Mechanism Of TSPO Involved In Early Cognitive Decline Through Mitochondrial-mediated Astrocyte Apoptosis

Part Ⅰ Analysis of TSPO expression in hippocampus of aMCI ratsObjectiveTo investigate the expression and expression of TSPO in hippocampus of aMCI model rats induced by D-gal.MethodsMale Sprague-Dawley rats were randomly divided into control group(n=6)and D-gal group(aMCI group,n=6).The aMCI group was continuously injected with D-gal 1000 mg·kg-1·d-1 dissolved in sterile saline for 7 days;The Control group injected the corresponding volume of solvent.The dose of D-gal injection was based on the results of the previous experiments.After the end of D-gal injection,a 5-day water maze positioning navigation experiment was started,and then a space exploration experiment was performed 1 day after the end.After the behavioral test,the brain was taken and pathological HE staining was performed separately.Hippocampus was taken from the remaining rats in each group,and the expression of TSPO protein in hippocampus was detected by Western-Blotting.Results1.MWM behavior test:as shown in the track chart,the movement patterns of rats in the aMCI group were mostly "edge type→random type",and occasionally "trend type→linear type";Rats in the Control group were more frequently presented with "trend type" and "straight-line type".The photographic records showed that the aMCI group rats made fewer stopover observation movements and less crossing the middle quadrant.In the positioning navigation experiment,the escape latency time of the aMCI group was significantly prolonged compared with the Control group(F=57.956,P<0.001).In the space exploration experiment,compared with the Control group,rats in the aMCI group spent less time in the target quadrant of the original platform of the water maze and less swimming distance in the target quadrant.Compared with the Control group,the aMCI group traversed the platform less frequently(F=18.443,P=0.002).2.Pathological sections were observed by HE staining and it was found that compared with the Control group,the density of vertebral cells in the CA1 area of the hippocampus in the aMCI group decreased significantly,and the arrangement of vertebral cells was obviously loose and disordered.Some of the cells showed apoptosis,and there was a certain degree of nucleus fixation and deep staining.3.Compared with the Control group,the expression level of TSPO in the hippocampus of the aMCI group was increased(F=202.596,P<0.001).ConclusionD-gal can induce aging in the brain and lead to early impairment of cognitive function.The possible mechanism is associated with increased expression of TSPO protein and its associated downstream regulatory protein activation.Part Ⅱ The protective effect of TSPO ligands on D-gal-induced amnesic mild cognitive impairment in ratsObjectTSPO ligand PK11195 inhibits d-gal induced astrocyte apoptosis in rats and its possible mechanism.MethodsRats were randomly randomized according to body weight:Control group(n=10),D-gal group(aMCI group,n=10),PK11195+D-gal group(n=10),PK11195 group(n=10).PK11195+D-gal group and PK11195 group were intraperitoneally injected with PK11195(3mg/kg).Three days after PK11195 injection,the D-gal group and the PK11195+D-gal group began to inject D-gal into the head and neck for 7 consecutive days.The D-gal injection dose is the same as in the first part,and PK11195 uses the dose reported in the reference.The 5-day water maze navigation experiment was started 1 day after d-gal injection,and the space exploration experiment was conducted 1 day after the completion.After the behavioral test,the brain was taken and pathological staining(HE staining and silver staining)and immunofluorescence were performed.The hippocampus tissues were taken from the remaining rats in each group.Western blotting was used to detect the expression of TSPO protein and mitochondria-induced apoptosis proteins Cyt C and AIF in hippocampus.Results1.In MWM training experiment,the trajectory diagram shows that the aMCI group mainly adopts "edge→random→trend";The Control group and PK11195 group were mainly "trend→straight" or "random→trend→straight".In PK11195+D-gal group,"edge→random or random→trend→straight" was the main expression.The results of positioning experiments showed that during the first day of training,the aMCI group rats took longer to locate the platform.On the fifth day of training,the aMCI rats had to swim a longer distance to locate the platform.There was no significant difference between the Control group and PK11195+D-gal group(P>0.05).Compared with the Control group,the latency of escape was prolonged in the aMCI group(t=-5.337,P<0.05).Compared with the Control group,the PK11195 group showed a shorter escape latency(t=2.255,P<0.05).In the space exploration experiment,compared with the Control group,the number of crossing the platform was significantly lower in the aMCI group(P<0.05).Compared with aMCI group,rats in PK11195+D-gal group had more crossing times(P=0.019,P<0.05).There was no significant difference in crossing times between the PK11195 group and the Control group(P>0.05).2.Pathological staining results showed that the aMCI group showed obvious pathological changes of pyramidal cells under the microscope after HE staining.Silver-plated staining showed that the AS arrangement density around the hippocampus of the aMCI group decreased and some cells underwent apoptosis.3.Immunofluorescence results showed that compared with the Control group,the expression level of TSPO in the hippocampus CA1 of the aMCI group was significantly increased(F=30.547,P<0.001).There was no significant difference in TSPO protein expression between PK11195+D-gal group and Control group(P>0.05).There was no significant difference in TSPO protein expression between the PK11195 group and the Control group(P>0.05).4.Compared with the Control group,the expression levels of TSPO(P<0.05),Cyt C(P<0.001)and AIF(P<0.05)in the hippocampus of the aMCI group were increased.Compared with aMCI group,the expression levels of TSPO(P<0.05),Cyt C(P<0.001)and AIF(P<0.05)in PK11195+D-gal group were decreased.ConclusionPK11195 may protect against early mild cognitive impairment caused by short-term high-dose injection of D-gal.The possible mechanism is that PK11195 down-regulates the TSPO expression in the hippocampus CA1 region,reduces the accumulation of mitochondrial apoptosis-related proteins Cyt C and AIF,and finally alleviates the apoptosis of astrocytes.