The Protective Effects Of Dl-3-n-butylphthalide Against Mitochondrial Injury Caused By HSPB8K141N Mutation

Background:Charcot-Marie-Tooth disease (CMT), which is also called hereditary motor and sensory neuropathy is the most common inherited peripheral nerve disorders. According to the pathological and electrophysiological characteristics CMT can be divided into three types:demyelinating form (CMT1type), axonal form (CMT2type) and intermediate form (ICMT type). Point mutations (423G�'T) in small heat shock protein B8(HSPB8) gene was shown to cause CMT2L. Our previous study has shown that mutant HSPB8caused mitochondrial aggregation and the reduction of mitochondrial resistance to stress.dl-3-n-butylphthalide (NBP), a novel agent extracted from seeds of Apium graveolens, has protective effects against ischemic damage in brain by promoting cerebral blood flow, improving mitochondrial function, increasing ATPase activity in neurons’ mitochondria and reducing neuronal injury to protect neuronal function. We speculate that NBP may have a protective effect on mitochondrial injury caused by HSPB8K141N mutation.Objective: To investigate the effect of mutant HSPB8on mitochondria, use NBP pretreat at the same time and evaluate the protective effects of NBP against mitochondrial injury caused by HSPB8K141N mutation.Methods:(1) The distribution of mitochondria in sural nerve from CMT2L patient was observed by transmission electron microscopy (TEM)(2) Overexpressed wild type (WTHSPB8) and K141N mutant HSPB8(K141NHSPB8) in SH-SY5Y cells, after pretreatment with NBP of different concentrations (1、10、100μmol/L), the cell viability was measured by CCK-8.(3) Overexpressed WTHSPB8and K141NHSPB8in SH-SY5Y cells and pretreated with10μmol/L NBP or not, the distribution of mitochondria was observed by TEM.(4) Overexpressed WTHSPB8and K141NHSPB8in SH-SY5Y cells and pretreated with10μmol/L NBP or not, the reactive oxygen species (ROS) formation was measured when cells were exposed to H2O2stimulation by flow cytometry.(5) Spinal motor neurons infected with WTHSPB8and K14INHSPB8and pretreated with10μmol/L NBP or not, the number of neurite was observed by immunocytochemistry.Results:(1) TEM showed abnormal mitochondrial aggregation in sural nerve from CMT2L patient.(2) NBP can inhibit the decrease of cell viability caused by HSPB8K141N mutation in SH-SY5Y cell. Cell viability increased after pretreated with NBP. A pretreatment with10μmol/L NBP could significantly inhibit the decrease of cell viability caused by HSPB8K141N mutation (P<0.001).(3) The mitochondria distribution was normal in SH-SY5Y cells overexpressed WTHSPB8. NBP can inhibit mitochondrial aggregation caused by K141NHSPB8.(4) The mitochondrial resistance to oxidative stress was impaired by K141N mutation in HSPB8. ROS formation was higher in cells overexpressed K141NHSPB8than WTHSPB8when cells were exposed to an oxidative stress induced by H2O2(P<0.05). A pretreatment with10μmol/L NBP could significantly reduce ROS formation caused by K141NHSPB8(P<0.05).(5) The number of neurite was decreased in motor neurons overexpressed K141NHSPB8. A pretreatment with10μmol/L NBP could inhibit the decrease of neurite number caused by K141N HSPB8in spinal motor neurons (P<0.01).Conclusions:NBP can inhibit the decrease of cell viability caused by HSPB8K141N mutation and promote neurite outgrowth. NBP exhibits protective effects through increasing mitochondrial resistance to an oxidative stress, inhibiting mitochondrial aggregation to against mitochondrial abnormality caused by HSPB8K141N mutation.