Objective: To explore the expression characteristics and role of Krüppel-like factor 4 (KLF4) in macrophage inflammatory response and its effects on inflammatory response and organ injury in septic mice, so as to lay a theoretical foundation for targeted treatment of burns and trauma sepsis. Methods: The method of experimental research was used. Mouse RAW264.7 macrophages and primary peritoneal macrophages (PMs) isolated from 10 male C57BL/6J mice aged 6-8 weeks were used for the experiments. RAW264.7 macrophages and PMs were treated with endotoxin/lipopolysaccharide (LPS) for 0 (without treatment), 1, 2, 4, 6, 8, 12, and 24 h, respectively, to establish macrophage inflammatory response model. The mRNA expression of interleukin 1β (IL-1β), IL-6, CC chemokine ligand 2 (CCL2) and tumor necrosis factor-α (TNF-α) were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), and the LPS treatment time was determined for some of the subsequent experiments. RAW264.7 macrophages were treated with LPS for 0 and 8 h, the localization and protein expression of KLF4 were detected by immunofluorescence method, transcriptome sequencing of the cells was performed using the high-throughput sequencing technology platform, and the differently expressed genes (DEGs) between the two time points treated cells were screened by DESeq2 software. RAW264.7 macrophages and PMs were treated with LPS for 0, 1, 2, 4, 6, 8, 12, and 24 h, respectively, and the mRNA and protein expressions of KLF4 were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively. RAW264.7 macrophages were divided into negative control (NC) group and KLF4-overexpression group according to the random number table, which were treated with LPS for 0 and 8 h respectively after transfection of corresponding plasmid. The mRNA expressions of KLF4, IL-1β, IL-6, CCL2, and TNF-α were detected by real-time fluorescence quantitative RT-PCR, while the protein expression of KLF4 was detected by Western blotting. The number of samples in aforementioned experiments was all 3. Forty male C57BL/6J mice aged 6-8 weeks were divided into KLF4-overexpression group and NC group (with 20 mice in each group) according to the random number table, and the sepsis model of cecal ligation perforation was established after the corresponding transfection injection was injected respectively. Twelve mice were selected from each of the two groups according to the random number table, and the survival status within 72 hours after modeling was observed. Eight hours after modeling, the remaining 8 mice in each of the two groups were selected, the eyeball blood samples were collected to detect the levels of IL-1β and IL-6 in serum by enzyme-linked immunosorbent assay, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum by dry chemical method. Subsequently, the heart, lung, and liver tissue was collected, and the injury was observed after hematoxylin-eosin staining. Data were statistically analyzed with independent sample t test, Cochran & Cox approximate t test, one-way analysis of variance, Dunnett test, Brown-Forsythe and Welch one-way analysis of variance, Dunnett T3 test, log-rank (Mantel-Cox) test. Results: Compared with that of LPS treatment for 0 h, the mRNA expressions of IL-1β in RAW264.7 macrophages treated with LPS for 6 h and 8 h, the mRNA expressions of IL-6 in RAW264.7 macrophages treated with LPS for 4-12 h, the mRNA expressions of CCL2 in RAW264.7 macrophages treated with LPS for 8 h and 12 h, and the mRNA expressions of TNF-α in RAW264.7 macrophages treated with LPS for 4-8 h were significantly up-regulated (P<0.05 or P<0.01), while the mRNA expressions of IL-1β and CCL2 in PMs treated with LPS for 4-8 h, the mRNA expressions of IL-6 in PMs treated with LPS for 2-24 h, and the mRNA expressions of TNF-α in PMs treated with LPS for 2-12 h were significantly up-regulated (P<0.05 or P<0.01). Eight hours was selected as the LPS treatment time for some of the subsequent experiments. KLF4 mainly located in the nucleus of RAW264.7 macrophages. Compared with those of LPS treatment for 0 h, the protein expression of KLF4 in RAW264.7 macrophages treated with LPS for 8 h was obviously decreased, and there were 1 470 statistically differentially expressed DEGs in RAW264.7 macrophages treated with LPS for 8 h, including KLF4 with significantly down-regulated transcriptional expression (false discovery rate<0.05, log₂ (fold change)=-2.47). Compared with those of LPS treatment for 0 h, the mRNA expressions of KLF4 in RAW264.7 macrophages treated with LPS for 6-24 h, the protein expressions of KLF4 in RAW264.7 macrophages and PMs treated with LPS for 1-24 h, and the mRNA expressions of KLF4 in PM treated with LPS for 4-24 h were significantly decreased (P<0.05 or P<0.01). Compared with those in NC group, the mRNA (with t' values of 17.03 and 8.61, respectively, P<0.05 or P<0.01) and protein expressions of KLF4 in RAW264.7 macrophages treated with LPS for 0 h and 8 h in KLF4-overexpression group were significantly increased, the mRNA expressions of IL-6 and CCL2 increased significantly in RAW264.7 macrophages treated with LPS for 0 h (with t values of 6.29 and 3.40, respectively, P<0.05 or P<0.01), while the mRNA expressions of IL-1β, IL-6, CCL2, and TNF-α decreased significantly in RAW264.7 macrophages treated with LPS for 8 h (with t values of 10.52, 9.60, 4.58, and 8.58, respectively, P<0.01). The survival proportion of mice within 72 h after modeling in KLF4-overexpression group was significantly higher than that in NC group (χ²=4.01, P<0.05). Eight hours after modeling, the serum levels of IL-1β, IL-6 and ALT, AST of mice in KLF4-overexpression group were (161±63), (476±161) pg/mL and (144±24), (264±93) U/L, respectively, which were significantly lower than (257±58), (654±129) pg/mL and (196±27), (407±84) U/L (with t values of 3.16, 2.44 and 4.04, 3.24, respectively, P<0.05 or P<0.01) in NC group. Eight hours after modeling, compared with those in NC group, the disorder of tissue structure of heart, lung, and liver, inflammatory exudation, and pathological changes of organ parenchyma cells in KLF4-overexpression group were obviously alleviated. Conclusions: The expression of KLF4 is significantly down-regulated in LPS-induced macrophage inflammatory response, which significantly inhibits the macrophage inflammatory response. KLF4 significantly enhances the survival rate of septic mice and alleviates inflammatory response and sepsis-related organ injury.
Male ; Mice ; Animals ; Mice, Inbred C57BL ; Lipopolysaccharides ; Tumor Necrosis Factor-alpha ; Kruppel-Like Factor 4 ; Interleukin-6 ; Wound Infection ; Sepsis

OBJECTIVE: To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. METHODS: Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCA^TM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. RESULTS: The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2^-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2^-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2^-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2^-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. CONCLUSIONS The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
Animals ; Brain Diseases ; Cells, Cultured ; Fibroblasts ; Humans ; Kruppel-Like Factor 4 ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Sepsis