在細胞和動物模式下金銀花茶及其誘發表現的微小RNA let-7a對登革二型病毒的抑制效果

Abstract

Dengue virus (DV), a flavivirus of Flaviviridae, a positive strand RNA virus with envelope, causes diseases from mild dengue fever to severe dengue hemorrhagic fever and dengue shock syndrome worldwide. The study of WHO showed that dengue virus causes severe infection per year. Limited to different sero-types and antibody-dependent enhancement, there is no effective vaccine for prevention and antiviral agent for therapy of dengue diseases. Therefore, it is noteworthy to find a natural compound for prevention or therapy of dengue virus infection. It is known that blood let-7a was up-regulated in 9-week-old C57BL/6 male mice with feeding honeysuckle (Hs). Does Hs inhibit DV2 infection via let-7a induction? MicroRNAs are on average 22 nt long small RNA molecules. MicroRNAs are able to control viral infection has been reported. It is interesting to identify the microRNA which can inhibit DV2 infection. A Chinese herb honeysuckle (Lonicera japonica; Hs) is generally used to treat wind-heat, sores, and epidemic diseases. Hs also found to inhibit HSV, HIV, RSV, influenza A virus, and SARS. let-7a ,one of up-regulated microRNAs after Hs treatment, was predicted to target DV2 NS1 gene(3313-3333) via Vita website. let-7a induction in blood of 6-day-old suckling mice was stimulated by Hs treatment, indicating that Hs inhibited DV2 replication via let-7a induction. Our study demonstrated that let-7a decreased luciferase activity of the reporter plasmid with DV2 NS1 target sequence. Our Huh7 cell line experiment showed that let-7a inhibited DV2 NS1 RNA (40%) and protein synthesis (20%), as well as DV replication determined by decreased negative strand RNA synthesis. It indicates that let-7a suppressed DV2 replication. In animal studies, Hs pretreatment followed by intracranially inoculated(i.c.) with DV2(2.5 x 105 PFU), showed that Hs delayed disease formation, decreased disease severity. DV2 NS1 RNA (20%) and protein expression (70%), and DV2 titer (45%) was decreased in the brain. In Hs therapy study, mice were i.c. injected with DV2 before feeding Hs. Our data showed that Hs prolonged survival time, delayed disease symptoms, and decreased disease severity of the infected mice. DV2 NS1(78%) and protein expression (52%) , and viral titer (44%) were decreased in mice brain. Above data demonstrated that Hs has an inhibitory effect on DV2 replication. The suckling mice were injected let-7a twice by i.c. after infected with DV2. These showed that let-7a delayed disease formation, reduced DV2 NS1 RNA (26%) and protein expression (50%), and had more than 90% inhibition on viral titer. Above data showed that Hs inhibitory effect on DV replication was related to let-7a stimulation. In conclusion, we are the first to show that honeysuckle can inhibit DV2 infection possibly via let-7a induction. Moreover, let-7a inhibits DV2 replication by directly binding its target site in CDS of DV2 NS1 gene. Our finding may shed light on developing new DV2 prevention and healthy food for dengue virus prevention and therapy.