| ObjectiveTo explore the relationship between inflammatory response and autophagy in the process of acute kidney injury,and to explore the effect and mechanism of the tetramethylpyrazine ameliorating acute kidney injury through two aspects of in vivo and in vitro.MethodsEight-week-old male Sprague-Dawley rats were purchased.They were randomly divided into three groups.These three groups are respectively sham-operated group,ischemical reperfusion injury(I/R)group and TMP treatment group.The serum creatinine(SCr)and blood urea nitrogen(BUN)was measured using creatinine assay kits and urea assay kits.These two can be used to evaluate renal function.The test results were used to determine whether the model of acute renal injury was successful.HE staining and analysis showed severe morphological kidney injury.Cell death was demonstrated by TUNEL staining and analysis.Immunohistochemical staining,Western Blot analysis and Real-time PCR analysis showed the expression of NOD2,CD68,TNF-a,IL-6,MCP-1,autophagy-related protein Beclinl and ratio of LC3A/B-Ⅱ/Ⅰ in protein and mRNA levels.In vitro experiments were carried out by two models to mimic hypoxia conditions.One is made by incubating NRK-52E cells in different concentration of CoCl2.Another was conducted by OGD treatment in which NRK-52E cells were incubated in hypoxic environment.After the success of the cell model was constructed,Western Blot analysis showed the expression of inflammation-related protein and autophagy-related protein Beclinl and ratio of LC3A/B-II/I in protein.Cell death was demonstrated by TUNEL staining and analysis.Cell vitality was demonstrated by CCK-8 and analysis.ResultsCompared with sham-operated group,rats after I/R displayed higher elevation of serum creatinine(SCr)and blood urea nitrogen(BUN),which could be reduced by TMP treatment.Hematoxylin-eosin(HE)staining and analysis showed severe morphological kidney injury with loss of the brush border and tubule dilatation after I/R injury,which were lighten in TMP treatment groups;TMP also reduced cell death,as demonstrated by TUNEL staining and Caspase3/cleaved Caspase3 immunohistochemistry staining.TMP inhibited levels of proinflammatory mediators in renal tissue after I/R injury by real-time PCR analysis.Western Blot analysis showed that protein level of CD68 were also inhibited by TMP.Real-time PCR analysis and Western Blot analysis together with immunohistochemical staining showed the upregulation of NOD2 expression after I/R injury was inhibited by TMP treatment in mRNA and protein levels.In vitro experiments,under different concentration of CoCl2 treatment,upregulation of NOD2 was shown by Western Blot.The protein level of Beclinl and ratio of LC3A/B-II/I increased with lower concentration of CoCl2 and reduced with higher concentration.Western Blot analysis showed that TMP at 50 μM could inhibit the upregulation of NOD2 expression.The protein level of autophagy-related protein Beclinl and ratio of LC3A/B-Ⅱ/Ⅰ after CoCl2 treatment with highter concentration increased significantly with TMP treatment.The inhibitory effect of TMP treatment on protein level of NOD2 was weaken when autophagy was inhibited by Western Blot analysis.Real-time PCR analysis showed TMP treatment inhibited levels of proinflammatory mediators,which were blocked when autophagy was inhibited.After autophagy was inhibited,the effect of TMP on reducing cell death was weaken by TUNEL assays.Cell counting kit-8(CCK-8)assays also showed that cell viability in response to TMP decreased after autophagy was inhibited by CQ.ConclusionThe upregulation of NOD2 expression in acute kidney injury,together with a series of inflammatory mediators,exacerbate the renal ischemia-reperfusion injury.The induction of autophagy is restrained at the same time.The application of tetramethylpyrazine can induce autophagy and reduce inflammation reaction.The induced autophagy can reduce the inflammatory reaction mediated by NOD2 to protect the kidney in the process of acute kidney injury.This provides a new way to prevent the deterioration of renal function and to treat acute renal injury. |
