| Objective:In this study,Tribulus Terrestris(TT)was administrated to elderly spontaneously hypertensive rats(SHR).The hypothalamic IKK-β/NF-κB pathway was taken as the assumed targets,to evaluate the central effect of TT on blood pressure regulating and to alleviate the degree of arteriosclerosisin the treatment of isolated systolic hypertension(ISH)in the elderly;in addition,we observed the pharmacodynamics of TT on cultured hypothalamic neurons in vitro and of the change of mRNA and protein expression of IKK-β/NF-κB signaling path way via blocking or activating IKK-β.Thus,we hoped to explore the pharmacological mechanism of TT on isolated systolic hypertension in the elderly in the molecular level and provide new treatment ideas to essentially improve the elderly ISH.Methods:1.The pharmacodynamics of TT in the treatment of elderly spontaneously hypertensive rats(SHR):40 male SHR rats of 16-month old were randomly divided into the high dose group of TT(17.2mg.kg-1·d-1),the low dose group of TT(8.6mg·kg-1·d-1),valsartan group(13.35mg·kg-1·d-1)and model group(normal saline 2ml·d-1);10 male normotensive WKY rats with the same age as normal control group(the same volume of saline).They were all given drugs or saline intragastrically for 4 weeks.Blood pressure was measured regularly.After 4 weeks of the observation,the blood,hypothalamus,thoracic aorta,common carotid artery and mesenteric artery were taken for morphological study.The level of serum Ang II,AVP,CRP,NPY and PAI was assayed using ELISA kits;morphological changes of hypothalamus,thoracic aorta,common carotid artery and mesenteric artery were observed by HE staining;the rat thoracic aorta in each group were observed by scanning electron microscope,and the subcellular structure of hypothalamus and thoracic aorta were observed by transmission electron microscope;collagen deposition of thoracic aorta were oberved by hydroxyproline assay.The distribution of AT1 and NPY on hypothalamus,as well as AT1 and TGF-β on thoracic aorta were observed by immunohistochemical staining.2.The pharmacological study of TT in the treatment of isolated systolic hypertension in the elderly:the study was divided into 2 parts,the first part was the pharmacodynamics study of hypothalamic neurons in vitro.The hypertension cell model was developed in primary hypothalamic neuron cells induced by AngⅡ.Cells were divided into 5 groups:model group,the low dose of TT group,the high dose of TT group and valsartan group.While cells were divided into 6 groups for the following pharmacological research:control group,model group,TT group,TPCA-I group,TNF-a group and TT+TNF-α group.The number of neurons in the hypothalamus was counted.The cell survival rate,apoptosis rate,the length of neuron axon and the area of cell body were detected;preliminary screening the levels of mRNA expression of IκBα,IKK-α,IKK-β,NF-KBp65,JAK2,STAT3,ATI,GnRH,PKC,PI3K was conducted by real-time quantitive real-time PCR(qRT-PCR);there after,the level of mRNA and protein expression of IκBα,IKK-α,IKK-β,NF-κB p65 was determined by qRT-PCR and Western Blot.Results:1.The pharmacodynamics of TT in the treatment of elderly SHR:blood pressure of all medication groups was decreased in different degree after drug administration for 4 weeks and among them,the high dose group of TT demonstrated the best efficacy(P<0.05);The level of serum AVP,CRP,NPY and PAI was decreased significantly P<0.05);HE staining and transmission electron microscopy observation of hypothalamus displayed that the high dose of TT significantly improved hypothalamic morphology and neuron number.Masson staining,HE staining,scanning electron microscopy,transmission electron microscopy and hydroxyproline assay showed that the high dose of TT improved arterial morphology,and reducing collagen deposition as well.The density of ATI in hypothalamus of the high dose of TT was decreased significantly,whereas the density of NPY was increased significantly(P<0.05);and the density of AT1 and TGF-β in aorta decreased significantly(P<0.05).2.The pharmacological research of TT:The results in vitro showed that the high dose of TT obviously decreased the survival rate,apoptosis rate,the absolute number of cells,cell size,while increased the length of axons of hypothalamic neurons induced by AngⅡ at each time point(P<0.05).The mRNA expression of IKK-β,NF-KBp65,JAK2,AT1,PKC of the high dose group of TT was decreased significantly,while IKK-a,IkBa,GnRH increased significantly(P<0.05).Further study showed the mRNA and protein expression levels of IKK-β,NF-KBp50,NF-κBp65 decreased significantly,whereas IκBα increased significantly(P<0.05).The further study in vivo displayed that TT decreased the mRNA and protein expression levels of IKK-β,NF-KBp50,NF-KBp65 while IκBα increased significantly(P<0.05)in hypothalamus and thoracic aorta.Conclusion:TT can steadily reduce the aged SHR rats systolic and diastolic blood pressure and pulse pressure;decreased the level of plasma CRP,AVP,NPY,PAI to improve neuroendocrine activity and the level of inflammation,regulate the activity of coagulation fibrinolytic system;through the study on the pathological changes of rat hypothalamus and all levels of artery TT can improve the cell morphology of hypothalamus and arteries vascular;in vivo and in vitro experiments have confirmed that the TT can improve hypothalamic neuronal cell morphology and status,down-regulating the mRNA levels and protein expressionswhich relative to IKK-β/NF-κB pathway about IKK-β,NF-κBp50,NF-KBp65 while up-regulation of the expression of IKBa in hypothalamus and thoracic aorta.The possible mechanism is that TT can achieve the purpose of reducing blood pressure via the inhibition of hypothalamic IKK-β/NF-κB pathway which control blood pressure regulation and central inhibition of vascular remodeling,... |
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