A Study On The Protective Effects Of HSYA On Podocyte Injury By Regulating M1/M2 Polarization Induced By High Glucose Based On Gene Expression Of Macrophage Phenotype

1 Objective:1.1 Gene expression regulated by M1/M2 transcriptional signals in macrophagesMany studies show that the occurrence of DN(diabetic nephropathy)is closely related to the inflammatory process in which macrophages polarization plays important roles.The M1 subtype(classical activated macrophages)with pro-inflammatory phenotype participates in inflammatory reaction and causes tissue damage.The M2 subtype(alternatively activated macrophages)inhibits inflammation and repairs tissue damage,and it is called anti-inflammatory phenotype.The phenotypes of M1/M2 in renal tissue of DN are out of balance,and infiltration in kidney is mainly from M1.Intervention with M1/M2 phenotypes is a research hotspot in DN treatment.Based on GEO(gene expression omnibus)database,our study will first explore the critical gene expression when macrophages polarize to different types.1.2 The protective effects of HSYA on podocyte injury by regulating high glucose-induced RAW264.7 polarizationAccording to current literatures,HSYA(Hydroxysafflor yellow A)can treat diabetes clinically.Our previous work showed that HSYA inhibits the M1 subtype induced by LPS(lipopolysaccharide).In the development of DN,podocyte injury is most severe.M1 and M2 subtypes display totally different effects on podocyte injury.What is the effect of HSYA on podocyte injury induced by high glucose?Which role of HSYA on M1/M2 will be play on podocyte injury?Based on our previous work and the results from the first part of this study,the podocyte injury model in vitro and M1/M2 induced by high glucose in vivo were established.The direct and indirect effects of HSYA via M1/M2 on podocyte injury were explored then.2 Methods:2.1 Gene expression regulated by M1/M2 transcriptional signals in macrophages(1)In Array Express(https://www.ebi.ac.uk/arrayexpress),the data sets were searched by the following keywords:macrophage differentiation,macrophage polarization,macrophage phenotype,macrophage subtype,M0(resting state of macrophages),M1 and M2.(2)The obtained data sets from(1)were screened by Accession,Organism,Cell type,Treatment and so on.The screening criteria of data sets were as follows:a.Organism was from mouse,b.Cell type was from bone marrow-derived macrophages,c.In treatment groups,M0 was used as control against M1 and M2.(3)The final selected data set from(2)was linked to the corresponding GEO database(www.ncbi.nlm.nih.gov/geo/),and the DEGs(differentially expressed genes)of M1/M2 typing were analyzed by GEO2R(http://www.ncbi.nih.gov/geo/geo2r),in which M0 was the control and M1/M2 were the experimental groups.The DEGs with adj.P<0.01 were statistically significant.The log FC>0 was regarded as up-regulated,and Log FC<0 down-regulated.(4)Based on DEGs from(3),the GO(The Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)were used to analyze the biological processes and functions of DEGs.2.2 The protective effects of HSYA on podocyte injury by regulating high glucose-induced RAW264.7 polarization(1)Griess assay was used to determine the glucose concentration which can induce macrophages to M1.(2)CCK8 method was used to define the safe concentration of HSYA for macrophages cultured in high concentration of glucose.(3)Western Blot and qPCR measured the protein and mRNA expression of TNF-α(tumour necrosis factor-α),iNOS(inducible nitric oxide synthase),the biomarker of M1 polarization,and the expression of CD206(mannose receptor 1)and Arg-1(arginase 1),the biomarker of M2 polarization.The effects of HSYA were studied on abovementioned biomarkers,and kaempferol was used as positive control.(4)The podocytes were cultured with high concentration of glucose and(high glucose)MS(high glucose treated macrophage supernatants),and the survival rates of podocytes in different culture conditions were determined by MTT assay.Finally,the contents of TNF-α and IL-1β in podocyte supernatant were detected by Elisa.3 Results:3.1 Gene expression regulated by M1/M2 transcriptional signals in macrophages(1)According to the screening criteria,the data set GEO77425 titled“Control of the inflammatory macrophage transcriptional signature by miR-155”was obtained for further analysis.(2)The comparison between M1 and M0 showed that IL-1β and TNFR(TNF receptor)family were all up-regulated.According to the value of log FC,IL-1β was listed as the second and IL-1α the tenth in all upregulated biomarkers for M1.The main up-regulated biomarkers for M2 were Arg-1,Retnla,Rnase2A,etc.According to the log FC value,Arg-1 was ranked as the first in all up-regulated biomarkers.(4)The results of GO and KEGG showed that M1/M2 polarization-related DEGs were enriched in the cytoplasm of CC(cell component),protein binding of MF(molecular function),cell cycle and inflammatory response of BP(biological process),and the TNF signaling pathway of KEGG3.2 The protective effects of HSYA on podocyte injury by regulating high glucose-induced RAW264.7 polarization(1)The 33.3 mM glucose induced RAW264.7 cells polarized to M1 subtype.(2)Compared with the 11.1 mM control and 33.3 mM glucose model,HSYA at 100-300 μM did not significantly affect the growth rates of RAW264.7 cells in 33.3 mM glucose(P<0.05).(3)Compared with the 11.1 mM control,33.3 mM glucose significantly increased the protein and mRNA expression of iNOS,TNF-α and decreased the protein and mRNA expression of Arg-1 and CD206(All:P<0.05).Compared with the 33.3 mM glucose model,HSYA could significantly increased the protein and mRNA expression of CD206(P<0.05),and significantly decreased the protein and mRNA expression of iNOS and TNF-α(Both:P<0.05).(4)The survival rate of podocyte in 33.3 mM glucose model decreased significantly compared with that in 11.1 mM glucose control((80.1±11.2)%vs(100.0±0.0)%,P<0.05).Compared with the 33.3 mM model,the podocyte survival rate of HSYA at 100μM was(92.5±10.5)%,which was significantly higher(P<0.05).(5)The survival rate of podocyte decreased to(57.3±1.7)%when cultured with(high glucose)MS,which was significantly lower than that of(control)MS(95.7 ± 4.0)%(P<0.05).Compared with(high glucose)MS,the survival rates of(HSYA 100 μM)MS and(HSYA 200 μM)MS)increased,among which(HSYA 100 μM)MS)showed significantly increased effect(P<0.05).(6)Compared with the control group,high concentrations of glucose and(high glucose)MS significantly increased the contents of TNF-α and IL-1β in podocytes(All:P<0.05).The 100 μM and 200 μM HSYA,(HSYA 100 μM)MS and(HSYA 200 μM)MS)significantly decreased TNF-α and IL-1β compared with high glucose and(high glucose)MS(All:P<0.05).4 Conclusions:4.1 Gene expression regulated by M1/M2 transcriptional signals in macrophages(1)IL-1 and TNF are critical biomarkers for M1 subtype,and Arg-1,etc.for M2.(2)M1 and M2 phenotypes are related to inflammatory and immune processes in vivo.4.2 The protective effects of HSYA on podocyte injury by regulating high glucose-induced RAW264.7 polarization(1)The 100 μM HSYA showed a direct protective effect on podocyte injury induced by 33.3 mM glucose.(2)The 100-200 μM HSYA played indirect protecting roles in podocytes injury via regulating the protein and mRNA expression of iNOS,TNF-α,Arg-1 and CD206 in M1/M2 polarization induced by 33.3 mM glucose.(3)The above-mentioned direct and indirect effects were all related to the inhibitory effects of HSYA on TNF-α and IL-1β contents.