Pharmacodynamics And Proteomics Research Of TBN In SOD1^G93A Mouse Amyotrophic Lateral Sclerosis Model

Research objectivesTo investigate the therapeutic effect of ligustrazine derivative TBN on amyotrophic lateral sclerosis in SOD1G93A transgenic mice.Pole-climbing test,hangingtest and grip strength testwere used to determine the drug’s ability to exert influences.The time of onset of the mice and the survival time of the mice were recorded,and the pathophysiological changes of the mice after administration were analyzed by proteomics.Study MethodsPart 1:Exploring the therapeutic effects of TBN and Edaravone on SOD1G93A transgenic mice.In this experiment,a human-derived SOD 1G93A(SOD 1 mutation)transgenic mouse model was used.Female mice were dosed from 13 weeks and male mice were dosed from 12 weeks.The test mice were divided into seven groups.Normal control group(Wild type+saline),normal administration group(Wild type+TBN 30 mg/kg),hSOD1G93A model group(hSODlG93A+saline),TBN low-dose group(hSOD1G93A+TBN 10 mg/kg),TBN medium-dose group(hSOD1G93A+TBN 30 mg/kg),TBN high-dose group(hSOD1G93A+TBN 60 mg/kg),positive control drug Edaravone group(hSOD1G93A+Edaravone 15 mg/kg)group.Mice in each group were administered for 4 weeks.Before the drug administration(female 13 weeks old,male 12 weeks old),and after the end of the administration(4 weeks later),Pole-climbing test,hangingtest and grip strength test were used to detect exercise ability and muscle strength of the limbs of mice.After the experiment,the spinal cords of mice were taken for proteomics experiments to investigate the effects of TBN and Edaravone on the molecular level of mice.The survival of mouse spinal anterior horn motor neurons was evaluated by Nissl staining.Part 2:Exploring the effects of TBN and Riluzole and their combination on the survival rate of hSOD1G93A transgenic mice.In the experiment,a human-derived SOD1G93A(SOD1 mutation)transgenic mouse model was also used.The experiment was completed after 14 weeks of drug administration from 6 weeks of age to observe the death of the mice.The weight of the mice was recorded every three days,and the exercise ability and muscle strength of the limbs were measured by pole-climbing test,hanging test and grip strength test.The mice were checked for morbidity every three days.Mice were divided into 5 groups:normal control group(Wild type+Saline),SOD1 model group(hSOD1G93A+Saline),TBN medium-dose group(hSOD1G93A+TBN 30 mg/kg),and positive control drug riluzole group(hSOD1G93A+Riluzole 5mg/kg),combined administration group(hSOD1G93A+Riluzole 5mg/kg+TBN 30mg/kg).Experimental resultsFirst part:Behavioral evaluation:before drug administration,the behavioral indicators of the normal group were not significantly among different groups.Treatment with TBN(30mg/kg and 60 mg/kg)and Edaravone significantly improved the motor coordination ability of mice and muscle atrophy process.Survival of motor neurons in the anterior horn of the spinal cord:Statistics of the number of motor neurons in the anterior horn of the unilateral spinal cord indicate that there were 6.387±0.1269(n=16)motor neurons in the anterior horn of the spinal cord examined in wild-type mice.In untreated hSOD1G93A mice,the number was only 2.846±0.1019(n=16).Compared with the untreated group,the survival rate of motor neurons in SOD1G93A mice treated with TBN increased by 41.70%(10 mg/kg),55.23%(30 mg/kg),and 67.15%(60 mg/kg).Edaravone treatment also increased the survival rate of motor neurons by 63.13%.The experimental results show that TBN has a protective effect on motor neurons in the spinal cord anterior horn of hSOD1G93A mice.Proteomics analysis:Compared with the model group(hSOD1G93A),normal wild-type mice have obvious difference in biological processes such as muscle contraction,muscle system processes,and striated muscle development.Compared with the hSOD1G93A model group,the TBN 30 mg/kg group has a larger difference in the biological processes of inflammatory response and glucose metabolism.Through Heat map and String analysis,we speculated that TBN may treat hSOD1G93A mice by decreasing oxidative stress,inflammatory response,and inhibiting apoptosis.The second part:Body weight:Compared with the normal control group(Wild type+Saline),the body weight of the model group(hSOD1G93A+Saline)decreased significantly after the onset of hSOD1G93A mice.The treatment of TBN 30 mg/kg had no effect on the body weight of hSOD1G93A mice.Behavioral evaluation:There was no significant difference in every groups before administration.After the onset of mice,the behavioral group of TBN 30 mg/kg and co-administration group(hSOD1G93A+Riluzole 5mg/kg+TBN30mg/kg)was better than the model group(hSOD1G93A+Saline).Survival rate:TBN 30 mg/kg,Riluzole 5mg/kg and the combined administration group had a longer onset time than the model group(hSOD1G93A+Saline),but TBN 30 mg/kg had no effect on the survival time of hSOD1G93A mice.Experimental conclusion and significanceTBN 30 mg/kg and 60 mg/kg have a better improvement in the exercise ability of hSOD1G93A mice,which can delay the progress of mouse atrophy and improve the survival rate of spinal motor neuron.TBN 30 mg/kg can delay the onset time of mice,but TBN 30 mg/kg had no effect on the survival time of hSOD1G93A mice.TBN may treat amyotrophic lateral sclerosis by regulating inflammation,oxidative stress,and inhibiting neuronal apoptosis.TBN can increase the expression of Tpm2,Myh3,Tnnc2 and Casq2 in hSOD1G93A mice to treat hSOD1G93A mice.The study compared the efficacy of TBN and two marketed drugs(Riluzole and Edaravone)and analyzed the possible mechanism of TBN treatment of ALS.Providing reference value for further preclinical research and clinical research.