Leflunomide inhibits the apoptosis of human embryonic lung fibroblasts infected by human cytomegalovirus

Abstract

Background: The immunomodulatory drug leflunomide (LEF) is frequently used for treating human cytomegalovirus (HCMV), but its antiviral mechanism is still unclear. In this study,we therefore investigated the effects of the active LEF metabolite A771726 on the HCMV lifecycle in human embryonic lung fibroblasts. We clarified the mechanism of LEF antiviral infection, and provide a new way to treat immune dysfunction patients with HCMV infection.

Methods: The experiment was divided into four groups: the control group, the HCMV group, the ganciclovir+HCMV group as well as the LEF+HCMV group. MTT was used for assessment of the cell inhibitory rate. Apoptosis was measured by staining with fluorescein isothiocyanate Annexin V and propidium iodide. Statistical significance was determined by paired t-test using SPSS software.

Results: The results of the study showed that cell proliferation was significantly inhibited by HCMV at 24 hours and 48 hours. With increasing HCMV concentration, the value-added inhibition of the cells was significantly decreased compared with the control group, and was statistically significant (P<0.01). Ganciclovir can increase proliferation of cells infected with HCMV; compared with the control group it was statistically significant (P<0.05). Meanwhile, with LEF treatment cell proliferation was significantly improved at 24 hours and 48 hours, with statistical significance (P<0.05). The apoptosis rate of human embryonic lung fibroblasts infected with HCMV increased significantly at 24 hours, 48 hours and 72 hours, and as time goes on the apoptosis rate increases statistically significantly (P<0.01) compared with the control group The apoptosis rate of the HCMV infection group decreased by adding LEF,and was statistically significant (P<0.05).

Conclusions: In this study we show that LEF is an exciting new drug for cytomegalovirus infection. LEF significantly inhibited HCMV infection-induced apoptosis and proliferation, playing an important role in the treatment of patients infected by HCMV. In this study we explored the potential usefulness of LEF for cytomegalovirus infection and found it to be a cost-effective new treatment for cytomegalovirus infection that deserves further study.

Figure 1

Cell changes in cell morphology at 24 hours and 48 hours. (A) Control group (normal human embryonic lung fibroblast at 24 hours, microscope × 200). (B) Control group (normal human embryonic lung fibroblast at 48 hours, microscope × 400). (C) Human cytomegalovirus (HCMV) group (human embryonic lung fibroblast infected with HCMV at 24 hours, microscope × 200). (D) HCMV group (human embryonic lung fibroblast infected with HCMV at 48 hours, microscope × 400). (E) Ganciclovir (GCV) + HCMV group (GCVadded to the human embryonic lung fibroblast infected with HCMV at 24 hours, microscope × 200). (F) GCV + HCMV group (GCV added to the human embryonic lung fibroblast infected with HCMV at 48 hours, microscope × 400). (G) Leflunomide (LEF) + HCMV group (LEF added to human embryonic lung fibroblast infected with HCMV at 24 hours, microscope × 200). (H) LEF + HCMV group (LEF added to the human embryonic lung fibroblast infected with HCMV at 48 hours, microscope × 400).

Figure 2

Leflunomide inhibition rate of cytomegalovirus infection in each group at 24 hours.

Figure 3

Leflunomide inhibition rate of cytomegalovirus infection in each group at 48 hours. In this study, we found that the leflunomide (LEF) inhibition rate of cytomegalovirus (CMV) infection has obvious effects at 24 hours and 48 hours. Compared with the control group, the CMV infection group was statistically significant (P <0.05).

Figure 4

Apoptosis in each group at 24 hours, 48 hours and 72 hours.

Figure 5

Apoptosis rate of cells at 24 hours, 48 hours and 72 hours. At 24 hours, 48 hours and 72 hours, leflunomide (LEF) has a significant anti-human cytomegalovirus (anti-HCMV) infection. Compared with the control group, the apoptotic effect of cytomegalovirus (CMV) infectionwas significant (P <0.05).