| 1 Objective:Methylprednisolone(MP)is a commonly used glucocorticoid in clinical practice.It is currently widely used in various immune-mediated diseases such as membranous nephropathy(MN),Ig A nephropathy(Ig AN),ANCA-associated glomerulonephritis(AAGN),systemic lupus erythematosus(SLE),etc.As inflammatory factors,tumor necrosis factor(TNF)-α and interleukin(IL)-1β play a key role in the inflammatory process of AAGN,Ig AN and LN.Studies have shown that MP can inhibit the release of TNF-α and IL-1β through a variety of pathways,thereby reducing the inflammatory response in the body.MP exerts its powerful anti-inflammatory effect mainly through genomic effect and non-genomic effects.The genomic effect takes effect slowly but has a strong effect.In contrast,non-genomic effects were of short duration and weak effect.The genomic effect is the main way for MP to exert its pharmacological effects.The anti-inflammatory biological information of MP obtained from the gene expression omnibus(GEO)database will be the basis of this study.Macrophage is an important immune cell in the human body,which has a variety of functions.It is an important object for the study of cellular phagocytosis,cellular immunity and molecular immunity.Resting macrophages(M0)can polarize to a classically activated macrophage(M1)or an alternatively activated macrophage(M2)phenotype depending on the microenvironment.The two different phenotypes of macrophages showed opposite results on inflammatory factors such as TNF-α,IL-1β and inducible nitric oxide synthase(iNOS).Similarly,when various factors cause changes in the renal microenvironment,the innate macrophages in the kidney will also polarize into different phenotypes and participate in or suppress the inflammatory response.In summary,based on the GEO database,we aimed to investigate the effects of MP administration on M1 and M2 macrophage-associated biomarkers in normal rat kidneys at16 time points from 0.25 to 72 hours;Furthermore,an in vitro model of M1-type macrophages was established by lipopolysaccharide(LPS)to study the effect of MP on the phenotypic switching of M0/M1 macrophages and the expression of TNF-α,IL-1β and iNOS.2 Methods:2.1 To analyze the biological information related to the classification of macrophages in the kidney of normal rats treated with methylprednisolone(1)In the Array Express(https://www.ebi.ac.uk/arrayexpress),with methylprednisolone and kidney as keywords,retrieve the related data sets.(2)The selection criterion was the data set of normal kidney tissues treated with MP.(3)After selecting the data set that met the research criteria,the relevant data were imported into the GEO database and grouped and analyzed by GEO2 R.All the differentially expressed genes(DEGs)of normal rats at 16 time points from 0.25 to 72 hours after using MP were obtained compared with those after not using MP.DEGs defined as P < 0.05,and | log FC(a fold change)| > 0.(4)gene ontology(GO)database was used to analyze the biological processes involved in DEGs at different time points and the biomarkers when M0 polarization to M1/M2.The Kyoto Encyclopedia of Genes and Genomes(KEGG)website was used for signal pathway enrichment analysis.In the process of analysis,we mainly focused on the effect of MP on inflammation-related GO in normal rat kidney tissue.Among the inflammation-related biological information,we focused on whether MP could affect the possible polarization of M0 macrophages to M1/M2 macrophages.2.2 In vitro effect of methylprednisolone on LPS-induced typing of RAW264.7macrophages(1)CCK-8 was used to determine the toxicity of LPS at 0.01,0.05,0.10,0.25,0.50,1.00 μg/m L and MP at 5,10,20,and 40 μM to RAW264.7 macrophages,respectively.(2)Griess method was used to detect the concentration of NO in RAW264.7 cells cultured with 0.01,0.05,0.10,0.25,0.50,1.00 μg/m L LPS for 4,6,8,12,18,and 24 h,and to determine the optimal concentration and action time of LPS for M1 polarization of macrophages.(3)The M0 and M1 macrophages were treated with MP.PCR and Western Blot were used to detect the expression changes of m RNA and protein of the markers TNF-α,iNOS and IL-1β of M1 macrophages in different groups,and the expression of m RNA and protein of mannose receptor 1(CD206)and arginase 1(Arg-1)of M2 macrophages.(4)The supernatant of each group was collected,and the contents of IL-1βand TNF-α were detected by Elisa.3 Results:3.1 Methylprednisolone on DEGs associated with macrophage typing in normal rat kidneys(1)A total of 5 datasets were retrieved according to the screening criteria.It is entitled "Transcription profiling of rat kidney samples following treatment with the corticosteroid methylprednisolone over.several time intervals " dataset GSE1721 was included in this study.(2)In GSE1721,52 male Wistar rats with adrenal glands removed were divided into a control group(n = 4)and an experimental group(n = 48).The 48 rats in the experimental group were randomly divided into 16 groups(n = 3).Animals were sacrificed at 16 time points(0.25,0.5,0.75,1,2,4,5,5.5,6,7,8,12,18,30,48 and 72 hours),and bilateral kidneys were obtained for whole RNA collection and high-throughput analysis.(3)In the observation time of 72 hours,the number of DEGs was the highest at 4 hours,which was1203;The second was 748 at 6 hours.At 8 hours,it ranked third,with 402.The number of DEGs began to increase from 0.5 hours after MP treatment,gradually increased to the peak at 4 hours,and continued to increase until 12 hours,then leveled off,and stabilized below300 at 5 time points up to 72 hours.3.2 Effect of methylprednisolone on markers associated with M1 and M2 types in normal rat kidneysAfter the above DEGs data and literature analysis,TNF-α,iNOS,CD68,IL-1β,and IL-6 were screened as markers of M1 polarization,and Arg-1,IL-4,and IL-13 were markers of M2 polarization.In the study,the expression of each gene in the normal control group was set to 1 at each time point,and the results compared with the normal control group were as follows:(1)The highest expression fold of the M1 polarization markers TNF-α,IL-1β,IL-6,and iNOS occurred within 4 hours of MP administration: The gene expression of IL-1β,IL-6,TNF-α and iNOS increased 19.66,7.89,1.94 and 1.94 folds at0.5 h,0.5 h,0.5 h and 4 h,respectively.(2)The lowest expression levels of M1 markers were found at 5.5 hours after MP treatment: TNF-α decreased to 0.63-fold at 5.5 hours,IL-1β decreased to 0.98-fold at 5.5 hours,IL-6 decreased to 0.50-fold at 30 hours,iNOS decreased to 0.60-fold at 48 hours.(3)The highest expression fold of Arg-1,IL-4 and IL-13,the markers of M2 polarization,appeared 12 hours after MP treatment,and the increased fold was less than 1.72.(4)The lowest expression of M2 polarization markers was found within 6 hours after MP treatment: Arg-1 and IL-4 decreased to 0.63-fold and0.31-fold at 6 hours,and IL-13 decreased to 0.25-fold at 4 hours.(5)The gene folds of TNF-α,IL-1β,iNOS and Arg-1 were 0.71-fold,1.28-fold,1.51-fold and 0.64-fold at 18 hours,respectively.3.3 Results of GO biological process studies based on DEGsAccording to the findings of DEGs,information related to inflammation in the biological process of GO was analyzed 4,6,8,12,18,48,and 72 h.The results showed that:(1)The "Response to LPS" appeared for the first time at 4 hours and again at 48 hours.(2)Information related to the inflammatory response was described as "positive regulation of inflammatory response" at 6 hours and "cellular response to IL-1" at 12 hours.(3)The description of "Signal transduction" appeared at 4,48 and 72 h,respectively,and the P value was the lowest and the gene proportion was the highest at these three time points.(4)Descriptions of "Apoptotic processes","negative regulation of apoptotic processes" and "positive regulation of apoptotic processes",appeared within the first 8 hours of MP administration,respectively.3.4 Results of in vitro effects of methylprednisolone on M0 and LPS-induced M1macrophages(1)The optimal condition of RAW264.7 cells polarization to M1 induced by LPS at0.5μg/m L for 18 hours was determined.(2)The safe concentration of methylprednisolone was 5-20 μM.After RAW264.7 cells were treated with MP for 2,4,12,18,24,and 48 hours,the content of NO in the cell supernatant was detected by Griess method,and the content of NO was not significantly increased compared with the control group(P = ns).(3)Compared with the normal group,0.5μg/m L LPS significantly increased the protein and m RNA expression of TNF-α,iNOS and IL-1β(P < 0.05),and decreased the protein and m RNA expression of Arg-1 and CD206(P < 0.05).(4)Compared with the LPS model group,MP at 5,10 and 20 μM significantly decreased the protein and m RNA expressions of TNF-α,iNOS and IL-1β(P < 0.05),and significantly increased the protein and m RNA expressions of Arg-1 and CD206(P < 0.05).(5)In the cell culture supernatant,the content of IL-1β and TNF-α in the model group was significantly increased(P < 0.05),and the content of Il-1β and TNF-α was significantly decreased after MP treatment(P < 0.05).4 Conclusion:(1)The study based on the dataset GSE1721 showed that the number of DEGs in the kidney tissue of rats was the highest from 4 hours to 8 hours after MP administration,and leveled off after 12 hours to 72 hours.(2)The gene expression of M1 markers TNF-α,iNOS and IL-1β and M2 markers Arg-1,IL-4 and IL-13 in normal rat kidney tissue showed a different trend of increase or decrease with the time of MP treatment.It is noteworthy that at 6 h after MP administration,the expression of M1 type increases,while the expression of M2 decreases.(3)MP couldn’t induce the transformation of RAW.264.7 macrophages from M0 to M1 under the present study conditions.(4)MP could significantly reduce the m RNA and protein expression of TNF-α,iNOS and IL-1β,and significantly increase the m RNA and protein expression of Arg-1 and CD206 in LPS-induced RAW.264.7 macrophages,and MP could significantly inhibit the production of TNF-α and IL-1β by M1 macrophages.The above results appeared when MP was applied to M1 for 18 hours.(5)In summary,our conclusions are as follows: Firstly,the expression profile of M0 gene by MP above was not consistent with the results of the corresponding in vitro studies.Gene expression profiling showed an increase in M1 expression and a decrease in M2 expression within 6 hours after MP administration,but in vitro studies showed that MP did not have an effect on inducing M0 to M1 polarization.Secondly,MP could inhibit LPS-induced generation of TNF-α and IL-1β by M1 subtype,which was related to the effect of MP on regulating M1/M2 subtype. |
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