN (cyr61, CTGF, and nov) family of immediate early response genes. It acts as a downstream mediator of TGF-β1,It also can induce cell proliferation and extracellular matrix (ECM) deposition. To date, litle is known about the functional role of CTGF in the pulmonary fibrosis. In this paper, we discuss the association of CTGF with TGF-β1 and ERK1/2 signaling way in pulmonary fibrosis.Curcumin is one of the effective element extracted from curcuma. It owes various biological functions, including antiflammation, anticoagulation, decreasing lipid, antioxidation, and inhibiting hepatic fibrosis. From the theory above, we postulate that curcumin may do precautionary and therapeutic effect on pulmonary fibrosis. It have been testified from Experimental studies in vivo we have done that curcumin also have inhibiting pulmonary fibrosis.Methods and Results1 In first part,we investigate the effect of curcumin on proliferation inhibition and expression of collagen and the cell cycle distribution of human embro fibroblasts (MRC-5),Method: The human embryo fibroblasts (MRC-5) were allocated into seven groups:①normal group,②10μmol/L curcumin group,③20μmol/Lcurcumin group,④30μmol/L curcumin group,⑤40μmol/L curcumin group,⑥50μmol/L curcumin group;⑦60μmol/L curcumin group;24 hours later, the proliferation activity of human embro fibroblasts was detected by MTT assay,and the expression of collegan was observed by sirius red staining method, the cell cycle phase was analyzed by flow cytometry. The results indicated that curcumin can suppress the proliferation activity of human embryo fibroblasts and the expression of collagen, It also made cell cycles arrested at G0/G1 phase which implies curcumin may resist the pulmonary fibrosis by suppressing the proliferation activity of human embro fibroblasts and decreasing the expression of collagen.2 In the second part,we established a cellular model of pulmonary fibrosis in vitro.Method:Cultured HLF were divided into four groups①control groups②TGF-β1groups③CTGF groups④TGF-β1+CTGFgroups; Western Blotting were performed to detect the levels of colⅠ,colⅢand FN; RT-PCR were performed to detect the levels ofα-SMA, ELISA were performed to detect the levels of TIMP-1, sirius red staining were performed to detect the levels of the expression of collegan. And the proliferation activity of human embryo fibroblasts was detected by MTT assay.The result indecated that TGF-β1and CTGFcould significantly reduce the COLⅠ,COLⅢ,and FN formation.It also can actived HLF, promote the transdiferentiation of human embro fibroblasts towards myofibroblast and further promote ECM accumulation..So it can concluded that model of human embryonic lung fibroblast activation stimulated with TGF-βand CTGF can reflect more intact HLF activation effects..3 In the third part,we observed the expression and regulation of CTGF by TGF-β1stimulates, and study the interations betwen ERK1/2 signaling pathway and the expression of CTGF on HLF.Method: (1)Cultured HLF were stimulated by TGF-β1. Western-blotting were performed to detect the levels of CTGF protein after treatment. HLF without stimulation were used as control(2) Cultured HLF were incubated with l0ng/ml of TGF-β1,for 30min,1h,3h,6h,12h,24h,ELISA were performed to detect the levels of p- ERK1/2 and total- ERK1/2Result: (1) TGF-β1up-regulated the protein level of CTGF.(2) ERKI/2 signaling way were activated by TGF-β1,(3) Blockade of ERKI/2 activation reduced the level of CTGF protein.4 In the forth part,we examined the effect of curcumin on the expression of CTGF protein.and the ERKI/2 signaling wayMethod: cultured HLF were divided into five groups:①the control group,②TGF-β1stimulated group③PD98059 pretreated ,then TGF-βstimulated group④TGF-β1and 60umol/L curcumin group⑤TGF-β1and 40umol/Lcurcumin , immunohistochemistry and western-blotting were performed to detect the levels of CTGF protein of each group,RT-PCR were performed to detect the levels of CTGF mRNA.of each group, western-blotting were performed to detect the levels of p- ERK1/2 and总- ERK1/2.The results indicated that curcumin can partly inhibit the expression of CTGF and p- ERK1/2 and total- ERK1/2.Conclusions1 Curcumin can suppress the proliferation activity of human embro fibroblasts and the expression of collagen, thus exertive prevention and the treatment pulmonary fibrosis function2 The cellular model of pulmonary fibrosis in vitro we have established use TGF-β1and CTGF stimulates HLF is well reflect the chang of fibroblasts proliferation and the fomation of ECM.3 CTGF is partly induced by TGF-β1.4 Curcumin can partly suppress the expression of CTGF by ERK1/2 signaling way.