Research On The Neuroprotective Effects Of Cannabidiol In Experimental Models Of Amyotrophic Lateral Sclerosis

Objective:.Amyotrophic lateral sclerosis(ALS)is a human neurodegenerative disease characterized by selective loss of motor neurons in the motor cortex of the brain and spinal cord,and it is both idiopathic and fatal.Currently,the range of drug options for treating ALS is limited,and cannabidiol(CBD),a promising candidate compound for treating neurodegenerative and psychiatric disorders,has garnered significant attention.However,there have been few reports on its neuroprotective effects in ALS models.Therefore,investigating the neuroprotective effects of CBD on ALS disease models and further elucidating its underlying mechanisms would be beneficial for the treatment of neurodegenerative diseases,including ALS.Some studies have reported a certain degree of neuroprotective effects of CBD in multiple sclerosis and ischemic brain injury.We aim to further investigate whether CBD can exert neuroprotective effects in ALS and explore potential mechanisms of action.Methods:(1)Established a cellular oxidative stress model in NSC34 mouse spinal motor neuron cells by stimulating with hydrogen peroxide(H2O2).The effects of CBD on cell viability after H2O2-induced injury were assessed using the MTT cell proliferation assay kit and Cell Counting Kit-8(CCK-8)cell viability assay kit.The protective effects of CBD against neuronal oxidative stress were evaluated using the malondialdehyde(MDA)assay kit,reactive oxygen species(ROS)assay kit,MitoSOX assay kit,and tetramethylrhodamine ethyl ester(TMRE)assay kit.(2)We selected transgenic SOD1G93A mice as an in vivo model for ALS and administered 10 mg/kg CBD via intraperitoneal injection daily starting at day 63 before the onset of symptoms.We recorded the body weight of the mice prior to daily injections and conducted weekly behavioral experiments,including the rotarod test,pole test,and open field test,to evaluate the effects of CBD on delaying the progression of ALS and enhancing motor function.We evaluated the neuroprotective effects of CBD using immunofluorescence(IF)and Nissl staining.Additionally,we measured changes in serum malondialdehyde(MDA)levels using an ELISA assay.(3)The changes in PGC-1α and AMPK pathway-related proteins in mouse cortex and spinal cord tissues were assessed using Western blot analysis.In the H2O2-induced oxidative stress model of NSC34 cells,the impact of CBD on the levels of JNK pathway-related proteins was examined using Western blot analysis.Results:(1)NSC34 mice motor neurons were stimulated with H2O2 to establish a cellular oxidative stress model.CBD(1 μM)could increase cell activity and reduce cell toxicity.(2)CBD(1 μM)was found to reverse the elevated levels of reactive oxygen species(ROS)and mitochondrial superoxide induced by H2O2 in NSC34 cells.Additionally,it was able to reverse the H2O2-induced decrease in mitochondrial membrane potential(MMP).(3)In the SOD1G93A mouse model,daily intraperitoneal injection of CBD(10 mg/kg)improved the latency to fall in the rotarod test and reduced the climbing time in the pole test.(4)Nissl staining and immunofluorescence results demonstrate that intraperitoneal injection of CBD(10 mg/kg)can reduce neuronal damage in layer 5 of the motor cortex and in the L4-L5 region of the spinal cord in SOD1G93A mice;simultaneously,it can also lower the level of MDA in the serum of SOD1G93A mice.(5)Intraperitoneal injection of CBD(10 mg/kg)upregulated the expression of PGC-1α、Nrf2 and HO-1 in the cortex and spinal cord of SOD1G93A mice,promoted AMPK phosphorylation,and downregulated JNK phosphorylation.(6)Western blot showed that CBD could increase the phosphorylation level of AMPK,decrease the phosphorylation level of JNK,and promote the expression of HO-1 in the H2O2-induced oxidative stress model of motor neurons.Conclusion:CBD exhibits the ability to inhibit H2O2-induced oxidative stress,thereby reducing neuronal damage.Furthermore,intraperitoneal injection of CBD enhances motor function and slows down disease progression in SOD1G93A mice.It is speculated that the neuroprotective effects of CBD against ALS model-induced neuronal damage may be correlated with PGC-1α and the JNK signaling pathway.