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.