The Effect And Mechanism Of Biochanin A On Dopaminergic Neuron Injury Induced By AngⅡ

Parkinson’s disease(PD)is a neurodegenerative disease characterized by degeneration,apoptosis,and loss of dopamine(DA)neurons in the substantia nigra pars compacta.The renin-angiotensin system(RAS)plays an important role in regulating neuroinflammatory responses and progression of dopaminergic(DA)neuronal degenerative diseases.Angiotensin II(Ang II)is closely related to the course of PD by angiotensin II type 1 receptor(AT1R)-induced microglial activation-mediated neuroinflammation,which affects the function of DA neurons.Endophilin A2(EP2)is involved in rapid endothelin-mediated endocytosis(FEME)and rapid endocytosis of several G-protein coupled receptors(GPCRs),while AT1 R belongs to the GPCR family.Therefore,EP2 may exert endocytosis of AT1 R regulating activation of microglia.Biochanin A(Bioch A)is an isoflavone phytoestrogens.Previous studies showed that Bioch A can inhibit LPS-induced microglia activation,however,it induced microglia activation by AngII is unclear.Objective: To study the protective effects and mechanism of Bioch A on AngII-induced DA neuronal injury.Methods: SD rats were randomly divided into 6 groups,including sham operation group,AngII(5μg/3μl)model group,Losartan control group and chickpea A different concentration group(AngII+Bioch A(16mg/kg)AngII+Bioch A(32 mg/kg),AngII+Bioch A(64 mg/kg)).The DA neuron injury model was prepared by injecting AngII into the substantia nigra pars compacta of the rat with a stereotaxic instrument.The sham operation group was injected with an equal volume of normal saline.After24 hours of injection,the Bioch A group was intragastrically administered according to the corresponding concentration gradient,and the Losartan group was also administered by intragastric administration.On the 7th day after the injection,the changes in behavioral indicators of each group were observed uniformly.Next,apomorphine was injected intraperitoneally,and the rotation of each group was observed,and then sacrificed,and the tissue was taken for examination.Immunohistochemistry(IHC-P)was used to observe the activation of microglia in the substantia nigra pars compacta and the damage of dopaminergic neurons.Nucleotide binding oligomerization domain-like protein 3(Nucleotide binding oligomerization domain-like protein 3)Receptor family pyrin domain containing 3,NLRP3),Endophilin A2(EP2).Western Blot was used to detect α-synuclein and Cysteinyl aspartate specific proteinase-1(Caspase-1)in the substantia nigra pars compacta.Apoptosis-associated speck-like protein containing CARD(ASC),gp91 phox,p22phox,IL-1β(Interleukin 1β,L-1β),IL-6(Interleukin-6),IL-18(Interleukin-18,IL-18),TNF-α(Tumor necrosis factor-α,TNF-α)expression.Result:1)Behavioral observations: Compared with the sham group,the spontaneous activity and exploration behavior of the AngII group were reduced to some extent;compared with the AngII group,the spontaneous activity and exploration behavior of the Bioch A group were Increased to varying degrees.2)Rotational experiment observation: The intraperitoneal injection of apomorphine in the AngII group showed a phenomenon of rotation to the opposite side,and the number of rotations in the Bioch A group was less than that in the AngII group.3)Immunohistochemical observation: In the sham group,the DA neurons in the substantia nigra pars compacta were neatly arranged and the number was large,the number of microglia was small and most of them were at rest;while the AngII group,DA could Most of the neurons were degenerated and the cell bodies were atrophied,and the activation of microglia was significantly increased,showing an amoebic appearance.Compared with the AngII group,the Bioch A group reduced the number of microglia activation and decreased DA neuron damage.degree.4)Immunohistochemical observation: NLRP3 protein expression was increased in AngII group,while NLRP3 expression was decreased in Bioch A group.Western Blot observation showed that Caspase-1 and ASC protein expression levels were increased in AngII group,and Caspase was significantly decreased in Bioch A group.-1 and ASC protein expression levels.5)Western Blot observation: The expression levels of gp91 phox and p22 phox were increased in AngII group,but the expression levels of gp91 phox and p22 phox were decreased in Bioch A group.6)Western Blot observation: The expression levels of IL-1β,IL-6,IL-18 and TNF-αwere increased in AngII group,and IL-1β,IL-6,IL-18 and TNF-α were decreased in Bioch A group.7)Immunohistochemical observation: The expression of EP2 was decreased and the expression of AT1 R was increased in AngII group,but the expression of EP2 protein was promoted in Bioch A group,and the expression level of AT1 R was decreased.Conclusion :Bioch A has a protective effect on AngII-induced DA neuronal injury in rats.The mechanism may be that Bioch A inhibits AngII-induced microglia activation,and its mechanism may be further inhibited by inhibiting the activity of NADPH oxidase and NLRP3 inflammatory bodies.It is related to the interaction between EP2 and AT1R.