| Part1Anti-inflammatory activity and regulation on inflammatory signal transduction pathway of fluorofenidone in HK-2cellsBackground:Renal fibrosis is the final common pathway and main pathogenic basis of a wide variety of chronic kidney diseases. The pathogenesis of renal fibrosis is a progressive process that ultimately leads to substantial renal fibrosis, renal damage and renal failure whatever the causes of chronic kidney disease. Therefore, renal fibrosis continues to remain a challenging and perplexing problem for both researchers and clinicians. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD), a novel anti-fibrotic agent, is developed by our team. Our data showed that AKF-PD exerts a strong antifibrotic effect, as shown in experimental liver fibrosis, lung fibrosis and renal fibrosis in vivo. However, the mechanism underlying the antifibrotic effect of AKF-PD in renal disease remains to be elucidated. Our recent results showed that AKF-PD significantly reduced the expression and activity of NADPH oxidase, and inhibited ROS generation, the apoptosis of renal tubular epithelial cell, as well as the proliferation of renal fibroblast. In addition, AKF-PD significantly downregulated the expression of pro-fibrotic cytokines, and consequent reduced interstitial matrix deposition. Accumulated evidence indicates that inflammation is reported to be essential for the development of renal fibrosis and is the early event in renal fibrosis. And the effect of fluorofenidone on inflammation in renal local cells is unkown. Renal fibrosis is a complex pathophysiological process, involving in the participation and interaction of various cells, including renal resident cells and inflammatory cells. On the renal damages induced by a variety of risk factors, renal resident cells exhibit proliferation, activation and release cytokines/chemokines from non-lethal damage; on the other hand, renal resident cells induce necrosis, apoptosis from lethal damage which activate the inflammatory cells around them, further promote the release of cytokines/chemokines, amplify inflammation and aggravate renal injury.Tubular epithelial cell has not only biological but also immunomodulatory actions. As the main part of renal resident cells, tubular epithelial cells play an important role in renal microenvironment. The expression of proinflammatory cytokine TNF-a is increased in renal interstitial fibrosis and the increase of TNF-a level aggravate the inflammatory response and renal damages.Objective:The aim of this study was to investigate the anti-inflammatory effect of fluorofenidone on IL-6, IL-8and MCP-1expressions in HK-2cells and the underlying mechanism.Methods:Firstly, HK-2cells were respectively pretreated with2 mM AKF-PD or1μM dexamethasone for1hour and then exposed to10ng/ml TNF-a for24hours. Cells supernatants were collected and stored at-80℃until used for cytokine determination. The levels of IL-6, IL-8and MCP-1of HK-2cells were determined using commercially available ELISAkits.HK-2cells were respectively pretreated with2mM AKF-PD for24hours,10μM MAPK inhibitors (PD98059, SP600125, SB-203580) for1hour, and then exposed to TNF-a (lOng/ml) for15min before collection cell protein. The expression of p-ERK1/2, p-p38and p-JNK were determined by Western Blot.HK-2cells were respectively pretreated with2mM AKF-PD for24hours,10μM NF-kB inhibitor BAY11-7082for1hour, and then exposed to TNF-a (lOng/ml) for60min before collection cell protein. The expression of p-IκBa was determined by Western Blot.HK-2cells were pretreated with2mM AKF-PD for24hours, and then exposed to TNF-a (lOng/ml) for60min before collection nuclear protein. The expression of P65was determined by Western Blot.After transfected with reporter plasmid pNF-icB-Luc, HK-2cells were respectively pretreated with2mM AKF-PD or1μM dexamethasone for24hour and then exposed to10ng/ml TNF-a for60min. After treatment, luciferase activity was assessed using a luciferase assay substrate according to manufacturer’s instructions. Result:In TNF-a stimulated HK-2cells, expression of IL-6, IL-8and MCP-1in supernatant were significantly increased (p<0.05). AKF-PD and DEX markedly attenuated the elevation of IL-6, IL-8and MCP-1expression (P<0.05).TNF-a significantly increased the expression of p-ERK1/2after15min stimulation, AKF-PD and inhibitor PD98059inhibited ERK1/2phosphorylation (p<0.05). TNF-a significantly increased the expression of p-P38after15min stimulation, AKF-PD and inhibitor SB203580inhibited P38phosphorylation (p<0.05). TNF-a significantly increased the expression of p-JNK after15min stimulation, AKF-PD and inhibitor SP600125inhibited JNK phosphorylation (p<0.05).TNF-a significantly raised the expression of p-IκBa after60min stimulation, inhibitor BAY11-7082inhibited the phosphorylation IκBa (p <0.05), AKF-PD exerted no effect on the phosphorylation of IκBa.After stimulation of TNF-a for60min, the expression of P65in nucleus elevated obviously. AKF-PD significantly inhibit the nuclear translocation of P65(p>0.05)After stimulation of TNF-a for60min, the luciferase level of NF-kB in HK-2cells was increased. Both AKF-PD and DEX significantly inhibit NF-kB transcriptional activity.Conclusion:(1) AKF-PD attenuated TNF-a-stimulated IL-6, MCP-1and IL-8protein expression in HK-2cells. (2) AKF-PD attenuates inflammation in HK-2cells via MAPK signaling pathway.(3) AKF-PD attenuates inflammation in HK-2cells via NF-kB signaling pathway. Part2Anti-inflammatory activity and regulation on inflammatory signal transduction pathway of fluorofenidone in macrophagesBackground:The pathogenesis of renal fibrosis is complex. Accumulation of leukocyte in the renal interstitium is a prominent feature of renal inflammation and subsequent renal fibrosis. Macrophages are a kind of multi-functional inflammatory cells and are central mediators of immune defense and immune dysfunction, contributing to renal inflammation. It was demonstrated that renal fibrosis is associated with the number of macrophages infiltration. There are also a large number of reports that inhibition of macrophages infiltration is found to reduce interstitial fibrosis.Renal resident cells death must occur in the inflammatory sites. Renal resident cells induce necrosis from lethal damage during the initiation phase of renal fibrosis. Necrotic cells and their products will activate the inflammatory cells around them, further promote the release of cytokines/chemokines, amplify inflammation and aggravate renal injury. But the effect of AKF-PD on necrotic cells-induced cytokines/chemokines expressions in macrophages remains unknown.Objective:The aim of this study was to investigate the anti-inflammatory effect of fluorofenidone on TNF-a and MCP-1expressions in macrophages and the underlying mechanism.Methods:Firstly, macrophages were stimulated with necrotic cells with concentrations from1.5×106to12×106cells/ml for18hours. And then the level of TNF-a in the medium was detected using ELISA.Macrophages were pretreated with2mM AKF-PD for1hour and then incubated with necrotic cells (6×106cells/ml) or lipopolysaccharide (LPS,500ng/ml) for18hours. After treatment, cells supernatants were collected and the levels of TNF-a and MCP-1were determined using ELISA.Macrophages were pretreated with2mM AKF-PD for24hours, and then exposed to necrotic cells (6×106cells/ml) for15min before collection cell protein. The expression of p-ERK1/2and p-p38were determined by Western Blot.Macrophages were pretreated with2mM AKF-PD for24hours, and then exposed to necrotic cells (6×106cells/ml) for60min before collection cell protein. The expression of IκBa was determined by Western Blot.Macrophages were pretreated with2mM AKF-PD for24hours, and then exposed to necrotic cells (6×106cells/ml) for60min before collection nuclear protein. The expression of P65was determined by Western Blot.Result:Necrotic cells triggered a large amount of TNF-a, and peak induction of TNF-a was observed at6×106cell/ml in macrophages (P<0.05).In necrotic cells (6×106cells/ml) or LPS (500ng/ml) stimulated macrophages, expression of TNF-a and MCP-1in supernatant were significantly increased (p<0.05). AKF-PD markedly attenuated the elevation of TNF-a and MCP-1expressions (P<0.05).Necrotic cells (6×106cells/ml) significantly increased the expression of p-ERK1/2after15min stimulation in macrophages, and AKF-PD inhibited the phosphorylation ERK1/2(p<0.05); Necrotic cells (6×106cells/ml) significantly increased the expression of p-P38after15min stimulation in macrophages, and AKF-PD inhibited the phosphorylation P38(p<0.05).Necrotic cells (6×106cells/ml) significantly reduced the expression of IκBa after60min stimulation in macrophages (p<0.05), AKF-PD exerted no effect on the degradation of IκBa.After stimulation of necrotic cells (6x106cells/ml) for60min, the expression of P65in nucleus elevated obviously in macrophages. AKF-PD significantly inhibit the nuclear translocation of P65(p>0.05).Conclusion:(1) AKF-PD attenuated necrotic cells-stimulated TNF-a and MCP-1protein expression in macrophages.(2) AKF-PD attenuates inflammation in macrophages via MAPK signaling pathway.(3) AKF-PD attenuates inflammation in macrophages via NF-κB signaling pathway. |
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