TSG-6 Inhibited The HCMV Infection-induced Expression Of TNF-α And IL-6

Objective To detect the expression levels of tumor necrosis factor a stimulated gene 6(TSG-6) in cells which were infected by human cytomegalovirus (HCMV); To reaserch the role of TSG- 6 in the inflammatory responses induced by HCMV and explore whether TSG-6 can change the localization of nuclear factor-kappaB(NF-κB). Try to provide new ideas for mechanisms of HCMV immune evasion and the treatment of inflammatory diseases caused by HCMV.Methods (1) The proliferation of HCMV on human fibroblasts were identified by assays of PCR,IFA and electron microscopy, then the the virus titers were detected by experiments of plaque forming assay.(2) Human lung fibroblast cells (MRC-5) and human retinal pigment epithelial cells (ARPE-19) were infected by HCMV. A normal cell control group and an inactivated virus infection group were set up at the same time. At the time-points of 24 h, 48 h,72 h,96 h and 120 h post infection, total RNA and protein were extracted from the infected cells. RT-qPCR analysis and Western blot assay were performed to measure the expression of TSG-6 at mRNA and protein levels in each group to detect whether TSG-6 was involved in the inflammatory reactions caused by HCMV.(3) The recombinant human TSG-6 protein (rhTSG-6) was administrated to the HCMV-infected MRC-5 cells at the concentration of 100 ng/mL.At the same time, a normal cell control group and a virus control group were set up for comparison. The supernatants were harvested at 24 h,48 h,72 h,96h and 120 h post virus infection and the expression levels of TNF-a and IL-6 were measured by ELISA, and the results were verified that if TSG-6 had the anti-inflammatory effect in HCMV-induced inflammatory responses.(4) Comparing with the virus control group, The levels of nuclear localization and expression of the NF-κB after rhTSG-6 dosing for 6h were detected by IFA and Western Blotting experiments. The effects that whether TSG-6 can change the NF-κB nuclear localization at 24 h,48 h,72 h,96 h and 120 h post virus infection were discussed.Results:(1) After virus infection,the cytopathic effect was significantly observed on cells. The phenomenon of cells swelling round and the refraction were enhanced and HCMV UL83 and UL86 genes were detected by PCR; Also,HCMV pp65 antigen was detected by IFA and green fluorescence signals were observed under a fluorescent microscope; The typical morphological characteristics of HCMV were confirmed by Transmission electron microscopy.(2) The mRNA level of TSG-6 at in HCMV-infected MRC-5 cells group were 19 (tD=17.12, P<0.01) and 376 (tD=373.15, P<0.01)times of that in normal cell group at the time points of 72 h and 96 h post virus infection. But it began to decline at the time point of 120 h, and reduced to 113 (tD=111.25, P<0.01) times of that in cell control group.The mRNA level of TSG-6 in HCMV-infected ARPE-19 cells were 15 (tD= 14.25, P<0.01)and 354 (tD=352.42,P<0.01) times of that in normal cells at the time points of 96 h and 120 h post infection. The TSG-6 mRNA levels in normal cell group and inactivated virus group were rare, No significant changes were found at different time points. The Western blot assay further confirmed the above results of TSG-6 at the protein expression level for the corresponding time points.(3) Comparing with the virus control group, in the group of both rhTSG-6 and HCMV-treated MRC-5 cells, the levels of TNF-α and IL-6 in the supernatants of the cell culture decreased significantly (P<0.05)。(4) The results of IFA showed that rhTSG-6 protein could suppress the nuclear translocation of NF-κB; The result of Western Blot showed that the protein expression of NF-κB p65 was significantly reduced compared with virus control group in different time.Conclusion (1) TSG-6 was involved in the course of HCMV infection-induced inflammation responses and presented high expression in late stage of HCMV infection;(2) TSG-6 could inhibit the inflammation caused by HCMV and played a important role in reducing the expression of HCMV infection-related inflammatory factors; (3) TSG-6 could inhibit the nuclear translocation of NF-κB induced by HCMV.