| Parkinson's disease(PD)is the second most common neurodegenerative disease in the world.Its main pathological characteristics are the loss of dopaminergic neuronsand the accumulation of misfolded ?-synucleinthe in substantia nigra pars compacta.Autophagy is a highly conserved process of self degradation,which plays an irreplaceable role in the circulatory system of cells.Numerous studies showed that autophagy plays an important role in the occurrence and development of PD,but the specific mechanism of autophagy in PD is still unclear.Endophilin A is the member of Endophilin family.The study showed that the loss of Endophilin A2 can inhibit autophagy,while overexpression of Endophilin A2 can activate autophagy.At the same time,a large amount of evidence showed that the knockout of Endophilin A can cause a significant upregulation of the transcription factor Foxo3 a,which,as one of the regulatory factors of autophagy,plays an important role in regulating the transcription of autophagy related genes.In addition,our previous experiments found that the protein level of Endophilin A2 was significantly reduced in Ang?-induced dopamine neuron injury model.The above studies suggest that there may be a close association between FOXO3 a and Endophilin A2,and they are involved in the regulation of autophagy in PD.Biochanin A(Bioch A)is a natural plant estrogen,with various biological activities such as anti-oxidation,anti-inflammatory,anti-infection.The results of the previous research group showed that Bioch A has a good neuroprotective effect,but its mechanism was not completely clear.Objective: To study the changes of Endophilin A2,FOXO3 a and autophagy-related proteins in Ang?-induced dopaminergic neuron injury model,and the protective mechanism of Bioch A on damaged dopaminergic neurons.Methods: 1.SD rats were randomly divided into 5 groups: sham operation group,Ang? 1d group?Ang? 3d group?Ang? 5d group and Ang? 7d group.All the Ang?model groups were injected with Ang? into the substantia nigra pars of the mouse with a brain stereotactic injection instrument and the sham operation group was giventhe same volume of saline in the same way.Behavioral experiments were performed at 1,3,5,and 7 days after modeling to observe the changes in behavioral indicators of the mice.After the behavior experiment,the mice were sacrificed and brain tissue was taken for subsequent experiments.The immunofluorescence method was used to detect the damage of DA neuron and the activation of microglia,and the expression levels of FOXO3 a and LC3-?.The Western blot was used to detect the protein levels of Arg-1,i NOS,IL-1 ?(interleukin 1 ?),IL-6(interleukin-6),TNF-?(tumor junction factor-?),Endophilin A2,FOXO3 a,p-FOXO3 a and autophagic proteins LC3-?,Beclin1,p62.2.SD rats were randomly divided into 6 groups: sham operation group,Ang? model group,Candesartan group and Bioch A different concentration group[Ang?+Bioch A(18mg/kg)Ang?+Bioch A(36 mg/kg),Ang?+Bioch A(72 mg/kg)].All the above experimental groups except the sham operation group were injected with Ang? into the substantia nigra pars compacta of the mouse with a brain stereotactic injection instrument and the sham operation group was given the same volume of saline in the same way.After 24 hours of injection,Bioch A group were intragastric administration according to the corresponding concentration gradient for one week.The Candesartan group was also given 1.2mg/kg Candesartan by intragastric administration for one week.On the 7th day after the injection,behavioral experiments were performed to observe the behavioral changes of the mice.After the behavior experiment,the mice were sacrificedand brain tissue was taken for subsequent experiments.The immunofluorescence method was used to detect the damage of DA neuron and the activation of microglia,and the expression levels of FOXO3 a.The Western blot was used to detect the protein levels of Arg-1,i NOS,IL-1?,IL-6,TNF-?,Endophilin A2,FOXO3 a,p-FOXO3 a and p62.Result:1.The Variations of Endophilin A2,FOXO3a-autophagy signal in Ang?-Induced DA neuron injury model.1).Behavioral observations of Ang?-induced model mice: Compared with the sham operation group,the spontaneous activity aof Ang ? mice decreased with themodeling time.In addition,compared with the sham operation group,the swimming score of mice also decreased with the modeling time.The above behavioral results show that Ang ? can damage the spontaneous activity and motor ability of mice.2).Pathological observation of Ang?-induced model mice: In the sham operation group,the DA neurons in the substantia nigra pars compacta were full and large in number,and the microglia were basically in a resting state,and there was no significant change in morphology;while in the Ang? group,DA neurons gradually lost with the time of modeling,the activation of microglia increased significantly and the morphology gradually changed to an amoebic appearance.3).The expression of Arg-1,i NOS and inflammatory factors in SN of Ang?-induced model mice: Compared with the sham operation group,the expression of Arg-1 in the Ang? model group gradually decreased and the expression of i NOS gradually increased with the increase of modeling time.In addition,compared with the sham operation group,the protein levels of inflammatory factors IL-1?,IL-6 and TNF-? in the substantia nigra of mice all increased in varying degrees with the increase of Ang? modeling time.4).The expression of Endophilin A2 in SN of Ang?-induced model mice :Compared with the sham operation group,the expressions of Endophilin A2 in the Ang? 5d and 7d groups were significantly reduced.5).The expression of Foxo3 a and p-Foxo3 a in SN of Ang?-induced model mice : The results of immunofluorescence showed that compared with the sham operation group,the content of FOXO3 a in Ang? 3d,5d and 7d groups increased significantly;In addition,Western blot results showed that compared with the sham operation group,the ratios of p-FOXO3 a / FOXO3 a in Ang? 5d and 7d groups were significantly reduced.6).The expression of autophagy protein in SN of Ang?-induced model mice :The results of immunofluorescence showed that compared with the sham operation group,the protein levels of LC3-? increased with the increase of Ang? modeling time.In addition,Western blot results showed that compared with the sham operation group,the ratio of LC3-? to LC3-I and the expression level of Beclin1 graduallyincreased with the increase of Ang? days,while the protein level of P62 showed a trend of decline first and then increase with the increase of Ang? days.7).FOXO3 a and LC3-? Associate with DA neuron: Significant FOXO3 a probe localization was observed in the nucleus of TH positive neurons when FOXO3 a co-expressed with TH;moreover,significant co-localization of both probes was observed when the LC3-? co-expressed with TH.2.Protective effect and mechanism of Bioch A on angiotensin II-induced dopamine neuron damage1).Effect of Bioch A on the behavior of Ang?-induced model mice: Compared with the sham operation group,the spontaneous activity and swimming score of the Ang? group were reduced to some extent;Bioch A significantly improved the spontaneous activity and swimming score of mice compared with the Ang? group.2).Effect of Bioch A on the pathology of Ang?-induced model mice: Compared with the Ang? group,the loss of TH-positive neurons in each concentration of Bioch A group was reduced in varying degrees,and the DA neuronal body gradually became full,besides the degree of microglial activation was also significantly reduced.3).Effect of Bioch A on the level of Arg-1,i NOS and inflammatory factors:Compared with Ang?group,Bioch A significantly inhibited the decrease of Arg-1and decreased the expression of i NOS.In addition,the levels of IL-1 ?,IL-6 and TNF-?were significantly reduced by Bioch A.4).Effect of Bioch A on the level of Endophilin A2: Compared with the Ang?group,the treatment with Bioch A significantly increased the expression of Endophilin A2.5).Effect of Bioch A on the level of FOXO3 a,p-FOXO3a:The results of immunofluorescence showed that the levels of FOXO3 a protein in different dose groups of Bioch A were reduced to varying degrees compared with the Ang?group;Moreover,Western blot results showed that compared with Ang? model group,the ratio of p-FOXO3 a /FOXO3 a in different dose groups of Bioch A increased.6).Effect of Bioch A on autophagy protein: western blot results showed that the protein levels of P62 in the Bioch A group were significantly reduced compared with the Ang? group.Conclusion:1.Ang? can induce DA neuron damage in mice,the mechanism may be related to the loss of Endophilin A protein,the blocking of FOXO3a-autophagy flow.2.Bioch A inhibits Ang?-induced neuroinflammation and protects damaged DA neurons,The mechanism may be related to the restoration of FOXO3a-autophagy flow,and the further mechanisms may be related to regulating the expression of Endophilin A... |
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