| Background:Cerebrovascular remodeling during the development of hypertension is the pathological basis for stroke,and preventing the occurrence of cerebrovascular remodeling can effectively prevent stroke.The cerebral artery is composed of intima,media and adventitia.Among them,the media is the main structure of the arterial vascular wall,and the increase of the total volume of media plays an important role in cerebrovascular remodeling.The media is mainly composed of cerebrovascular smooth muscle cells(CSVMCs)and collagen fiber.In the process of hypertension,the increase of blood pressure can change the production and degradation of extracellular matrix and affect the proliferation and apoptosis of vascular smooth muscle cells.And,the increased of vasoconstriction contractility rather than the attenuated of the expanding ability,may be the cause of increased basilar resistance in chronic hypertension.Cerebrovascular remodeling involves CSVMCs proliferation,hypertrophy,apoptosis,inflammation,fibrosis and extracellular matrix(ECM)deposition,etc.Fibronectin(FN)is the structural component of ECM,and also transforms the cellular phenotype of CSVMCs.All the structure and dynamics properties of cerebrovascular are significant changed during the development of hypertension,and basilar artery is a typical muscular resistance vessel in the brain,which plays an important role in the process of developing hypertension.Therefore,this experiment aims to research the changes of the basilar artery in the pathogenesis of hypertension.Ligustrazine,the chemical structure is tetramethylpyrazine(TMP),with the extensive biological activities including the modulation of ion channels,inhibiting the oxidative stress,inhibiting apoptosis,promoting cell proliferation,inhibiting the excitatory amino acids released,promoting endogenous neural stem cell migration,promoting angiogenesis after cerebral ischemia and inhibiting platelet aggregation,etc.The clinical application of ligustrazine has been widely used in hypertension,stroke,angina,arrhythmia and other diseases,however,there is no detailed report on the study of the RHRSP rats,and the mechanism of ligustrazine on the basilar artery is not clear.Cerebrovascular stroke damage in sponetaneously hypertensive rats and stroke-prone spontaneously hypertensive rats are greatly influenced by genetic factors.So,the two hypertension modes are not purely reflect the effects of high blood pressure itself on cerebrovascular remodeling.RHRSP rats have stable hypertensive formation rate and spontaneous stroke rate,and the cerebrovascular damage is not affected by genetic factors.Therefore,RHRSP rats are used as the hypertensive rats in our study.Objective:In this study,the stroke-prone renalvascular hypertensive rats model were established,then the anti-hypertensive mechanism of ligustrazine and the reverse effect of ligustrazine on basilar vascular remodeling were investigated.Moreover,the inhibitory effect and mechanism of ligustrazine on BASMCs cell proliferation induced by ET-1 and Ang were ?also investigated.Methods:(1)Experimental animal modelTwo-kidney-two-clip method is adopted to establish the stroke-prone spontaneously hypertensive rats model.All the rats were randomly divided into four groups: sham group,model group,high TMP treatment group and low TMP treatment group.And blood pressure was measured every 2 weeks after surgery.(2)Histological changes of basilar artery tissuesBrain basal artery vessels were isolated from rats in different groups at 4w,8w and 12 w after the 2K2 C surgery.The histological changes of basilar artery tissues were detected by HE and SVG staining.The thickness,internal lumen diameter and external lumen diameter of media of brain basal artery were investigated by ?-SM actin immunohistochemical staining.The changes of ECM of brain basal artery were detected by FN immunofluorescence analysis.The changes of ultrastructure of brain basal artery were detected by transmission electron microscopy assay.The changes of BASMCs cell proliferation of brain basal artery were investigated by Brdu immunohistochemical staining and PCNA,Ki67 immunofluorescence analysis.(3)The reverse effect and mechanismof ligustrazine on basilar vascular remodelingThe ET-1,Ang and NO? levels in basilar artery tissues and plasma were determined with the corresponding kits.The PTEN expressions in basilar artery tissues were detected by immunohistochemical staining.The activation of PI3K/Akt pathway in basilar artery tissues were detected by Western blot.In addition,the ET-1-induced and Ang?-induced BASMCs cell proliferation cell model were establish;and cell proliferation was determined with CCK-8 assay kit and Brdu assay kit;cell cycle was determined by flow cytometer using the propidium iodide(PI)detection kit,and the expressions of cyclin D1 and P27 were detected by Western blot.The activation of PI3K/Akt pathway in BASMCs cells were detected by Western blot;The NO,ROS and SOD levels in BASMCs cells were determined with the corresponding kits.Results:(1)The anti-hypertensive mechanism of ligustrazine may involve lower plasmatic and basilar artery vascular ET-1 levels,basilar artery vascular Ang?and higher plasmatic and basilar artery vascular NO levels.1)The systolic pressure of RHRSP group was significantly higher than the sham group(P<0.05),however,the systolic blood pressure of TMP treatment group was significantly lower than RHRSP group(P<0.05).2)The plasmatic and basilar artery vascular ET-1 levels of RHRSP group were significantly higher than the sham group(P<0.05),the plasmatic and basilar artery vascular ET-1 levels of TMP treatment group were significantly lower than RHRSP group(P<0.05).3)The basilar artery vascular Ang levels? of RHRSP group were significantly higher than the sham group(P<0.05),the basilar artery vascular Ang? levels of TMP treatment group were significantly lower than RHRSP group(P<0.05).4)The plasmatic and basilar artery vascular NO levels of RHRSP group were significantly lower than the sham group(P<0.05),the plasmatic and basilar artery vascular NO levels of TMP treatment group were significantly higher than RHRSP group(P<0.05).(2)Ligustrazine reversed basilar vascular remodeling in RHRSP rats.1)Ligustrazine improved histological changes of basilar artery tissues in RHRSP rats.Media thickoess,and medial cross-sectional area of basilar artery tissues in RHRSP group were significantly increased,internal lumen diameter and external lumen diameter were significantly decreased than that in the sham group(P<0.05);and the histological changes of basilar artery tissues in TMP treatment group were improved than RHRSP group(P<0.05).2)Ligustrazine reduced ECM deposition of basilar artery tissues in RHRSP rats.Collagen area percent and FN expression of basilar artery tissues were significantly increased in RHRSP group than that in the sham group(P<0.05),Collagen area percent and FN expression of basilar artery tissues were significantly decreased in TMP treatment group than that in the sham group(P<0.05).3)Ligustrazine reduced BASMCs cell proliferation of basilar artery tissues in RHRSP rats.The expressiones of Brdu,PCNA and Ki67 of media of basilar artery tissues in RHRSP group were significantly increased(P<0.05),the expression of PTEN was significantly decreased than that in sham group(P<0.05),however,the expressiones of Brdu,PCNA and Ki67 of media of basilar artery tissues in TMP treatment group were significantly decreased,the expression of PTEN was significantly increased than that in RHRSP group(P<0.05).(3)Ligustrazine could remarkably alleviate BASMCs cell proliferation of basilar artery tissues and its protective effect may be related to suppress the activation of PI3K/Akt pathway.The expressiones of p-PI3 K and p-Akt of basilar artery tissues in RHRSP group were significantly increased(P<0.05),and there were postive correlation between p-PI3 K,p-Akt and systemic blood pressure,however,the expressiones of p-PI3 K and p-Akt of basilar artery tissues in TMP treatment group were significantly decreased(P<0.05).(4)Ligustrazine could remarkably alleviate ET-1-induced and Ang?-induced BASMCs cell proliferation and blocke BASMCs cell cycle at G0/G1 phase.BASMCs treatment with 10-8M ET-1 and 10-7M Ang?for 48 h respectively,cell proliferation was significantly increased(P<0.05)and cell cycle transitions was significantly increased(P<0.05),after treatment with(5,10,20?M)TMP.TMP decreased cell proliferation and cell cycle transitions in a concentration dependent manner(P<0.05).(5)Ligustrazine could remarkably alleviate ET-1-induced and Ang?-induced BASMCs cell proliferation by suppressing the activation of PI3K/Akt pathway and oxidative stress reaction.10-8M ET-1 and 10-7M Ang?for 48 h respectively,the expressiones of p-PI3 K and p-Akt of BASMCs were significantly increased(P<0.05),the release of ROS of BASMCs was significantly increased(P<0.05),the production of NO and SOD were significantly decreased(P<0.05),after treatment with(5,10,20?M)TMP,TMP could remarkably reduce the release of ROS,increase the production of NO and SOD,decrease the expressions of p-PI3 K and p-Akt induced ET-1 and Ang?in a concentration dependent manner(P<0.05).Conclusion:(1)The anti-hypertensive mechanism of ligustrazine may involve lower plasmatic and basilar artery vascular ET-1 levels,basilar artery vascular Ang?and higher plasmatic and basilar artery vascular NO levels.(2)Ligustrazine could remarkably inhibit cell proliferation and blocke cell cycle at G0/G1 phase,and its protective effect are related to suppress the activation of PI3K/Akt pathway and oxidative stress reaction.(3)Ligustrazine reversed basilar vascular remodeling in RHRSP rats. |
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