Objective:To investigate the interventional effects and mechanisms of spermine and spermine derivative 1 (SD1) on human cytomegalovirus (HCMV) infection by establishing an HCMV-infected human bronchial epithelial (HBE) cell model.Methods:HBE cells were used as the study carrier and divided into four groups: a blank control group without HCMV, a control group with HCMV, a spermine group with HCMV and 2 mmol/L spermine, and an SD1 group with HCMV and 2 mmol/L SD1. Viral titers were measured using the 50% tissue culture infectious dose (TCID50) method. The expression of related proteins and genes was detected by enzyme-linked immunosorbent assay (ELISA), Western-blot, and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and cellular reactive oxygen species (ROS) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The above data were compared in multiple groups using one-way analysis of variance, and further pairwise comparisons were conducted using SNK- q test. Results:Compared with the blank control group, the control, spermine, and SD1 groups showed increased viral titers [(0.00±0.00) vs. (8.38±0.50), (6.75±0.46), (5.63±0.52)-log10 TCID50/100 μl; q=21.85, 15.87, and 11.06; all P<0.05]. Compared with the control group, the spermine and SD1 groups exhibited reduced viral titers ( q=5.98 and 10.79; both P<0.05). The SD1 group showed a further reduction compared with the spermine group ( q=4.82, P<0.05). ELISA and RT-qPCR results demonstrated that compared with the blank control group, the control, spermine, and SD1 groups exhibited elevated levels of cyclic guanosine monophosphate-adenosine mompho-sphate (cGAMP), interferon-β (IFN-β), IFN-β mRNA, myxovirus resistance gene 1 (MX1) mRNA, and interferon-stimulated gene 15 (ISG15) mRNA [cGAMP: (0.20±0.03) vs. (0.92±0.10), (1.45±0.09), (2.03±0.15) pmol/ml; IFN-β: (0.13±0.02) vs. (2.34±0.12), (3.50±0.10), (4.50±0.14) ng/ml; IFN-β mRNA: 10.26±1.53 vs. 403.10±15.01, 602.35±11.02, 778.67±24.13; MX1 mRNA: 1.02±0.06 vs. 198.33±7.41, 304.61±9.98, 401.25±10.53; ISG15 mRNA: 1.04±0.08 vs. 273.84±13.71, 396.35±15.20, 489.57±17.46; q=6.93, 12.02, 17.60; 18.43, 28.10, 36.43; 12.52, 19.02, 24.66; 14.30, 21.36, 28.28; 15.12, 20.12, and 25.02; all P<0.05]. Compared with the control group, the spermine and SD1 groups showed further increases in these markers ( q=5.10, 10.68; 9.67, 18.00; 6.50, 12.14; 7.06, 14.00; 5.00, and 9.90; all P<0.05). The SD1 group exhibited even higher expression than the spermine group ( q=5.58, 8.33, 5.64, 6.92, and 4.90; all P<0.05). Western-blot results revealed that compared with the blank control group, the control, spermine, and SD1 groups showed increased expression of phosphorylated TANK-binding kinase 1 (p-TBK1) and phosphorylated interferon regulatory factor 3 (p-IRF3) (p-TBK1: 0.10±0.01 vs. 0.31±0.02, 1.04±0.04, 1.54±0.04; p-IRF3: 0.24±0.02 vs. 0.37±0.02, 0.58±0.03, 1.09±0.04; q=6.57, 29.41, 45.09; 3.38, 8.72, and 21.88; all P<0.05). Compared with the control group, the spermine and SD1 groups exhibited stronger expression ( q=22.84, 38.52; 5.34, and 18.50; all P<0.05). The SD1 group showed further enhancement compared with the spermine group ( q=15.68 and 13.16; both P<0.05). ROS levels were higher in the control group than in the blank control group (1 180.00±16.33 vs. 2 126.67±71.94; q=16.90, P<0.05). Compared with the control group, the spermine and SD1 groups showed reduced ROS expression (2 126.67±71.94 vs. 1 660.00±45.17, 1 473.23±55.58; q=7.30 and 11.66; both P<0.05). The SD1 group exhibited further reduction compared with the spermine group ( q=4.36, P<0.05). Conclusions:In the HCMV-infected HBE cell model, the addition of spermine and SD1 promoted the activation of the cGAMP synthase and type Ⅰ interferon signaling pathway, thereby enhancing innate immune suppression of HCMV infection. Meanwhile, it scavenged ROS to alleviate oxidative stress-induced cellular damage.

Objective:To investigate the interventional effects and mechanisms of spermine and spermine derivative 1 (SD1) on human cytomegalovirus (HCMV) infection by establishing an HCMV-infected human bronchial epithelial (HBE) cell model.Methods:HBE cells were used as the study carrier and divided into four groups: a blank control group without HCMV, a control group with HCMV, a spermine group with HCMV and 2 mmol/L spermine, and an SD1 group with HCMV and 2 mmol/L SD1. Viral titers were measured using the 50% tissue culture infectious dose (TCID50) method. The expression of related proteins and genes was detected by enzyme-linked immunosorbent assay (ELISA), Western-blot, and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and cellular reactive oxygen species (ROS) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The above data were compared in multiple groups using one-way analysis of variance, and further pairwise comparisons were conducted using SNK- q test. Results:Compared with the blank control group, the control, spermine, and SD1 groups showed increased viral titers [(0.00±0.00) vs. (8.38±0.50), (6.75±0.46), (5.63±0.52)-log10 TCID50/100 μl; q=21.85, 15.87, and 11.06; all P<0.05]. Compared with the control group, the spermine and SD1 groups exhibited reduced viral titers ( q=5.98 and 10.79; both P<0.05). The SD1 group showed a further reduction compared with the spermine group ( q=4.82, P<0.05). ELISA and RT-qPCR results demonstrated that compared with the blank control group, the control, spermine, and SD1 groups exhibited elevated levels of cyclic guanosine monophosphate-adenosine mompho-sphate (cGAMP), interferon-β (IFN-β), IFN-β mRNA, myxovirus resistance gene 1 (MX1) mRNA, and interferon-stimulated gene 15 (ISG15) mRNA [cGAMP: (0.20±0.03) vs. (0.92±0.10), (1.45±0.09), (2.03±0.15) pmol/ml; IFN-β: (0.13±0.02) vs. (2.34±0.12), (3.50±0.10), (4.50±0.14) ng/ml; IFN-β mRNA: 10.26±1.53 vs. 403.10±15.01, 602.35±11.02, 778.67±24.13; MX1 mRNA: 1.02±0.06 vs. 198.33±7.41, 304.61±9.98, 401.25±10.53; ISG15 mRNA: 1.04±0.08 vs. 273.84±13.71, 396.35±15.20, 489.57±17.46; q=6.93, 12.02, 17.60; 18.43, 28.10, 36.43; 12.52, 19.02, 24.66; 14.30, 21.36, 28.28; 15.12, 20.12, and 25.02; all P<0.05]. Compared with the control group, the spermine and SD1 groups showed further increases in these markers ( q=5.10, 10.68; 9.67, 18.00; 6.50, 12.14; 7.06, 14.00; 5.00, and 9.90; all P<0.05). The SD1 group exhibited even higher expression than the spermine group ( q=5.58, 8.33, 5.64, 6.92, and 4.90; all P<0.05). Western-blot results revealed that compared with the blank control group, the control, spermine, and SD1 groups showed increased expression of phosphorylated TANK-binding kinase 1 (p-TBK1) and phosphorylated interferon regulatory factor 3 (p-IRF3) (p-TBK1: 0.10±0.01 vs. 0.31±0.02, 1.04±0.04, 1.54±0.04; p-IRF3: 0.24±0.02 vs. 0.37±0.02, 0.58±0.03, 1.09±0.04; q=6.57, 29.41, 45.09; 3.38, 8.72, and 21.88; all P<0.05). Compared with the control group, the spermine and SD1 groups exhibited stronger expression ( q=22.84, 38.52; 5.34, and 18.50; all P<0.05). The SD1 group showed further enhancement compared with the spermine group ( q=15.68 and 13.16; both P<0.05). ROS levels were higher in the control group than in the blank control group (1 180.00±16.33 vs. 2 126.67±71.94; q=16.90, P<0.05). Compared with the control group, the spermine and SD1 groups showed reduced ROS expression (2 126.67±71.94 vs. 1 660.00±45.17, 1 473.23±55.58; q=7.30 and 11.66; both P<0.05). The SD1 group exhibited further reduction compared with the spermine group ( q=4.36, P<0.05). Conclusions:In the HCMV-infected HBE cell model, the addition of spermine and SD1 promoted the activation of the cGAMP synthase and type Ⅰ interferon signaling pathway, thereby enhancing innate immune suppression of HCMV infection. Meanwhile, it scavenged ROS to alleviate oxidative stress-induced cellular damage.

Bone mesenchymal stem cells (BMSCs) have been used worldwide to treat spinal cord injury, but their therapeutic mechanism is poorly understood. In this study, BMSCs were transplanted to aneurysm clip-injured rats to demonstrate their protective effect. We observed myelin sheaths through Luxol fast blue (LFB) staining, osmic acid staining, TUNEL and transmission electron microscopy (TEM). We performed Western blotting to analyze the expressions of brain-derived neurotrophic factor (BDNF) and caspase 3. BMSCs were transplanted at 1, 7 and 14 days after spinal cord injury. Hindlimb movement (Basso, Beattie and Bresnahan; BBB) score, CNPase (2', 3'-cyclic-nucleotide 3'-phosphodiesterase), myelin basic protein (MBP) and caspase 3 protein levels were detected. Immunofluorescence was used to test the differentiation of BMSCs after implanted into damaged spinal cord and co-expression of CNPase-caspase 3+. At 7 days after BMSCs transplantation, some injected BMSCs expressed neuronal and oligodendrocyte markers. And both locomotor skills and ultra-structural features of myelin sheaths were significantly improved. The expressions of BDNF were clearly increased by BMSCs transplantation, the expression of caspase 3 was the opposite. Compared with the 1 and 14 days transplantation after spinal cord injury, MBP and CNPase expressions were highest, caspase 3 expression was lowest in 7 days BMSCs transplantation. After BMSCs transplantation, CNPase-caspase 3+ cells scattered in the white matter of the spinal cord. Therefore, BMSCs had a tendency to differentiate into neurons and oligodendrocytes after transplantation, which could promote the secretion of BDNF. BMSCs protected neural myelin sheaths by inhibiting oligodendrocyte apoptosis via increased secretion of BDNF after SCI. The best therapeutic time was 7 days after spinal cord injury.

Thymic epithelial cells (TECs) are one of the most important components in thymic microenvironment supporting thymocyte development and maturation. TECs, composed of cortical and medullary TECs, are derived from a common bipotent progenitor, mediating thymocyte positive and negative selections. Multiple levels of signals including intracellular signaling networks and cell-cell interaction are required for TEC development and differentiation. Transcription factors Foxn1 and autoimmune regulator (Aire) are powerful regulators promoting TEC development and differentiation. Crosstalks with thymocytes and other stromal cells for extrinsic signals like RANKL, CD40L, lymphotoxin, fibroblast growth factor (FGF) and Wnt are also definitely required to establish a functional thymic microenvironment. In this review, we will summarize our current understanding about TEC development and differentiation, and its underlying multiple signal pathways.
Cell Communication ; genetics ; Cell Differentiation ; Epithelial Cells ; cytology ; metabolism ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Signal Transduction ; genetics ; Thymocytes ; cytology ; metabolism ; Thymus Gland ; cytology ; growth & development ; Transcription Factors ; genetics ; metabolism