Kumara Dennaya, Harsan Hayfa Salsabila, Septisetyani Endah Puji, Prasetyaningrum Pekik Wiji, Paramitasari Komang Alit, Syaifudin Mukh, Astirin Okid Parama, Ikawati Muthi, Meiyanto Edy
Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Mammalian Cell Engineering Research Group, Research Center for Genetic Engineering, National Research and Innovation Agency (BRIN), West Java, 16911, Indonesia.
Adv Pharm Bull. 2025 Mar 9;15(2):416-427. doi: 10.34172/apb.44060. eCollection 2025 Jul.
SARS-CoV-2 infection may lead to a worse prognosis in COVID-19 patients by inducing syncytia formation which implies intercellular transmission and immune evasion. Hesperidin (HSD) and hesperetin (HST) are two citrus flavonoids that demonstrate the potential to interfere with spike/human angiotensin-converting enzyme-2 (hACE2) binding and show an inhibitory effect in the SARS-CoV-2 pseudovirus internalization model. Here, we determined the effects of HSD and HST to inhibit syncytia formation using in vitro cell models.
We confirmed spike, hACE2, and transmembrane protease, serine 2 (TMPRSS2) ectopic expressions by immunofluorescence staining (IF) after transfection using polyethylene imine (PEI) in 293T cells. Then, the cells were transfected with a set of plasmids encoding spike/hACE2/TMPRSS2 or spike/hACE2 to induce syncytia formation. Cell treatment with HSD/HST was performed 4-5 h after transfection and then incubated for another 16-18 h. Syncytia were observed using an inverted microscope or a high content screening (HCS) platform. The data obtained from syncytia formation assays were statistically analyzed using ANOVA (Bonferroni).
We successfully observed spike, hACE2, and TMPRSS2 expression in 293T cells by IF staining. Furthermore, we showed that HSD 10 and 100 µM significantly inhibited the formation of small-to-medium-sized syncytia compared to the control cells by manual syncytia observation. In the HCS assay, 10 µM HSD showed an inhibitory effect of syncytia induced by spike WT. In contrast, 100 µM HSD, 10 and 100 µM HST, and 10 µg/mL citrus peel extract containing HSD prepared by the hydrodynamic cavitation method (HCV) inhibited syncytia formation induced by spike Omicron.
HSD and HST show the potential inhibitory activity of SARS-CoV-2 intercellular transmission. Further study is needed to confirm the mechanism of action of the antiviral activity.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染可能通过诱导合胞体形成导致冠状病毒病（COVID-19）患者预后更差，这意味着细胞间传播和免疫逃逸。橙皮苷（HSD）和橙皮素（HST）是两种柑橘类黄酮，它们具有干扰刺突蛋白/人血管紧张素转换酶2（hACE2）结合的潜力，并在SARS-CoV-2假病毒内化模型中显示出抑制作用。在此，我们使用体外细胞模型确定了HSD和HST对抑制合胞体形成的作用。
在293T细胞中使用聚乙烯亚胺（PEI）转染后，通过免疫荧光染色（IF）确认刺突蛋白、hACE2和跨膜蛋白酶丝氨酸2（TMPRSS2）的异位表达。然后，用一组编码刺突蛋白/hACE2/TMPRSS2或刺突蛋白/hACE2的质粒转染细胞以诱导合胞体形成。转染后4-5小时用HSD/HST处理细胞，然后再孵育16-18小时。使用倒置显微镜或高内涵筛选（HCS）平台观察合胞体。使用方差分析（Bonferroni）对从合胞体形成试验获得的数据进行统计分析。
通过IF染色，我们成功观察到293T细胞中刺突蛋白、hACE2和TMPRSS2的表达。此外，通过人工观察合胞体，我们发现与对照细胞相比，10和100 μM的HSD显著抑制了中小尺寸合胞体的形成。在HCS试验中，10 μM的HSD对刺突野生型（WT）诱导的合胞体有抑制作用。相比之下，100 μM的HSD、10和100 μM的HST以及通过水力空化法（HCV）制备的含10 μg/mL HSD的柑橘皮提取物抑制了刺突奥密克戎变异株诱导的合胞体形成。
HSD和HST显示出对SARS-CoV-2细胞间传播的潜在抑制活性。需要进一步研究以确认抗病毒活性的作用机制。
