Jalali Sedigheh, Farhadi Ali, Rafiei Dehbidi Gholamreza, Farjadian Shirin, Sharifzadeh Sedigheh, Ranjbaran Reza, Seyyedi Noorossadat, Namdari Sepide, Behzad-Behbahani Abbas
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Interdiscip Perspect Infect Dis. 2022 Jun 6;2022:1639990. doi: 10.1155/2022/1639990. eCollection 2022.
The nonstructural protein (NS1) of human parvovirus B19 (hPVB19) is considered to be a double-edged sword in its pathogenesis. NS1 protein promotes cell death by apoptosis in erythroid-lineage cells and is also implicated in triggering and the progression of various inflammation and autoimmune disorders.
We investigated the possible role of hPVB19 NS1 in the modulation of proinflammatory cytokines in nonpermissive HEK-293T cells.
A plasmid containing the fully sequenced NS1 gene (pCMV6-AC-GFP-NS1) was transfected into HEK-293T cells. Transfection efficiency was assessed by fluorescent microscopy over time. Mock (pCMV6-AC-GFP) transfected cells were used as controls. The percentage of apoptotic cells was measured by flow cytometry at 24, 48, and 72 h posttransfection. Interleukin 6 (IL-6) mRNA, as a pleiotropic cytokine, was measured by real-time PCR. Furthermore, cellular supernatants were collected to determine the type and quantity of cytokines produced by mock- and NS1-transfected cells using flow cytometry.
Fold change in the expression level of IL-6 mRNA in transfected cells after 72 hr of incubation was found to be 3.01 when compared with mock-transfected cells; however, cell apoptosis did not happen over time. Also, the concentration of cytokines such as IL-2, IL-6, IL-9, IL-17A, IL-21, IL-22, interferon (IFN)-, and tumor necrosis factor (TNF-) increased in NS1-transfected cells.
Overall, our results indicated that proinflammatory cytokine levels had increased following the expression of hPVB19 NS1 in HEK-293T cells, consistent with a role for NS1 expression facilitating the upregulation of inflammatory reactions. Therefore, hPVB19 NS1 function may play a role in the progression of some chronic and inflammatory diseases.
人细小病毒B19（hPVB19）的非结构蛋白（NS1）在其发病机制中被认为是一把双刃剑。NS1蛋白通过诱导红系细胞凋亡促进细胞死亡，还与多种炎症和自身免疫性疾病的触发及进展有关。
我们研究了hPVB19 NS1在非允许性HEK-293T细胞中对促炎细胞因子的调节作用。
将含有完整测序NS1基因的质粒（pCMV6-AC-GFP-NS1）转染至HEK-293T细胞。通过荧光显微镜随时间评估转染效率。以转染空载体（pCMV6-AC-GFP）的细胞作为对照。在转染后24、48和72小时，通过流式细胞术检测凋亡细胞的百分比。作为多效性细胞因子的白细胞介素6（IL-6）mRNA通过实时PCR进行检测。此外，收集细胞上清液，使用流式细胞术确定转染空载体和转染NS1的细胞产生的细胞因子的类型和数量。
与转染空载体的细胞相比，孵育72小时后转染细胞中IL-6 mRNA表达水平的倍数变化为3.01；然而，细胞凋亡并未随时间发生。此外，转染NS1的细胞中IL-2、IL-6、IL-9、IL-17A、IL-21、IL-22、干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α等细胞因子的浓度增加。
总体而言，我们的结果表明，hPVB19 NS1在HEK-293T细胞中表达后促炎细胞因子水平升高，这与NS1表达促进炎症反应上调的作用一致。因此，hPVB19 NS1功能可能在某些慢性和炎症性疾病的进展中起作用。
