Fractalkine Aggravates LPS-induced Macrophage Activation And Acute Kidney Injury Via Wnt/β-catenin Signaling Pathway

Part Ⅰ Transcriptome Analysis of Fractalkine Overexpression in J774 A.1 Cell by RNA SequencingObjective: A stable infection strain of J774 A.1 cell with Fractalkine(FKN,CX3CL1)gene overexpression was constructed by lentiviral vector,then RNA sequencing was used to detect the altered genes in J774 A.1 cell after FKN overexpression.The molecular signal pathways with significant differential expression were screened out by bioinformatics analysis for follow-up experiment.Methods: Mouse macrophages cells line J774 A.1 cell was used as the research object.The target gene of FKN was inserted into CV186 lentiviral vector Ubi-MCS-3FLAG-SV40-Cherry-IRES-puromycin.After amplification,restriction enzyme digestion,sequencing,construction of recombinant plasmid and detection of virus quality,lentiviral vector was obtained to infect J774 A.1 cell to construct J774 A.1/ LV-CX3CL1 stable cell line.After infection,the expression level of FKN protein in the cells was detected by Western Blot assay,the cells viability was detected by CCK-8 assay,and the apoptosis rate was detected by flow cytometry.RNA library was constructed by using normal group and FKN-overexpressing J774 A.1 cell,then RNA sequencing was carried out.The whole gene set was analyzed by Gene Set Enrichment Analysis(GESA)to select the signal pathway with significant difference.At the same time,the Gene ontology(GO)were analyzed to identify the significant differences in biological functions.Results: 72 hours after lentivirus infection,the expression of red fluorescent protein was observed in J774 A.1 cell in the transfection group by inverted microscope,and the transfection efficiency was more than 90%.Western Blot assay showed that the expression of FKN protein in selected J774 A.1/LV-CX3CL1 cells was significantly higher than that in normal control cells(P < 0.01).It was confirmed that the construction of J774 A.1/LV-CX3CL1 stable cell line was successful.The results of CCK-8 assay showed that the cell viability of FKN-overexpressing group was higher than that of normal control group.The results of flow cytometry showed that the apoptosis rate of FKN-overexpressing group was lower than that of normal control group.RNA sequencing results showed that 306 differentially expressed m RNAs(273 up-regulated and 33down-regulated)were detected after FKN overexpression.GESA enrichment showed that there were significant differences in Wnt/β-catenin signaling pathway.Adhesion and migration are significant difference in biological functions.Conclusion: In this study,a stable infection strain of J774 A.1 cell with FKN overexpression was successfully constructed,and it was found that the viability of J774 A.1 cell increased and the apoptosis rate decreased after FKN overexpression.RNA sequencing revealed that Wnt/β-catenin signaling pathway was activated after FKN overexpression,which laid a foundation for subsequent experiments to study the biological functions of J774 A.1 cell interfered with FKN based on Wnt/β-catenin signal pathway.Part Ⅱ Effects of Fractalkine on J774 A.1 Cell Activation Induced by Lipopolysaccharide via Wnt/β-catenin Signaling PathwayObjective: Taking the Wnt/β-catenin signaling pathway as the experimental starting point and J774 A.1 cell as the research object,the mechanism of FKN in lipopolysaccharide(LPS)-induced J774 A.1 cell injury was investigated.Methods: J774 A.1 cell were cultured in vitro,infected with lentiviral vectors FKN-overexpressing,and interfered with Wnt3 a and ICG-001.The cells were divided into 12 groups:(1)normal control group;(2)LPS group;(3)Wnt3a(Wnt/β-catenin signal pathway stimulator)group;(4)ICG-001(Wnt/β-catenin signal pathway inhibitor)group;(5)Wnt3a+LPS group;(6)ICG-001+LPS group.(7)FKN-overexpressed(EX-FKN)group,(8)EX-FKN+LPS group,(9)EX-FKN+Wnt3a group,(10)EX-FKN+ICG-001 group,(11)EX-FKN+Wnt3a+LPS group,(12)EX-FKN+ICG-001+LPS group.The protein contents of FKN,β-catenin,Wnt-4,c-myc,Cyclin D1,iNOS,TNF-α,IL-10 and ARG-1 in cells of each group were detected by Western Blot.The secretion of Cyclin D1,TNF-α and ARG-1 in the cells supernatant was detected by Enzyme-linked Immunosorbent Assay(ELISA).The apoptosis rate of cells in each group was detected by flow cytometry.The localization of FKN,β-catenin,Cyclin D1,iNOS and ARG-1 proteins in cells was detected by Immunofluorescence(IF).Results: LPS stimulation increased the contents of FKN,β-catenin,Wnt-4,c-myc,Cyclin D1,iNOS,TNF-α,IL-10 and ARG-1 and the apoptosis rates of J774 A.1 cell.FKN overexpression facilitated the contents of FKN,β-catenin,Wnt-4,c-myc,Cyclin D1,M2 type polarizing factor(IL-10 and ARG-1)and inhibited the contents of M1 type polarizing factor(iNOS and TNF-α)and apoptosis rate in J774 A.1 cell induced by LPS.Combination with Wnt3 a enhanced the effects of FKN overexpression.Combination with ICG-001 down-regulated the contents of FKN,β-catenin,Wnt-4,c-myc,Cyclin D1,M2 polarizing factors(IL-10 and ARG-1)while enhanced the contents of M1 polarizing factors(iNOS and TNF-α)and the apoptosis rates.Conclusion: FKN-overexpressing enhanced the viability of LPS-induced J774 A.1cell activation and promoted their transformation to M2 subtype via Wnt/β-catenin signaling pathway.Part Ⅲ Fractalkine Participates in the Activation of Renal Macrophages in LPS-induced Acute Kidney Injury via Wnt/β-catenin Signaling PathwayObjective: To study the molecular mechanism of FKN regulating renal macrophages activation in LPS-induced acute kidney injury via Wnt/β-catenin signaling pathway,established a novel therapeutic target for LPS-induced acute kidney injury.Methods: C57BL/6 mice from WT and FKN-KO were divided into 12 groups:(1)Control group;(2)LPS group;(3)Wnt3a group;(4)ICG-001 group;(5)Wnt3a+LPS group;(6)ICG-001+LPS group;(7)FKN-KO group;(8)FKN-KO+LPS group;(9)FKN-KO+Wnt3a group;(10)FKN-KO+ICG-001 group;(11)FKN-KO+Wnt3a+LPS group;(12)FKN-KO+ICG-001+LPS group.The Blood urea nitrogen(BUN),Serum creatinine(Scr)and 24-hour urinary protein were detected,and the pathological changes of renal tissue were detected by Hematoxylin-Eosin(HE)staining and Periodic Acid-Schiff(PAS)staining.The protein contents of FKN,β-catenin,Wnt-4,c-myc,Cyclin D1,iNOS,TNF-α,IL-10 and ARG-1 in kidney tissue were detected by Western Blot,and the localization of F4/80,iNOS or ARG-1 protein in kidney tissue were detected by IF.Results: Compared with LPS group,the abundance of BUN,Scr,urinary protein,FKN,β-catenin,Wnt-4,c-myc,Cyclin D1,iNOS,TNF-α,IL-10 and ARG-1 in FKN knockout group were decreased,which improved the pathological injury of kidney.the above-mentioned indexes were significantly decreased and renal pathological injury was significantly improved by combined with ICG-001,while combination with Wnt3 a showed the effects of reversing FKN knockout.Conclusion: FKN deficiency ameliorates LPS-induced acute kidney injury by prevent macrophages proliferation and M2 subtype transformation via Wnt/β-catenin signaling pathway.