The Effect And Underlying Mechanisms Of Angiotensin Ⅱ On The Apoptosis Of Dopaminergic Neuron In Vitro And In Vivo

With the aggravation of global aging progress,Parkinson’s disease(PD)has seriously affected the life quality of the elderly and exerted huge burden to the society.PD is a progressive disease,clinically manifested by bradykinesia,resting tremor,rigidity and postural abnormalities.The neuropathological hallmark of PD is a progressive loss of dopaminergic neurons in the substantia nigra(SN).At present,its pathogenesis is unclear,and there is no effective measure to prevent the disease.Recently there is cumulative evidence demonstrating that the level of angiotensin Ⅱ(Ang Ⅱ),a chief component of rennin angiotensin system(RAS),is significantly higher in the brain of PD patients and PD animal models,suggesting that the pathogenesis of PD is closely related to the excessive activation of the intracerebral RAS,Therefore,to explore the relationship between Ang Ⅱ and the pathological process of PD has become the research focus.RAS is mainly responsible for regulating water and salt balance and the body blood pressure,thus maintaining the environmental homeostasis.Similar to the circulatory system,independent RAS existes in multiple tissues and organs including the brain,and stable expression of its main ingredients like Ang Ⅱ,Ang Ⅱ type 1 receptor(AT1R)and Ang Ⅱ type 2 receptor(AT2R)could be observed as well.Many previous studies including ours have reported that Ang Ⅱ was closely related to the injury of dopaminergic neuron.In addition,using angiotensin converting enzyme inhibitors(ACEI)or AT1R antagonist(ARB)could block the signaling pathway mediated by Ang Ⅱ,thus further reducing the apoptosis of dopaminergic neurons in basal ganglia region.More importantly,in tissues such as the heart and kidney,Ang Ⅱcould aggravate apoptosis of myocardial cells and kidney podocytes by enhancing autophagy,thus further strengthening damage of the heart and kidney.This suggested that apoptosis and autophagy may be interrelated.Therefore,in the first part of this study,we utilized Cath.a dopaminergic cell line which could stably express AT1R and AT2R as object of the research.We observed the effect of Ang Ⅱ on neuronal apoptosis and autophagy along with the relationship between the two.We also explored which kind of receptors was involved in the process of the aforementioned effect of Ang Ⅱ.All these findings could help us clarify the effect and underlying mechanisms of Ang Ⅱ on dopaminergic neurons in vitro.ARB has been proved to play a protective role on dopaminergic neurons,and up to now there are few researches about the influence and underlying mechanisms of ARB on pathology and ethology of PD animal models.Therefore,in the second part of this study,we first used rotenone-induced PD rat model to observe the expression of Ang Ⅱ,AT1R and AT2R in the substantia nigra,exploring whether Ang Ⅱ and its receptors were involved in the pathological process of PD.Afterwads,by injection of exogenous Ang Ⅱ and(or)losartan,we studied the effect of Ang Ⅱ and losartan on apoptosis of dopaminergic neuron in the substantia nigra of normal rats.Finally,by means of gavage of Azilsartan(a new type of AT1 receptor antagonist)to PD rat models,we clarified the influence of Azilsartan on dopaminergic neurons and Parkinsonian symptoms of PD animal models,and explored the protective role of ATI receptor antagonist in the treatment of PD.In conclusion,this study not only revealed the effect of Ang Ⅱ on apoptosis of dopaminergic neurons in vitro and in vivo,but also clarified partial pathogenesis of PD,therefore providing new targets and theoretical basis for the prevention and treatment of PD.PART Ⅰ The effect of Ang Ⅱ on apoptosis and autophagy in a dopaminergic neuronal cell line along with the underlying mechanisms.Aims:To determine the effect of Ang Ⅱ on apoptosis and autophagy in Cath.a cells along with the underlying mechanisms.Materials and Methods:By using CATH.a cells,a dopaminergic neuronal cell line stably expressing Ang Ⅱ type 1 receptor(AT1R)and Ang Ⅱ type 2 receptor(AT2R),we firstly investigated the effect of Ang Ⅱ on apoptosis and autophagy.Next,we observed the activity of apoptosis in Ang Ⅱ and 3-MA(a kind of autophagy inhibitor)co-treated cells to explore the association between apoptosis and autophagy.Finally,after respective administration of AT1R antagonist losartan and AT2R antagonist PD123319,we determined which receptor was involved in the aforementioned influence of Ang Ⅱ.Results:First,we showed Ang Ⅱ triggered apoptosis in CATH.a cells through a dose-dependent manner.Meanwhile,Ang Ⅱ also led to the activation of autophagy in the same cell line.Furthermore,after Ang Ⅱ-induced autophagy in cells was fully inhibited by 3-MA,we found that the apoptosis activity caused by Ang Ⅱ was completely abolished as well.More importantly,by co-incubation with Ang Ⅱ and AT1R antagonist losartan or AT2R antagonist PD123319,we revealed that the AngⅡ-induced apoptosis and autophagy in CATH.a cells were fully reversed by losartan,while PD123319 had no effect on the both two activation.Conclusion:These findings suggest that Ang Ⅱ activates autophagy and thus triggers apoptosis in CATH.a cells.In addition,the above effects of Ang Ⅱ on CATH.a cells are mediated by AT1R rather than AT2R.PART Ⅱ The effect of Ang Ⅱ on apoptosis of dopaminergic neurons and parkinsonian behaviors in a rat model of Parkinson’s disease along with the underlying mechanisms.Aims:To determine the effect of Ang Ⅱ on apoptosis of dopaminergic neurons and parkinsonian behaviors in a rat model of Parkinson’s disease along with the underlying mechanisms.Materials and Methods:First,by using a successful rat model of PD induced by rotenone,we investigated whether Ang Ⅱ and its receptors were involved in the pathogenesis of PD.Afterwards,by infusion into the right SN of normal rats with exogenous Ang Ⅱ and(or)AT1R antagonist losartan,we observed the effect of AngⅡ/AT1R axis on dopaminergic neurons in the right SN of rats.Finally,PD rats were orally administered with vehicle or a novel AT1R blocker azilsartan.By examining the effect of Ang II and azilsartan on apoptosis of dopaminergic neurons and parkinsonian behaviors,we intended to translate the above findings into a clinically relevant context.Results:First,we showed the levels of Ang Ⅱ and AT1R(rather than AT2R)in the SN of PD rats were markedly increased compared with that of control rats.Moreover,we also found that exogenous Ang Ⅱ triggered apoptosis of dopaminergic neurons via an AT1R-dependent manner in the SN of normal rats.Most importantly,by oral administration of a novel ATIR antagonist azilsartan to PD rats,we provided in vivo evidence that AT1R antagonist prevented neuronal losses via inhibiting apoptotic signaling and rescued characteristic parkinsonian behaviors.Conclusion:These findings suggest that overactivation of brain Ang Ⅱ/AT1R axis contributes to the progression of PD.More importantly,a newly developed AT1R blocker azilsartan plays a protective role in a rat model of PD induced by rotenone.