| Parkinson’s disease(PD)is a common neurodegenerative disease with clinical manifestations of movement disorders such as bradykinesia,rigidity and resting tremor.Its pathological features are mainly the formation of Lewy bodies(LBs)and the loss of substantia nigra(SN)dopamine(DA)neurons.Circular RNA(circ RNA),as a new type of non-coding RNA,is abundantly expressed in the nervous system and is widely involved in the occurrence and development of neurological diseases.However,the specific functions and regulatory mechanisms of circ RNAs in PD still need to be further studied.The early research of our group found that knocking down circ-Pank1 in cells can reduce the expression of α-syn,and the misfolding and aggregation of α-syn is an important sign of PD pathological changes.Therefore,we speculate that circ-Pank1 may play an important role in dyskinesia in PD model mice.Objective: This experiment aims to study the expression changes and possible mechanism of circ-Pank1 in PD model mice,and to provide new ideas and experimental basis for the treatment of PD in the future.Methods:1.Establish PD model mice and observe the expression changes of circ-Pank1.Male ICR mice(25-30 g,3-4 months old)were randomly divided into 2groups: control group(Veh group),rotenone administration group(Rot group),8 mice in each group,Mice in the Rot group were given intragastric administration at 30 mg/kg/day for 28 consecutive days,and the Veh group was given the same dose of solvent solution.28 days after the administration,the open field experiment,pole climbing experiment,and step measurement methods were used to observe the changes in the exercise ability of PD model mice;Using immunohistochemistry,Western Blot and other techniques to detect the expression changes of tyrosine hydroxylase(TH),α-syn and its oligomers in the substantia nigra of PD model mice;The q RT-PCR technology was used to detect the expression changes of circ-Pank1 in the substantia nigra of PD model mice.2.The effect of knocking down circ-Pank1 in substantia nigra brain area on the exercise ability of PD model mice.The PD model mice were randomly divided into 2 groups: PD model mice + GFP adeno-associated virus control group(Rot+AAV-GFP group)and PD model mice + knockdown circ-Pank1 adeno-associated virus group(Rot+AAV-sh-circ-Pank 1 group),8 in each group.Using stereotactic injection of brain,adeno-associated virus vector(AAV-sh-circ-Pank1)with specific knockout of circ-Pank1 and adeno-associated virus vector(AAV-GFP)of control GFP were injected into the substantia nigra of PD model mice,respectively.Using q RT-PCR technology to detect the knockdown efficiency of circ-Pank1.Through open field experiment,pole climbing experiment and step measurement method,the changes of exercise ability of Rot+AAV-GFP group mice and Rot+AAV-sh-circ-Pank1 group mice were detected.3.After knocking down circ-Pank1,the expression changes of TH,α-syn and their oligomers in the substantia nigra of PD model mice.Using immunohistochemical techniques,the changes in the positive expression of TH and oligomers in the substantia nigra of mice in the Rot+AAV-GFP group and Rot+AAV-sh-circ-Pank1 group were detected;Western Blot and Dlot Blot technology,detect the protein expression changes of TH,α-syn and its oligomers.Results:1.The expression of circ-Pank1 in the substantia nigra of PD model mice is increased.The results of the open field experiment showed that compared with the Veh group,the time spent in the central area,the total distance and the average speed of the Rot group were reduced;the results of the climbing pole experiment showed that the time spent climbing the full length of the rod in the Rot group was compared with that of the Veh group Increased;the step distance measurement results showed that the step distance of the front paw and hind paw of the Rot group was shortened compared with that of the Veh group.The results of immunohistochemistry showed that,compared with the Veh group,the expression of TH in the substantia nigra of the Rot group was reduced,and the expression of oligomers was increased;The results of Western Blot and Dlot Blot showed that,compared with the Veh group,the expression of TH protein in the substantia nigra of the Rot group was reduced,the protein expression of α-syn increased,and the expression of oligomers increased.The q RT-PCR results showed that the expression of circ-Pank1 in the substantia nigra in the Rot group was significantly higher than that in the Veh group.2.Injection of AAV-sh-circ-Pank1 into the substantia nigra brain area can improve the motor dysfunction of PD model mice.After injecting AAV-sh-circ-Pank1 into the substantia nigra brain area in stereotactic brain,q RT-PCR results showed that compared with the Rot+AAV-GFP group,the substantia nigra of mice in the Rot+AAV-sh-circ-Pank1 group The expression of circ-Pank1 in the brain area was significantly reduced.The results of the open field experiment showed that compared with the Rot+AAV-GFP group,the Rot+AAV-sh-circ-Pank1 group had no change in the central area residence time,the total distance increased,and the average speed increased;The rod climbing experiment results showed that the time spent climbing the full length of the rod in the Rot+AAV-sh-circ-Pank1 group was reduced compared with that in the Rot+AAV-GFP group;The step measurement results showed that in the Rot+AAV-sh-circ-Pank1 group,compared with the Rot+AAV-GFP group,the step length of the front paw of the mice increased but the step length of the hind paw did not change.3.After AAV-sh-circ-Pank1 was injected into the substantia nigra brain area,the expression of TH in PD model mice increased,and the expression ofα-syn and its oligomers decreased.The results of immunohistochemistry showed that,compared with the Rot+AAV-GFP group,the expression of TH in the substantia nigra of mice in the Rot+AAV-sh-circ-Pank1 group increased,and the expression of oligomers decreased;The results of Western Blot and Dlot Blot showed that compared with the Rot+AAV-GFP group,the expression of TH protein in the substantia nigra of mice in the Rot+AAV-sh-circ-Pank1 group increased,and The protein expression of α-syn and its oligomers is reduced.Conclusions:1.The expression of circ-Pank1 in the substantia nigra of PD model mice is elevated.2.Knockdown of circ-Pank1 in the substantia nigra brain area can improve the motor dysfunction of PD model mice.3.Knockdown of circ-Pank1 in the substantia nigra of PD model mice can increase the expression of TH,and decrease the expression of α-syn and its oligomers. |
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