| ObjectiveThe increasing resistance of bacteria has become one of the biggest public concerns worldwide.There are about 15 million deaths associated with bacterial infection every year,in which most of them occur in developing countries.However,the development of new antibacterial drugs lags behind the emergency of drug resistance.Enhancing host immune responses has become a new focus in the field of anti-infection.The rising number of immunocompromised patients,such as those with transplantation or cancer,further increases the incidence and mortality of serious infections.Colistin is the last line of defense against the multidrug-resistant Gram-negative bacteria.China Food and Drug Administration has approved the colistin injection in April,2018.It has been predicted that the clinical use of colistin in China would be rapidly increased.However,the colistin-mduced nephrotoxicity will become an obstacle for clinicians.Therefore,it is urgently necessary to reduce or eliminate its toxicity.In the previous research,we have found that colistin may activate the innate immunity and prevent infection of Caenorhabditis elegans.Accordingly,we aimed to investigate the immunomodulatory properties and nephrotoxicity of colistin and explore the key signaling pathways in higher organisms in order to expand the application of colistin in immunomodulation and reduce its nephrotoxicity.Methods(1)The immunomodulatory properties of colistin were investigated.We detected the expressions of miRNAs between colistin-stimulated and unstimxulated primary macrophages of rats and predicted potential target genes using miRNA microarray.Moreover,we evaluated the potential biological functions and the important signaling pathways for eolistin using GO enrichment analysis and pathway bioinformatic analysis.(2)The immune activation for colistin was evaluated at the animal level.We established a non-invasive rat model though endotracheal instillation with pulmonary infection.Colistin was applied to assess the effect of colistin on rats by detecting the total amount of leukocytes,differences in serum inflammation factors,bacterial loading of lung tissue and lung histopathological changes.IL-1β、IL-6、TNF-α in the lung tissue were determined using immunohistochemistry.Based on the previously certified colistin immunostimulatory effects associated with the p38/MAPK pathway,we compared whether the immunostimulatory effects of colistin at the animal level were altered by p38 inhibitors.(3)We conducted a mechanism research of colistin-caused nephrotoxicity.We used miRNA microarray and high-throughput sequencing technology to detect the expression levels of miRNAs and mRNAs in colistin-stimulated or unstimulated rat renal tubular epithelial cells.In addition,we identified target genes of differentially expressed miRNAs,which predicted the differences in expressions of target genes and mRNA sequencing.The genes were subjected to GO analysis and pathway analysis,and bioinformatic analysis was conducted to study the relevant pathways involved in nephrotoxicity induced by colistin.(4)The changes of autophagy-related proteins in rat renal tubular epithelial cells NRK-52E after colistin stimulation were evaluated at the cellular level.Cell viability was assessed after treatment with different concentrations of colistin using the MTT method.The levels of autophagy markers(LC3-Ⅱ and LC3-Ⅰ)and autophagy-related proteins(beclin-1,Atg5 and caspase-3)were detected by Western blotting analysis.In addition,the autophagy-related phosphorylation of LC3-Ⅱ,LC3-I,m-TOR,beclin and Atg5 was assess by using the phagocytic rapamycin and autophagy inhibitor chloroquine.The effects of colistin on autophagy of NRX-52E cells were also studied.Relevant effects and preliminary discussion on its mechanism of action were given.Results(1)A total of 28 miRNAs were significantly altered between colistin-stimulated and unstimulated rat primary macrophages.GO analysis showed that gene functions,such as MAPK pathway,inflammatory response,autophagy and interleukin activity,were significantly affected.Pathway analysis also found that mTOR signaling pathway,B cell receptor signaling pathway,MAPK signaling pathway and T cell receptor signaling pathway were also significantly affected.(2)The results showed that the total number of white blood cells,neutrophil counts,and the level of serum inflammatory cytokine IFN-γ were increased in the colistin pneumonia group eompared with the pneumonia eontrol group,while the levels of IL-6 and IL-1β were decreased.Moreover,the amount of bacterial load on the lung tissue was significantly decreased.Pathological morphology after HE staining showed that the hyperemia,edema and neutrophil infiltration in the alveolar space and interstitial space of the colistin-pneumonia group were alleviated compared with the pneumonia control group.Immunohistochemistry after HE staining showed that the protein expression levels of IL-6 and TNF-α were significantly reduced in colistin pneumonia group compared with the pneumonia control group.After the p38 inhibitor treatment,the amount of bacteria in the lungs of rats administered with colistin was not significantly different from that in the colistin pneumonia group.(3)A total of 856 differentially expressed genes were identified between colistin-stimulated and unstimulated rat renal tubular epithelial cells,including 21 differentially expressed miRNAs.GO analysis and KEGG analysis showed that MAPK pathway,mTOR signaling pathway,Wnt signaling pathway and autophagy regulation were significantly changed.(4)After the colistin treatment,the ratio of autophagic markers LC3-Ⅱ/LC3-Ⅰ in NRK-52E cells was significantly increased at 3-6 h.Their protein expressions were decreased to the same level as the control group at 12-24 h.The autophagy-related protein beclin-1 was significantly decreased at 24 h,and Atg5 did not change at all time points.The level of apoptosis-related protein cleaved-caspase-3 was significantly increased at 12-24 h.Administration of autophagy inducer rapamycin and autophagy inhibitor chloroquine showed that LC3-Ⅱ/LC3-Ⅰ in each group was significantly higher than that in the control group.Moreover,the level of Phosphorylated m-TOR was significantly decreased in colistin group and rapamycin group,and the expressions of beclin and Atg5 at the protein levels were not significantly changed.Conclusions(1)After colistin stimulation,the inflammatory response,MAPK pathway,T cell,B cell regulation,and partial interleukin signaling were significantly altered.The MAPK pathway interacted with many inflammatory and immune response pathways,the MAPK pathway was the critical pathway downstream of the entire network,and the MAPK pathway might be one of the key pathways for colistin-induced immunomodulatory effects.(2)Colistin could increase the levels of white blood cells and neutrophils in rats,regulate some pro-inflammatory cytokines,activate rat autoimmune function to clear pathogens,and improve lung tissue damage caused by MRS A.However,the effect of p38 inhibitor SB203580 was not evident in rat animal experiments,suggesting that the p38/MAPK pathway was not the only pathway in higher animals.(3)After colistin stimulation in NRK.52E cells,significant changes were observed in MAPK pathway,mTOR signaling pathway,Wnt signaling pathway and autophagy regulation,suggesting that the nephrotoxicity of colistin was closely related to the autophagy induced by mTOR pathway and the inflammatory effect induced by MAPK pathway as well as the Wnt pathway.(4)Colistin induced autophagy in NRK-52E in a short time(3-6 h)in a dose-and time-dependent manner.After long-term colistin treatment,autophagy was attenuated which might be associated with inhibition of mTOR phosphorylation,and cleaved-caspase-3 level was increased.Our findings suggested that the nephrotoxicity caused by colistin was related to autophagy.Colistin might have protective effect on early autophagy,which could prevent the process of apoptosis,decrease the level of autophagy after prolonged action,and trigger the cascade reaction of caspase activation,leading to kidney injury. |
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