Protective Effect Of TBK-1 On Rotenone-induced Dopaminergic Neurons In Rats With Parkinson’s Disease And Its Related Mechanism

Objective: In this study,we pretreated rats by injecting the TBK-1 gene overexpression vector into the substantia nigra of rats,and explored the protection of TBK-1 against rotenone(Rotenone,Rot)induced dopaminergic neurons in Parkinson’s disease rats The role and related molecular mechanisms provide more experimental basis for finding targeted drugs for the treatment of Parkinson’s disease.Methods: There are 48 SPF Wistar male rats in four groups(n=12):(1)control group;(2)PD model group(Rot group);(3)adeno-associated virus empty group(AAV-GFP+Rot group);(4)Adeno-associated virus TBK-1 overexpression group(AAV-GFP-TBK-1+Rot group);the brain substantia nigra part of the latter two groups of rats were separated by the brain stereotaxic injection instrument Inject 2 μl of AAV-GFP blank vector and AAV-GFP-TBK-1 overexpression vector,and start subcutaneous injection of rotenone working solution in the back of the rat’s neck 3days later,at the 7th,14 th,21st and 28 th of the rotenone working solution.Open field experiments were conducted on rats in each group to observe the effect of TBK-1protein on behavioral characteristics;the rats were then sacrificed and the substantia nigra tissue was taken for molecular experiments to detect small TH+ neurons(dopamine)in the substantia nigra of rats Neurons)number,α-synuclein,glial cell activation marker Iba-1,inflammatory factors IL-1β,TNF-α expression levels,and RIPK-1 protein expression.Results:1.The open field test showed that compared with the PD model group,the number of times the rats in the TBK-1 overexpression group traversed the central area in the open field experiment within 5 minutes was significantly more on the 24 th day(P<0.05),indicating that TBK-1 protein can improve rotenone-induced autonomic behavior in Parkinson’s disease rats.2.Immunohistochemical staining and real-time fluorescence quantitative PCR related results show that compared with PD model group rats,overexpression of TBK-1protein can induce rotenone-induced loss of dopaminergic neurons in the substantia nigra of Parkinson’s disease rats.To a certain degree of protection,it also has a certain degree of inhibitory effect on rotenone-induced abnormal deposition of α-synuclein protein in the substantia nigra of Parkinson’s disease rats.3.Immunohistochemical staining and real-time fluorescence quantitative PCR related results show that: compared with PD model group,overexpressed TBK-1 protein can significantly reduce rotenone-induced activation of microglia in the substantia nigra of Parkinson’s disease rats Level.4.Real-time fluorescence quantitative PCR and Elisa protein quantitative detection results show that: compared with PD model group,overexpressed TBK-1 protein can significantly reduce rotenone-induced substantia nigra inflammation factor TNF-αexpression in Parkinson’s disease rats Levels,slightly reducing the expression level of the inflammatory factor IL-1β.5.Western-blot detection and real-time fluorescence quantitative PCR results show that: compared with PD model group,overexpressed TBK-1 can only slightly inhibit rotenone-induced midbrain substantia nigra RIPK-1 protein in Parkinson’s disease rats Expression level,but it can significantly inhibit the phosphorylation level of RIPK-1 protein-related sites,that is,the expression level of ser166-RIPK-1 protein was significantly reduced(P<0.05).Conclusions: 1.TBK-1 protein can improve the autonomic function of rotenone-induced Parkinson’s disease rats.2.TBK-1 has a certain degree of protection against rotenone-induced loss of dopaminergic neurons in the substantia nigra of Parkinson’s disease rats,and has a certain degree of inhibition on the abnormal deposition of α-synuclein protein.But it needs to be confirmed by increasing the sample size.3.TBK-1 protein can significantly inhibit rotenone-induced microglia activation in the substantia nigra of Parkinson’s disease rats,and reduce the expression level of TNF-α protein.4.The mechanism by which TBK-1 protein reduces rotenone-induced inflammation in Parkinson’s disease rats may be achieved by inhibiting the phosphorylation level of ser166-RIPK-1 protein.