Isoflurane and Netrin-1 combination therapy enhances angiogenesis and neurological recovery by improving the expression of HIF-1α-Netrin-1-UNC5B/VEGF cascade to attenuate cerebral ischemia injury

Ischemic stroke (IS) causes many morbidities and deaths worldwide. However, the current monotherapy strategy is not satisfactory. Therefore, it is urgent to explore possible combined treatment methods. Although both isoflurane (ISO) and Netrin-1 (NT-1) have angiogenesis and neuroprotective effects, it is still unclear whether combining ISO with NT-1 will provide a positive effect and the possible mechanism of action. In this study, we used a photochemical (PTI) method to establish a mouse ischemic stroke model. ISO and NT-1 were used to treat the mice for 1 week. The adhesive removal test, Morris water maze test, modified neurological severity scores and triphenyl tetrazolium chloride staining were performed to test the treatment effect. Western blotting was performed to assess protein expression, immunofluorescence staining (IF) and immunohistochemical staining (IHC) was used to evaluate angiogenesis. The results suggested that combining ISO with NT-1 resulted in a better therapeutic effect than ISO or NT-1 treatment after PTI injury (all P < 0.01). The protein expression of VEGFA and CD34 in the ISO + NT-1 group was significantly increased compared with that in the other groups (all P < 0.05). IF and IHC also showed that the ISO + NT-1 group significantly improved angiogenesis (all P < 0.01). YC-1 (an HIF-1α inhibitor) and Unc5B siRNA were used to inhibit the expression of HIF-1α and UNC5B before and after combination ISO and NT-1 treatment. The combined inhibition group not only expressed the least VEGFA and CD34 but also expressed the least HIF-1α, UNC5B, FAK, and β-catenin in all groups (all P < 0.05). Most importantly, angiogenesis and neurological recovery were also significantly decreased by inhibiting HIF-1α and UNC5B (all P < 0.05). In conclusion, our results suggested that ISO combined with NT-1 could promote angiogenesis, recover long-term neurobehavioral function, and attenuate cerebral ischemia injury by activating the HIF-1α-Netrin-1-UNC5B/VEGF cascade.