| Research Background:Acute kidney injury(AKI)refers to a serious clinical syndrome with a sudden decline in renal function,which is caused by a variety of factors.The occurrence of AKI is often accompanied by prolonged hospitalization,increased medical costs,increased morbidity and mortality.There are many causes of AKI,and sepsis is one of the most common causes.The pathogenesis of septic AKI is very complicated.Many previous researches have found that its mechanism including,renal hemodynamics changes,insufficient renal blood perfusion,release of inflammatory mediators,oxidative stress,microcirculatory dysfunction and apoptosis.In the above pathogenesis of septic AKI,excessive release of inflammatory mediators is a major risk factor for AKI in sepsis.Therefore,inhibition of inflammatory response may be one of the strategies for improving sepsis-induced AKI.Zinc finger E-box binding homeobox 2(ZEB2)is an important gene transcriptional repressor and contains most of the functional domains that interact with various transcriptional co-effectors.ZEB2 is not only expressed in the nucleus but also in the cytoplasm.ZEB2 is involved in the inflammatory response of cells and the process of metabolic regulation of cell formation,growth,differentiation,apoptosis,and embryo development.ZEB2 is abnormally expressed in human tumors such as liver cancer,breast cancer,gastric cancer and ovarian cancer.Inflammatory factors(such as tumor necrosis factor-?,TNF-?)promote the development of septic AKI.TNF-?,IL-6,TGF-? and some signaling pathways are also involved in the expression of ZEB2.Our previous study showed that ZEB2 could inhibit the release of inflammatory factors such as TNF-? and IL-6 from RAW264.7 and mouse primary macrophages.Objective:To investigate the expression of ZEB2 in septic AKI and its effects and mechanisms on LPS-induced secretion of inflammatory cytokines in HK-2 cells.Methods and results:In this study,we developed a model of septic AKI by intraperitoneal injection of LPS into C57BL/6 mice,and LPS stimulated HK-2 cells as a cell model.The content of this study is as follows:1.Expression of ZEB2 in LPS-induced septic acute kidney injury in miceC57BL/6 mice were routinely reared for 1 week and then randomly divided into two groups,Control group(10 mice)and LPS group(20 mice).LPS group mice were established with a septic AKI mouse model by intraperitoneal injection of LPS(15 mg/kg),and Control group mice were intraperitoneally injected with an equal amount of PBS.After 24 hours,the mice were anesthetized and blood were collected from the heart to detect the levels of Scr and BUN.HE staining of renal histopathology was performed for analysis of pathological changes.The expression of mRNA and protein of ZEB2,KIM-1,TNF-? and IL-6 were detected by qPCR and Western blot.Our results showed the serum levels of Scr and BUN were higher expression in LPS-treated mice.Histopathological results showed that treatment with LPS resulted in serious kidney injury,such as diffuse dilatation and congestion of small blood vessel in renal interstitial,renal capillary atrophy,glomerular balloon dilatation,diffuse epithelial edema in renal proximal convoluted tubule,star-shaped or obstructed renal tubular lumen.Meanwhile,the KIM-1 mRNA expression in kidney tissue from LPS-treated mice was significantly increased.These results indicated that LPS-induced mice septic acute kidney injury model was successfully established.Moreover,the mRNA and protein expressions of ZEB2 in LPS group were significantly decreased,while the mRNA and protein expressions of inflammatory factors TNF-? and IL-6 were significantly increased.2.The effect of ZEB2 on the secretion of inflammatory factors in LPS-induced HK-2 cells and its possible mechanismHK-2 cells were stimulated with different concentrations of LPS(0.1,1,10 ug/ml).After 24 hours,qPCR and Western blot were used to detect the mRNA and protein expression of ZEB2,TNF-? and IL-6 in HK-2 cells.siRNA and plasmid were used to silence and overexpress ZEB2.After that,we stimulate HK-2 cells with LPS(1 ?g/ml)for 24 hours,the mRNA and protein expression of TNF-? and IL-6 were detected by qPCR and Western blot.In addition,to further investigate the possible mechanism of ZEB2 on LPS-induced inflammatory factor secretion in HK-2 cells,Western blot was used to detect p-p65 and p-I?B? protein levels.The results showed that the mRNA and protein expression of ZEB2 was significantly decreased in LPS-stimulated HK-2 cells,while the mRNA and protein expressions of TNF-? and IL-6 were significantly increased.When ZEB2 was silenced,mRNA and protein expression of TNF-? and IL-6 were increased in HK-2 cells,and the levels of p-p65 and p-I?B? was also increased.When ZEB2 was overexpressed,the mRNA and protein expression of TNF-? and IL-6 were decreased in HK-2 cells,and the protein expression of p-p65 and p-I?B? is also decreased.Conclusion: 1.The expression of ZEB2 is decreased in septic AKI,which may be involved in the regulation of inflammatory factor secretion.2.ZEB2 attenuate LPS-induced secretion of inflammatory factors TNF-? and IL-6 in HK-2 cells by inhibiting NF-?B signaling pathway. |
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