Molecular Mechanism Of Autophagy In MDBK Cells By High Glucose And Lipopolysaccharide

Transport stress syndrome of beef cattle(TSSBC)is a systemic,multi-system disease,involving lung,intestine,kidney and other tissues and organs,with a morbidity of up to 80% and a fatality rate of over 40%,which seriously endangers the efficiency of beef cattle breeding.The occurrence of TSSBC is mainly related to long-distance transportation stress,which will cause the decline of body resistance,cause gramnegative bacteria infection,produce a large amount of lipopolysaccharide,and enter various tissues and organs along with blood transportation,eventually leading to multiple organ inflammatory injuries such as pneumonia and nephritis.In addition,the stress of long-distance transportation can also cause the increase of blood glucose concentration in beef cattle.Kidney is one of the important organs affected by the concentration of blood glucose and lipopolysaccharide.Whether the mechanism of renal inflammatory injury in beef cattle with TSSBC is related to the increase of blood glucose concentration caused by long-distance transportation stress and the increase of lipopolysaccharide concentration caused by secondary infection,that is,the double action of hyperglycemia(high concentration of glucose)and lipopolysaccharide remains to be confirmed.Autophagy is one of the important mechanisms of inflammation induced by high glucose/lipopolysaccharide.However,the effect of the combination of high glucose and lipopolysaccharide on autophagy remains unclear.MDBK cells are bovine renal epithelial cells,which are commonly used in cell experiments.Based on this,MDBK cells were treated with high glucose and lipopolysaccharide.Based on AMPK/m TOR,ERK1/2/m TOR,PI3K/AKT/m TOR and other cellular autophagy signaling pathways,the effects of high glucose,lipopolysaccharide and their combined effects on MDBK cells were studied.And the relationship between Notch3 receptor activation and m TOR protein expression;Transcriptomics was used to further verify the effects of high glucose and lipopolysaccharide and their combined effects on the related biological functions of MDBK cells,autophagy related genes and signaling pathways,and to initially reveal the molecular regulatory mechanism of high glucose and lipopolysaccharide on autophagy.1.Molecular mechanism of autophagy in MDBK cells induced by high glucoseThe effects of glucose concentration(5.5,10.5,15.5 and 25.5 m M)and appropriate glucose concentration time(0,3,6,12 and 24 h)on the expression of autophagy related genes and proteins in MDBK cells were studied.Results:(1)The expressions of Notch3,AMPK,ERK1/2,LC3 and Beclin1 were up-regulated in MDBK cells under 10.5,15.5and 25.5 m M glucose,and the expressions of PI3 K,AKT,m TOR and p62 were inhibited.Autophagy of MDBK cells could be induced after treatment with 25.5 m M glucose for 3h.(2)DAPT inhibited the expression of Notch3 protein,LC3 and Beclin1 gene and protein in MDBK cells induced by high glucose,and promoted the expression of m TOR and p62 gene and protein in MDBK cells to improve cell viability.(3)Com C and GDC-0994 inhibited the expression of LC3 B and Beclin1 gene in MDBK cells induced by high glucose,respectively,and promoted the expression of p62 gene.In conclusion,the autophagy of MDBK cells can be induced by the treatment of 25.5 m M glucose for more than 3h.The mechanism is related to high glucose promoting the up-regulation of Notch3 gene and protein,AMPK and ERK1/2 gene expression,and inhibiting the gene and protein expression of PI3 K,AKT and m TOR in MDBK cells.Autophagy signaling pathway Notch3/m TOR activation.2.Molecular mechanism of lipopolysaccharide induced autophagy in MDBK cellsTo investigate the effects of lipopolysaccharide concentration(0,1,2 and 5 μg/m L)and time(0,3,6,12 and 24 h)on the expression of autophagy related genes and proteins in MDBK cells.Results:(1)Lipopolysaccharide treatment with 1,2 and 5 μg/m L could activate the expressions of Notch3,AMPK,ERK1/2,LC3 and Beclin1 in MDBK cells,and inhibit the expressions of PI3 K,AKT,m TOR and p62.Autophagy of MDBK cells occurred after treatment with lipopolysaccharide at 5 μg/m L for 6 h.(2)DAPT inhibited Notch3 protein expression,LC3 and Beclin1 gene and protein expression,and increased cell viability,m TOR and p62 gene and protein expression in MDBK cells induced by lipopolysaccharide treatment at 5 μg/m L.(3)Com C and GDC-0994 inhibited the expression of LC3 B and Beclin1 gene in MDBK cells induced by lipopolysaccharide at 5μg/m L,respectively,and promoted the expression of p62 gene.In conclusion,lipopolysaccharide treated with 5 μg/m L for 6 h can induce autophagy in MDBK cells,and its mechanism is related to lipopolysaccharide promoting the up-regulation of Notch3 gene and protein,AMPK and ERK1/2 gene expression,and inhibiting the expressions of PI3 K,AKT and m TOR gene and protein expression.Autophagy signaling pathway Notch3/m TOR activation.3.The co-action effect of high glucose and lipopolysaccharide on autophagy in MDBK cellsAutophagy of MDBK cells can be induced by high glucose or lipopolysaccharide alone.The effect of both factors on autophagy of MDBK cells remains unclear.MDBK cells were treated with different concentrations of lipopolysaccharide(0,1,2 and 5μg/m L)and glucose(5.5 and 25.5 m M),and treated with suitable concentrations of lipopolysaccharide(5 μg/m L)and glucose(25.5 m M).To explore the effect of high glucose and lipopolysaccharide on autophagy related gene and protein expression in DMBK cells.Results:(1)In MDBK cells treated with 25.5 m M glucose and 5 μg/m L lipopolysaccharide for 24 h,the gene and protein expressions of Notch3,AMPK,ERK1/2,LC3 and Beclin1 were increased,while the gene and protein expressions of PI3 K,AKT,m TOR and p62 were decreased.(2)DAPT inhibited Notch3 protein expression,LC3 and Beclin1 gene and protein expression in MDBK cells induced by the combination of 25.5m M glucose and 5 μg/m L lipopolysaccharide,and increased cell viability,gene and protein expression of m TOR and p62.(3)Com C and GDC-0994 inhibited LC3 B and Beclin1 gene expression in MDBK cells treated with 25.5 m M glucose and 5 μg/m L lipopolysaccharide for 24 h,and up-regulated p62 gene expression.In conclusion,the combination of 25.5 m M glucose and 5 μg/m L lipopolysaccharide can induce autophagy in MDBK cells.The mechanism of the combination of 25.5 mm glucose and 5 μg/ ml lipopolysaccharide can synergistic promote the gene and protein expression of Notch3,AMPK and ERK1/2,and synergistic inhibit the gene and protein expression of PI3 K,AKT and m TOR.Autophagy signaling pathway Notch3/m TOR activation.4.The effects of high glucose and lipopolysaccharide on the gene expression profile of MDBK cells were analyzed by transcriptomicsIn order to further verify the effects of the combination of high glucose and lipopolysaccharide on other related biological functions of MDBK cells and the expression of autophagy signaling pathway genes.Illumina platform was used to detect 5m M glucose(control group,CG),25.5 m M glucose(high glucose group,HGG)and 5μg/m L lipopolysaccharide(lipopolysaccharide group,CG)by RNA-seq technology.Effects of combination of 25.5 m M glucose and 5 μg/m L lipopolysaccharide(high glucose + lipopolysaccharide group,HLG)on gene expression in MDBK cells.PCA analysis,differential gene expression statistics and analysis,GO and KEGG enrichment analysis were used to analyze the effects of different treatments on the biological function of MDBK cells.Genes with differential expression of autophagy in transcription were screened,and the expression of genes related to autophagy signaling pathways in the pretrial transcriptomes was analyzed by one-way variance to further verify the effect of high glucose and lipopolysaccharide on autophagy in MDBK cells.Results: 1.The results of differential expression gene clustering and PCA analysis showed that the distance of samples within the same group was close,the gene expression profile was similar,and the distance of samples between the groups was far,indicating that high glucose and lipopolysaccharide separately or in combination had a significant effect on MDBK cells.2.Compared with CG,the differentially expressed genes of HGG and LPG were 1,975 and 2,535,respectively;compared with HLG,the differentially expressed genes of HGG and LPG were 66 and 1,291,respectively.3.GO enrichment analysis showed that high glucose or lipopolysaccharide,separately or in combination,mainly affected the bioregulatory function of MDBK cells,and the enriched genes were GRO1,TNF and MAPK13,etc.;Compared with HGG or LPG,HLG was mainly affected by the regulation of cellular processes and the response to stimulation,and the enriched genes were GPR4,WNT11 and SLC19 A,etc.4.KEGG enrichment analysis showed that compared with CG,the differentially expressed genes of HGG,LPG and HLG were mainly related to cellular inflammation and cellular immunity,and were mainly enriched in the signaling pathway of cytokine-cytokine receptor interaction(up-regulated)and calcium signaling pathway(down-regulated),and the common genes were CXCL8 and F2R;Compared with HGG or LPG,HLG differentially expressed genes were mainly related to inflammation genesis,cellular immunity and signaling transcription,IL-17 signaling pathway and TNF signaling pathway were down-regulated in MDBK cells with the combined effect of high glucose and lipopolysaccharide,enriched to shared genes were IL-6 and CXCL3,etc.5.The statistics of autophagy related genes in transcriptome data showed that compared with CG,the autophagy related differentially expressed genes in HGG were ATG16L2,ATG10,AMBRA1 and ULK2.Autophagy related differentially expressed genes in LPG were ATG16L2,ATG4 A,ATG10,AMBRA1 and ULK2.The autophagy related differentially expressed genes in HLG were ATG10 and AMBRA1.The expression trends of Notch3,AMPK,ERK1/2,PI3 K,AKT,m TOR,LC3,p62 and Beclin1 genes in MDBK cells with high glucose,lipopolysaccharide,and their combination were similar to those in the previous experiment.Transcriptome data also showed that MDBK cells with high glucose,lipopolysaccharide and their combined action can induce autophagy.In conclusion,high glucose,lipopolysaccharide,and their combined effects on MDBK cells activate inflammatory pathways and cause changes in inflammatory responses and related biological functions.Whether this is directly or indirectly related to autophagy induced by high glucose and lipopolysaccharide is still worthy of further study.Conclusion: 1.Autophagy of MDBK cells can be induced by the treatment of 25.5m M glucose for more than 3h or 5 μg/m L lipopolysaccharide for more than 6h,and the mechanism is related to the activation of autophagy signaling pathways(Notch3,AMPK,ERK1/2,PI3K/AKT and m TOR signaling pathways).2.The combination of 25.5 m M glucose and 5 μg/m L lipopolysaccharide can synergically promote autophagy of MDBK cells.3.Transcriptomic sequencing results showed that the differentially expressed genes of MDBK cells stimulated by high glucose and LPS separately or in combination were mainly related to cellular inflammation and cellular immunity,mainly enriched in the interaction signal pathway of cytokine-cytokine receptor(up-regulated)and calcium signaling pathway(down-regulated).The common genes were CXCL8 and F2 R.Moreover,high glucose promoted the expression of autophagy related differentially expressed genes in ATG16L2 and ATG10 cells,and inhibited the expression of AMBRA1 and ULK2.LPS promoted the expression of autophagy related different-expressed genes in ATG16L2,ATG4 A and ATG10 cells,and inhibited the expression of AMBRA1 and ULK2.The combination of high glucose and lipopolysaccharide promoted the expression of autophagy related differentially expressed genes and inhibited the expression of AMBRA1 in ATG10 cells.