| Background:Renal tubulointerstitial fibrosis (RIF) is the final common pathway of various kinds of chronic kidney diseases (CKD) developing to the end stage renal failure (ESRF). The main pathological manifestation is tubular interstitial extracellular matrix (ECM) deposition, tubular dilation, tubular epithelial cell necrosis and inflammatory cell infiltration, eventually leading to massive cell death, renal parenchymal collapse, fall, glomerular filtration rate of renal structural damage and renal failure. At present, it is difficult for RIF to discover, diagnose and treat early. The treatment measures focus on preventing kidney function from deteriorating further, but not completely reverse the process of fibrosis. Therefore, investigating pathogenesis of renal fibrosis and development of novel anti fibrotic drugs is the urgent task at present.Renal tubular epithelial cells (TECs) and tubulointerstitial play important roles in a variety of renal diseases. Inflammatory reaction and tissue free radical damage caused by Tubular interstitial inflammatory infiltration is considered to be the initiating factors of RIF. These inflammatory cells mainly cause kidney damage from three aspects:(1) the inflammatory cells can secrete inflammatory cytokines, such as interleukin (IL-1β), tumor necrosis factor alpha (TNF-a) and interferon (y-IFN), directly induce apoptosis of renal tubular epithelial cells. Furthermore, the oxygen free radicals and reactive oxygen species (ROS) produced by inflammatory cells cause oxidative stress injury. (2) The inflammatory cells also secrete chemokines, such as monocyte chemoattractant protein-1 (MCP-1), which can recruit the macrophages and lymphocytes from circulating system to the damaged tissue, further amplifying the inflammatory response signal that result in a vicious circle of kidney continued failure. (3) The inflammatory cell can secrete profibrotic cytokines, such as transforming growth factor (TGF-β1), that can induce renal tubular epithelial cells to initiate epithelial mesenchymal transition (EMT) and produce muscle fibroblasts (MyoF). The EMT process causes kidney structure damage, loss of renal parenchyma and glomerular filtration rate of collapse.The previous study shows that the renal tubular epithelial cells EMT-like phenotypic changes is the foundation for investigating the mechanism of RIF and discovering anti-fibrotic durg. the EMT-like phenotypic changes is that TECs lose epithelial markers and disappear adhesion structure.at the same time, TECs acquire mesenchymal cell markers and obtain the ability of migration and invasion. The generant MyoF can synthesize and secrete ECM components, such as collagen and fibronectin. ECM secretion increase and degradation decrease.a large number of ECM which deposit in the tubulointerstitial can not be degraded, as ultimately leads to the formation of RIF.MCP-1 as a chemotactic factor, not only can recruit and activate macrophages, also can directly drive the deposition of ECM induced by TGF-β1. Therefore, MCP-1 plays a key regulatory role in the process of tubulointerstitial inflammation. However, it is still not fully elucidated about how TGF-β1 stimulates cells to express MCP-1 and initiates EMT process. In many types of kidney cells, activation nuclear factor-κB (NF-κB) involve in the transcription and expression of MCP-1 gene. In addition, extracellular regulated protein kinase (ERK1/2) as a mitogen activated protein kinase (MAPK), plays an important role in the EMT process induced by TGF-β1. How to control EMT development with anti-fibrotic durg is key to the treatment of renal tubule interstitial fibrosis. Our research mainly explores the relationship between the EMT startup and production of MCP-1 through vivo and vitro experiments. ROS/ERK/NF-κB signaling pathway may be a new interpretation for EMT process and RIF induced by TGF-β1.Arctigenin (ATG), a bioactive constituent from dried seeds of Arctium lappa L. (Compositae) which has been widely used as a Traditional Chinese Medicine, was found to exhibit anti-inflammatory activities.In this study, we will also examine the effects of arctigenin on ROS/ERK/NF-kB activation and MCP-1 production in human renal tubular epithelial cells by TGF-β1 treatment and the rat RIF model by unilateral ureteral occlusion(UUO).The precise mechanism of these actions remain largely unknown and need to be defined. This investigation provides theoretical basis for finding new target in the prevention and treatment of RIF.Objective:investigating the interaction between production of MCP-1 and initiation of EMT-like phenotypic changes during the process of RIF. Arctigenin as a potential anti-fibrogenic candidate drug, intervenes EMT-like phenotypic changes during the RIF to provide a theoretical basis for finding a new target and new therapeutic candidate in the prevention and treatment of RIF.Methods:(1) Establishing the renal interstitial fibrosis models by UUO. observing the effect of ATG on renal cortex structure and function in rat RIF models, and investigating relationship between ATG intervention RIF and ATG reducing the inflammatory reaction and oxidative stress status in renal interstitial. At the same time, observing the effect of ATG on renal tubular epithelial cells EMT-like phenotypic changes during the rat RIF animal model to provide theoretical basis for ATG in the prevention and treatment of RIF. (2) Applying TGF-β1 stimulate renal tubular epithelial cells to simulate the RIF signal with different concentrations and different treatment time. in order to reveal the law of EMT in renal tubular epithelial cells in the case of induced by TGF-β1, we should examine the level of reactive oxygen species, the phosphorylation level of ERK 1/2, the activation level of transcription factor NF-κB, the protein expression and secretion level of MCP-1 and the change of EMT marker including a-SMA, E-cadherin, Fibronectin, Vimentin and ZO-1. (3) Investigating ATG whether or not inhibits the protein expression of MCP-1 and Reverses EMT-like phenotypic changes in vitro to reveal that ATG inhibits the overexpression of MCP-1, inhibits TGF-β1-drivened EMT and ultimately inhibits EMT through ROS/ERK1/2 MAPK/NF-κB pathway.Results:(1) In the present study, we first showed that The effects of ATG on RIF in rats subjected to UUO were also investigated. The mRNA levels of MCP-1,IFN-y, IL-1β and TNF-a increased notably in the obstructed kidney and significantly decreased in the presence of ATG. The expression of superoxide dismutase-2 (SOD2) were upregulated in the obstructed kidney and substantially suppressed by ATG treatment.Furthermore, the tubular EMT-like phenotypic changes could be counteracted after administration of ATG.(2) TGF-β1 induced ROS generation by up-regulating the expression of NADPH oxidase 2,4 and their regulatory proteins in human TECs. TGF-β1 induction of ROS production and hydrogen peroxide (H2O2)-induced oxidative stress contributed to the activation of ERK1/2/NF-κB signaling pathway and induction of NF-KB-directed MCP-1 overexpression. Moreover, overexpression of recombinant MCP-1 further worsened tubular EMT-like phenotypic changes compared with that in cells induced by TGF-β1 alone.(3) As a potential antioxidant and anti-inflammatory compound, ATG, exhibits protective effects against TGF-β1-drivened EMT mainly through inhibiting ROS/ERK1/2 MAPK/NF-κB pathway and consequently decreasing the overexpression of MCP-1.Conclusion:MCP-1 may be a potential therapeutic target for tubular interstitial inflammation and EMT-like phenotypic changes. ATG counteracted TGF-β1-induced EMT-like phenotypic changes in human TECs via inhibiting the MCP-1 overexpression.Arctigenin may serve as a new therapeutic candidate to inhibit or even reverse tubular EMT-like phenotypic changes during the process of renal RIF. |
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