Mechanism Of Podocyte Pyroptosis Mediated By Exosomal MicroRNA Derived From Glomerular Mesangial Cells In Diabetic Nephropathy And Intervention Of Tongluo Yishen Formula

Objective:To observe the effect of Tongluo Yishen Formula on the expression of glomerular mesangial cells(GMCs)derived exosomal microRNA(miRNA),and explore the mechanism of inhibiting podocyte pyroptosis by regulating the expression of exosomal miRNA in GMCs.Methods:Experiment 1:Differential miRNA expression profile analysis,target prediction,and functional enrichment of GMCs derived exosomes cultured in high glucose1.Rat GMCs were randomly divided into two groups,namely,the normal group(NG,5.6 mmol/L glucose)and the high glucose group(HG,30 mmol/L glucose).After 24 hours of culture,the supernatant of the two groups of cells was collected and exosomes was extracted by ultracentrifugation.Screening and analysis of differential miRNA expression profiles using high throughput sequencing technology.2.Prediction of target genes for differential miRNAs using miRanda and RNAhybrid databases for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis to explore the main biological functions and signaling pathways of differential miRNAs.3.The differential miRNAs obtained by sequencing were subjected to RT-PCR validation to ultimately obtain statistically significant miRNAs.4.The two groups of exosomes were labeled with PKH67 staining and co cultured with podocytes for 24 hours to observe the phagocytosis of the exosomes by podocytes.Experiment 2:Effects of GMCs derived exosomes on podocyte pyroptosis and intervention of Tongluo Yishen Formula1.Rat GMCs were randomly divided into: normal group: 5.6 mmol/L glucose+10%normal rat serum;high glucose group: 30 mmol/L glucose+10% normal rat serum;exosomes inhibitor group: 30 mmol/L glucose+exosomes inhibitor(GW4869 10 μM)+10% normal rat serum;Tongluo Yishen Formula group: 30 mmol/L glucose+10%Tongluo Yishen Formula medicated serum;miR-363-3p inhibitor group: 30 mmol/L glucose +miR-363-3p-inhibitor+10% normal rat serum;miR-200c-3p inhibitor group: 30mmol/L glucose+miR-200c-3p inhibitor+10% normal rat serum,after 24 h incubation,the cell supernatant was collected and the groups of exosomes were extracted by ultracentrifugation.2.Each group of exosomes was co cultured with podocytes at a concentration of 50μg/ml and divided into exosomes blank group(NO-EXO group): podocytes without exosomes;exosomes normal glucose group(NG-GMCs-EXO group): podocytes incubated with GMCs derived exosomes treated with 5.6 mmol/L glucose;exosomes high glucose group(HG-GMCs-EXO group): podocytes incubated with GMCs derived exosomes treated with high glucose;exosomes inhibitor group(GW4869+HG-GMCs-EXO group):podocytes incubated with high glucose GMCs derived exosomes treated with GW4869;exosomes Tongluo Yishen Formula group(TLYSF+HG-GMCs-EXO group): podocytes incubated with high glucose GMCs derived exosomes treated with Tongluo Yishen Formula;exosomes miR-363-3p inhibitor group(miR-363-3p-inhibitor+HG-GMCs-EXO group): podocytes incubated with high glucose GMCs derived exosomes treated with miR-363-3p inhibitor;exosomes miR-200c-3p inhibitor group(miR-200c-3p-inhibitor+HG-GMCs-EXO group): podocytes incubated with high glucose GMCs derived exosomes treated with miR-200c-3p inhibitor,collected after 24 hours of culture.3.Hoechst33342/PI fluorescence staining was used to observe the integrity of podocyte membrane.4.Transmission electron microscopy was used to observe the ultrastructure and podocyte pyroptosis.5.Detection of mRNA expression levels of NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β in rat podocytes by RT-PCR.6.Western Blot was used to detect the expression levels of NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β proteins in rat podocytes.Results:Experiment 11.A total of 10 differential miRNAs were obtained by sequencing,all of which were upregulated.2.Using biological information analysis,GO and KEGG enrichment analysis was performed on differential miRNA target genes.The GO results suggest that it may be enriched in processes such as metabolism,synaptic transmission,and cell differentiation that regulate the pathogenesis of diabetic nephropathy(DN);KEGG pathway analysis showed that DN pathogenesis is mainly related to histidine metabolism,synaptic vesicle cycle,apoptosis,type II diabetes,NF-κB signaling pathway,MAPK signaling pathway,Wnt signaling pathway,NOD-like receptor signaling pathway,TNF signaling pathway,cAMP signaling pathway,cGMP-PKG signaling pathway,Toll-like receptor signaling pathway,etc.3.The results of co culture of podocytes with PKH67 labeled exosomes showed that podocytes could successfully ingest exosomes.Experiment 21.The results of Hoechst33342/PI fluorescence staining showed that compared with the NO-EXO group,the number of PI positive cells in the HG-GMCs-EXO group was significantly increased(P<0.01),indicating an increase in the proportion of pyrolytic cells.Compared with the HG-GMCs-EXO group,the proportion of pyroptosis in the TLYSF+HG-GMCs-EXO group,NG-GMCs-EXO group,GW4869+HG-GMCs-EXO group,miR-363-3p-inhibitor+HG-GMCs-EXO group,and miR-200c-3p-inhibitor+HG-GMCs-EXO group significantly decreased(P<0.01).2.The results of transmission electron microscopy showed that in the NO-EXO group,the morphology and structure of cells were intact,the cell membrane was smooth and complete,the structure of organelles was basically normal,the morphology and structure of mitochondria were normal,and there were no cavity changes;In the HG-GMCs-EXO group,the typical morphological features were cell swelling and deformation,microvilli shedding on the surface of the cell membrane,membrane integrity destruction,accompanied by the outflow of cell contents,expansion of the rough endoplasmic reticulum,and formation of mitochondrial cavities,which were typical of pyroptosis.Compared with the HG-GMCs-EXO group,the TLYSF+HG-GMCs-EXO group had a normal cell structure,no swelling or deformation,and the cell membrane was basically intact,with a slight degree of mitochondrial damage;The podocyte damage in the NG-GMCs-EXO group,GW4869+HG-GMCs-EXO group,miR-363-3p-inhibitor+HG-GMCs-EXO group,and miR-200c-3p-inhibitor+HG-GMCs-EXO group was significantly reduced.3.The expression of NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β mRNA in rat podocytes detected by RT-PCR showed that compared with the NO-EXO group,the HG-GMCs-EXO group had NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β mRNA expression was significantly upregulated(P<0.01).Compared with the HG-GMCs-EXO group,the TLYSF+HG-GMCs-EXO group had NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β mRNA expression was down regulated(P<0.01);the NG-GMCs-EXO group,GW4869+HG-GMCs-EXO group,miR-363-3p-inhibitor+HG-GMCs-EXO group,and miR-200c-3p-inhibitor+HG-GMCs-EXO group NLRP3,GSDMD,Caspase-1,IL-18,IL-1β mRNA expression decreased to varying degrees(P<0.05 or P<0.01).4.Western Blot assay of NLRP3,GSDMD,Caspase-1,IL-18,IL-1β protein expression in rat podocytes showed that NLRP3,GSDMD,Caspase-1,IL-18,IL-1βprotein expression levels were significantly upregulated in the HG-GMCs-EXO group compared with the NO-EXO group(P<0.01).Compared with the HG-GMCs-EXO group,the expression levels of NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β protein expression were significantly downregulated in the TLYSF+HG-GMCs-EXO group(P<0.01);the NG-GMCs-EXO group,GW4869+HG-GMCs-EXO group,miR-363-3p-inhibitor+HG-GMCs-EXO group and miR-200c-3p-inhibitor+HG-GMCs-EXO group all had different degrees of reduction in NLRP3,GSDMD,Caspase-1,IL-18,IL-1β protein expression levels(P<0.05 or P<0.01).Conclusions:1.Crosstalk exists between GMCs and podocytes in high glucose environment,promoting DN progression,and the mechanism of action may be through upregulation of exosomal miR-363-3p and miR-200c-3p expression,promoting podocyte pyroptosis.2.Tongluo Yishen Formula downregulates NLRP3,GSDMD,Caspase-1,IL-18,and IL-1β mRNA and protein expression,improve podocyte pyroptosis,improve renal damage in DN,and protect renal function.