1.Knockout of MIF gene attenuates severe acute pancreatitis-associated lung injury in mice
Wanpeng WANG ; Bo CHENG ; Shujun YANG ; Yanna LIU ; Qiaofang WANG ; Yaodong SONG ; Changju ZHU
Chinese Journal of Emergency Medicine 2021;30(5):551-556
Objective:To investigate the role of macrophage migration inhibitory factor (MIF) in severe acute pancreatitis (SAP) associated lung injury in mice.Methods:Totally 32 mice were randomly divided into 4 groups ( n=8/per group): wild type control group (WT+CON group), wild type SAP group (WT+SAP group), MIF gene knockout control group (KO+CON group), and MIF gene knockout SAP group (KO+SAP group). SAP model was established by intraperitoneal injection of L-arginine (4 mg/g). The expression of serum amylase, IL-6, TNF-α and MIF were detected by ELISA. The pathological changes of pancreatic and lung tissues were observed by HE staining. The expression of IL-6 and TNF-α in lung tissue was detected by immunohistochemistry. The expression of NF-κB in lung tissue was detected by Western blot. For measurement data, t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. Results:Compared with the WT+CON group, pathological score of pancreatic and lung injury, serum amylase, TNF-α and IL-6 expression in serum and lung tissues were significantly increased in the WT+SAP group ( P<0.05), while the above indexes were significantly decreased in the KO+SAP group ( P<0.05). In addition, the expression of NF-κB protein in KO+SAP group was significantly lower than that in the WT+SAP group ( P<0.05). Conclusions:MIF gene knockout can alleviate severe acute pancreatitis associated acute lung injury in mice, and its mechanism may be related to NF-κB.
2.LncRNA Gm13568 regulates the activation of A1 astrocytes and affects the EAE process in mice
Ruixue LYU ; Yingyu CHEN ; Wanpeng CHENG ; Bo ZHANG ; Yifan WANG ; Jiaxin DENG ; Jinyu XIE ; Suping QIN ; Xiaomei LIU
Chinese Journal of Microbiology and Immunology 2022;42(2):121-127
Objective:To investigate the effects of long non-coding RNA (lncRNA) Gm13568 on the activation of A1 astrocytes and the progress of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:A recombinant lentiviral vector (LV-Inhibit-Gm13568) carrying astrocyte-specific promoter of glial fibrillary acidic protein (GFAP) was established to inhibit the function of endogenous Gm13568. A control vector (LV-ctrl) was established as well. The recombinant vectors were packaged. C57BL/6 mice were injected with 1×10 7 transforming units of viral suspension via the tail vein and 7 d after the injection, myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) was used to establish the mouse model of EAE. Four groups, PBS group, EAE group, LV-ctrl+ EAE group and LV-Inhibit-Gm13568+ EAE group, were included in this study. Clinical signs of the mice were monitored daily in a double-blinded manner. The mice were sacrificed 23 d after the EAE model was established and the spinal cord tissues were collected. The expression of Serping 1, C3, Srgn and H2-T23 at mRNA level was detected by real-time PCR. Changes in the expression of IL-6, TNF-α, macrophage chemotactic protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) were measured. Western blot was used to investigate the expression of GFAP and Notch1 in spinal cord tissues and the phosphorylation of signal transduction and transcription activator 3 (STAT3). The expression of Notch1 intracellular domain (NICD) and GFAP in spinal cord tissues was detected by immunofluorescence. Furthermore, the infiltration of inflammatory cells and the demyelination of spinal cord were observed using HE and Luxol fast blue (LFB) staining methods. Results:Compared with PBS group, A1 astrocytes were activated and Notch1 expression was significantly up-regulated in EAE group and LV-ctrl+ EAE group. The clinical score of mice in LV-Inhibit-Gm13568+ EAE group was decreased from an average score of 3.5 to less than 1 on 23 d after antigen induction and the clinical symptoms were alleviated as compared with the mice in LV-ctrl+ EAE group. Meanwhile, the activation of A1 astrocytes was down-regulated, and the production of inflammatory cytokines and chemokines was also reduced. The expression of Notch1, GFAP and NICD at protein level and the phosphorylation of STAT3 were significantly reduced. Moreover, the infiltration of inflammatory cells and demyelination of spinal cord tissues were alleviated significantly.Conclusions:LncRNA Gm13568 might regulate the activation of A1 astrocytes via the Notch1/STAT3 pathway, thus affecting the production of inflammatory cytokines and chemokines and participating in the process of EAE.
3.Regulatory effects of sphingosine kinase-2 on astrocyte function and EAE progression in mouse model
Jingjing GUO ; Ying YANG ; Qianwen CAO ; Zijun ZHAO ; Xiangyang LI ; Hui HUA ; Xiaocui LI ; Wanpeng CHENG ; Feng ZHOU ; Xiaomei LIU
Chinese Journal of Microbiology and Immunology 2020;40(10):780-786
Objective:To investigate the regulatory effects of sphingosine kinase-2 (SphK2) on the function of activated astrocytes and the progression of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:Primary mouse astrocytes were isolated from wild-type (WT) C57BL/6 mice and sphk2 gene knock-out ( sphk2 -/-) mice and stimulated in vitro with interleukin 17 (IL-17). Real-time PCR was used to measure the expression of inflammatory cytokines and chemokines at mRNA levels. Western blot and immunofluorescence were used to detect the expression of glial fibrillary acidic protein (GFAP) and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3). An EAE mouse model was constructed using myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) polypeptide. Western blot was used to detect the expression of GFAP and p-STAT3 at protein level and real-time PCR was used to detect the expression of inflammatory cytokines and chemokines at mRNA level in spinal cords. Hematoxylin-Eosin (HE) and Luxol fast blue (LFB) staining were used to observe the changes in inflammatory cell infiltration and demyelination in spinal cords. Results:Compared with the WT group, the phosphorylation of STAT3 was obviously reduced in in vitro activated mouse astrocytes of sphk2 -/- mice, and the expression of inflammatory cytokines and chemokines including monocyte chemotactic protein 1 (MCP-1), TNF-α and IL-6 at mRNA level was also significantly decreased. Compared with the WT EAE group, changes in the above-mentioned cytokines and relative proteins in sphk2 -/- EAE mice in vivo were similar to those in vitro. Moreover, inflammatory cell infiltration and demyelination were significantly reduced in spinal cords of sphk2 -/- EAE mice. However, no significant difference in in vitro or in vivo GFAP expression was observed between WT and sphk2 -/- mice. Conclusions:SphK2 might regulate the function of reactive astrocytes through STAT3 molecular pathway, thereby regulating the production of inflammatory cytokines and chemokines and participating in the pathological process of EAE.
4.Lipopolysaccharide regulates neutrophil inflammation through activating the LRG1/ROCK1 signaling
Qiao FENG ; Xin HAN ; Bohui YUAN ; Xuejiao ZHANG ; Hui HUA ; Wanpeng CHENG ; Suping QIN ; Feng ZHOU ; Xiaomei LIU
Journal of Xi'an Jiaotong University(Medical Sciences) 2024;45(4):597-602
【Objective】 To investigate the role of lipopolysaccharide (LPS) in regulating the inflammatory response of neutrophil through the leucine-rich α-2 glycoprotein 1 (LRG1)/ Rho-associated protein kinase (ROCK1) signaling. 【Methods】 HL-60 cells were treated with 1 μmol/L all-trans retinoic acid (ATRA) and 12.5 μL/mL dimethyl sulfoxide (DMSO) for 72 h and 96 h, and the morphological changes were observed by Wright-Giemsa staining. The expression of CD11b was detected by flow cytometry. LPS induced the activation of dHL-60 and human peripheral blood neutrophils. The transcription and secretion levels of LRG1, ROCK1 and inflammatory cytokines were detected by qPCR and ELISA, respectively. The expression levels of LRG1 and ROCK1 after the activation of dHL-60 were detected by Western blotting. Furthermore, dHL-60 was treated with the recombinant protein LRG1 and ROCK1 inhibitor Y-27632; the transcription levels of inflammatory cytokines were detected by qPCR. 【Results】 Neutrophils were activated by LPS. The expression levels of LRG1 and ROCK1 were significantly increased, and the transcription levels of inflammatory cytokines were significantly increased. The recombinant protein LRG1 activated dHL-60 in vitro, and the transcription levels of ROCK1 and inflammatory cytokines were significantly increased. Using the ROCK1 inhibitor Y-27632, the production levels of inflammatory cytokines were significantly reduced. 【Conclusion】 LPS can regulate the production levels of neutrophil inflammatory cytokines through activating the LRG1/ROCK1 signaling, thus exacerbating the inflammatory response.