Ameliorated biomechanical properties of carotid arteries by puerarin in spontaneously hypertensive rats

Abstract

Background: An emerging body of evidence indicates that puerarin (PUE) plays an important role in the treatment of angina pectoris, myocardial ischemia-reperfusion injury, hypertension and other cardiovascular diseases, but how PUE affects the vascular remodeling of hypertensive rats has not been reported yet. This study aimed to investigate the effect and mechanism of PUE on carotid arteries of spontaneously hypertensive rats (SHR) to provide the basis for the clinical application of PUE.

Methods: Thirty male SHR and six male Wistar Kyoto rats (WKY) aged 3 months were used in this study, SHR rats were randomly divided into 5 groups, PUE(40 or 80 mg/kg/d, ip) and telmisartan (TELMI) (30 mg/kg/d, ig) were administrated for 3 months. We use DMT myography pressure-diameter system to investigate biomechanical properties of carotid arteries, 10 μM pan-classical transient receptor potential channels (TRPCs) inhibitor SKF96365, 200 nM specific TRPC6 inhibitor SAR7334 and 100 μM Orai1 inhibitor ANCOA4 were used in the mechanical test.

Results: PUE can significantly decrease systolic and diastolic blood pressure, long-term administration of PUE resulted in a mild reduction of thickness and inner diameter of carotid artery. PUE ameliorate NE-response and vascular remodeling mainly through inhibiting TRPCs channel activities of VSMC.

Conclusion: PUE can ameliorate biomechanical remodeling of carotid arteries through inhibiting TRPCs channel activities of VSMC in spontaneously hypertensive rats.

Keywords: Biomechanical property; Hypertension; Puerarin; TRPC; Vascular remodeling.

Fig. 1

PUE significantly decreased SBP and DBP of SHR rats. A SBP of SHR group was significantly higher than WKY group, 80 mg/kg/d PUE could decrease SBP of SHR, systolic blood pressure: SBP; B DBP of SHR group was higher than WKY group, 80 mg/kg/d PUE could decrease DBP of SHR; diastolic blood pressure: DBP. C The chemical structure of puerarin. $ p < 0.05 vs WKY; * p < 0.05 vs SHR; # p < 0.05 vs SHR + TELMI. (Repeated measures ANOVA)

Fig. 2

Effect of PUE in common carotid artery with ultrasound image. A A representative ultrasound record of CCA at the end of 12 weeks. B Inner diameter changes of CCA with PUE during 0–12 weeks. C Histogram analysis of thickness changes of CCA with PUE at the end of 12 weeks. D Histogram analysis of inner diameter changes of CCA with PUE at the end of 12 weeks. † p < 0.05 vs WKY, * p < 0.05 vs SHR. (Repeated measures ANOVA)

Fig. 3

Effect of PUE on the morphological structure of common carotid artery. A Representative H&E-stained histological images of CCA in different groups; thickness of CCA of SHR group was significantly larger, and vascular smooth muscle cells were arranged in disorder, hypertrophy, and irregular in shape compared to WKY group; PUE led to a less damaged CCA. B Representative Masson-stained histological images of CCA in different groups. The collagen fibers stained blue-green of SHR group were obviously more than WKY group, and PUE could ameliorate CCA collagen fiber hyperplasia (Scale bar: 100 μm)

Fig. 4

Representative functional response of common carotid artery at P = 60 mmHg. A Representative original tracings of functional curves induced by NE and SNP. B The histogram analysis of percentage of NE-induced contraction extent, * p < 0.05 vs SHR. C The histogram analysis of percentage of SNP-induced relaxation extent, * p < 0.05 vs SHR. D The histogram analysis of out diameter changes in different groups by NE and SNP, data in PSS is taken as the control. * p < 0.05 vs the same group in PSS, † p < 0.05 vs SHR group in NE, # p < 0.05 vs the same group in NE. (One-way ANOVA with Bonferroni post-hoc test)

Fig. 5

PUE inhibited NE-induced passive vasoconstriction. A Representative original tracings of NE- reactivity curves of SHR rats fed with PUE, PUE could obviously reduce NE-induced passive vasoconstriction as the calcium concentration increasing. B The outer diameter of carotid rings of SHR rats incubated with various concentrations of PUE, 10^− 3 mol/L PUE could inhibit the outer diameter decreasing induced by increasing Ca²⁺

Fig. 6

Ca²⁺-induced pressure changes were associated with TRPCs channel. A, B, C, D the pressure- Ca²⁺ response curves of CCA rings of WKY rats with intact endothelium, 10 μM SKF and 200 nM SAR could obviously decrease CCA pressure with endothelium compared to the control; 100 μM ANCOA4 has no effect on CCA pressure. E The pressure-Ca²⁺ response of CCA rings in WKY rats without endothelium, SKF could obviously decrease CCA pressure without endothelium compared to the control. F, G The pressure-inner diameter curves of CCA of SHR rats incubated with SKF, SAR or ANCOA4 with intact endothelium or without endothelium. H Time course of [Ca2+]_i changes as seen in Fura2-loaded A7R5 cells, PUE were incubated for 24 h before experiment, 0.1 μM AngII was used in the experiment. I Fluorescent dye Fluo-3/AM staining of A7R5 cells, Scale bar: 1 mm. SKF: SKF96365, pan-TRPCs inhibitor; SAR: SAR7334, specific TRPC6 inhibitor; ANCOA4:Orai1 inhibitor, * p < 0.05 vs Ctrl. (Two-way ANOVA with Bonferroni post-hoc test)

Fig. 7

PUE ameliorated Ca²⁺-induced constriction with the similar role as SKF. A Representative original tracings of NE-reactivity curves of WKY rats, SKF could obviously inhibit the decrease of outer diameter of carotid rings induced by increasing extracellular Ca²⁺. B Representative original tracings of NE-reactivity curves of SHR rats incubated with 10^− 3 M PUE, PUE played the similar role as SKF. C Representative original tracings of NE-reactivity curves of SHR fed with 40 mg/kg/d of PUE. D Representative original tracings of NE-reactivity curves of SHR fed with 80 mg/kg/d of PUE; SKF has no obvious effect on inhibition the outer diameter decrease induced by increasing extracellular Ca²⁺

Fig. 8

Underlying effect and possible mechanism of PUE on blood pressure and vascular remodeling involving Ca²⁺ in hypertension. The mechanism by which PUE reduced blood pressure was mainly related to BK_Ca activation, VGCC inactivation and SERCA activation. The mechanism by which PUE ameliorated vascular remodeling was mainly related to TRPCs inactivation, regulating the Ca²⁺-CaN-NFAT pathway and Ca²⁺-PKC-NFκB pathway. BK_Ca: large-conductance voltage- and Ca²⁺-activated potassium channel; VGCC: Voltage gated calcium channels; SERCA: sarco endoplasmic reticulum Ca²⁺-ATPase; CaM: Calmodulin; CaN: Calcineurin; PKC: protein kinase C; GPCR: G protein coupled receptor; PLC: phospholipase C