| ObjectiveTo investgate the NF-κB/p65 short hairpin RNA has the capacity to ameliorate muscle pathologies in mdx mice and provide evidences for One-Step treatment of anti-inflammation and dystrophin gene replacement for Duchenne muscular dystrophy.MethodsThe construct of pAAV-D (+)-U6-NF-κB/p65 shRNA-CMV-ZsGreen plasmid DNA was made. The NF-κB shRNA virus and control shRNA virus were made according to a three-plasmid co-transfection and purified through CsCl density gradient ultracentrifugation. Finally the virus titers were tested by DNA dot-blot method. Mouse macrophage RAW cells were transfected with AAV plasmid DNAs by a Lipofectamine Kit according to a standard protocol.Then the cells were collected for western blot to check the different protein levels of p65 unit in all groups. The 1- and 4-month-old mdx mice were arranged to different groups (4 mice each group) and were i.m injected by 50μl AAV9-p65 shRNA-ZsGreen vectors into right GAS muscle and left one were treated by the same volume of PBS. In the control shRNA group the mice right GAS muscle were injected by 50μl AAV9-control shRNA-ZsGreen and left GAS muscle were done by the same volume of PBS as control. One month after virus treatment all the mice were sacrificed in CO2 container. The right and left GAS muscle were cut into 10μm crysection for histological analysis. qPCR and immunofluorescently stain were used to check the level of phosphor-p65. HE staining was used to compare the morphology of mice muscle directly.Mouse IgG staining showed the muscle nercosis and eMyHC staining showed the muscle regeneration in different group to investgate the role of NF-κB shRNA treated in mdx mice.Evans Blue Dye was injected to tail vein to test the integrity of myofibor membrance.ResultsThe AAV-NF-κB/p65 shRNA-ZsGreen construct was made and pure AAV9 vectors for experiments were manufactured and purified through CsC1 density gradient ultracentrifugation. Western blot test showed NF-κB/p65 shRNA can decrease the p-p65 level of mouse macrophage RAW cell even the inflammation was actived by LPS. HE staining suggested that there were fewer mononuclear cells (infiltrating immune cells) in the GAS muscles of the p65-shRNA-treated group compared to control shRNA group or PBS group. Moreover, the morphology of the p65-shRNA-treated mdx muscles was significantly improved when compared to the muscles of age-matched wild-type C57BL/6J mice. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67% to 34% compared to the ct-shRNA-treated or phosphate-buffered saline (PBS)-treated mdx mice. The results also demonstrated that NF-κB/p65 shRNA could down- regulate the p-p65 at the mRNA level and protein level. Both of muscle necrosis suggested by mouse IgG staining and muscle fibrosis showed by Masson Trichrom staining were significantly decreased by NF-κB/p65 shRNA, especially in young mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Importantly the NF-κB/p65 shRNA could reduce the Evans Blue Dye permeating into myofiber and it testified that NP-κB/p65 shRNA can improve membrane integrity of mdx muscle. ConclusionThe rAAV based NF-κB/p65 shRNA technology can ameliorate muscle pathologies through decreasing the chronic inflammation in mdx muscle. It can improve muscle morphology, reduce muscle necrosis and fibrosis.It is important to testify that NF-κB/p65 shRNA can defer the pathological process of myofiber degeneration, resulting in muscle regeneration increased in old mdx mice. More importantly, NF-κB/p65 shRNA can improve integrity of myofiber membrance. It may provide novel evidence to support the feasible therapy of anti-inflammation and dystrophin gene replacement in One-Step treatment.
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