Study On The Inhibitory Effects Of Src Tyrosine Protein Kinase Inhibitors On Aβ Toxicity In AD C.elegans

Alzheimer’s disease is an age-related neurodegenerative disease,associated with the presence of extracellular amyloid-β（Aβ）plaques and neurofibrillary tangles.Although the pathogenesis of AD remains unclear,the characteristic feature of AD was reported to be the buildup of Aβplaques.Aβaccumulation induced impairing cognitive and memory loss was proved in AD patient’s brain in clinical research.There are many people suffering from AD throughout the world,and the prevalence still keep alarmingly increase,which bring an enormous financial burden for patients.Therefore,it is of great importance to discovery effective drugs for anti-Alzheimer’s disease.The previous study showed that Src tyrosine protein kinase is a major signal enzyme of neuronal growth cone,and it is a key regulatory molecule in axon regeneration of damaged central nervous system.Moreover,Src tyrosine protein kinase has a potential regulation mechanism between Aβdeposition and Tau protein in AD and it can pass through the blood-brain barrier.This study aimed to investigate the inhibiory effects of Src inhibitor（SAFA320）on transgenic C.elegans CL4176,as well as its mechanism of action.The results showed that SAFA320 administration worms showed remarkably decreased in paralysis,less Aβplaque deposition comparing to that of the blank control group.The effect concentration of SAFA320 was in the descending order of 1μM<10μM<100μM.Therefore,100μM of compound 320 was chosen for the subsequent research.The results also showed that SAFA320 at the concentration of 100μM could improve the survival state of C.elegans N2,comparing with that of the blank control group.Results of tissue Src kinase activity spectrophotometry analysis showed that SAFA320 could reduce the activities of Src in nematodes.In terms of Aβtoxicity,the deposition of Aβplaque by Thioflavin T staining analysis indicated that it was much more in the head area of the untreated C.elegans than those in the SAFA320 treated CL4176.H₂DCF-DA fluorescence probe results demonstrated that compound 320significantly reduced the content of ROS in nematodes.In order to find the mechanism of SAFA320 on Aβtoxicity,Aβ,skn-1,daf-1 and hsf-1 gene expressions in m RNA levels were determined by real-time Quantitative PCR and actin-1 was used as keep-house gene.Genetic analyses showed that there was no significant difference in the relative expression of Aβm RNA level between SAFA320 and the blank control group.It suggests that SAFA320 reduced toxic protein deposition may regulate by post-transcription level,not transcription level.On contrary,the genes expression of skn-1and its downstream target gene gst-4 were significantly increased which revealed that SAFA320 inhibition of Aβtoxicity may involve in skn-1 signaling pathway in CL4176.In a word,the results showed that Src inhibitor（SAFA 320）could inhibit the toxicity of Aβ_1-42,significantly delay the paralysis,inhibit the deposition of Aβ.Moreover,this inhibition may be related to the ability of decrease the oxidative stress induced by Aβand may associate with skn-1 signaling pathway.In summary,Src inhibitor SAFA320 is a potent candidate agent Alzheimer’s disease.