Danhong injection reduces vascular remodeling and up-regulates the Kallikrein-kinin system in spontaneously hypertensive rats

Abstract

Although Danhong injection (DHI) is one of the most prescribed cardiovascular medicines in China, its therapeutic indications and mechanisms remain partially defined. We now identify molecular targets of DHI in resistance vasculatures and demonstrate its role in vascular function and blood pressure (BP) regulation. BP was determined in DHI, Losartan, and placebo- treated Spontaneously Hypertensive Rats (SHR) by both noninvasive and invasive measurements. Vasorelaxation was examined both in conduit and resistance vasculature by ex vivo aortic rings. Microarray analysis was performed and gene expression changes were verified by RT-qPCR and ELISA. Diastolic, systolic and mean BPs were significantly lower in DHI-treated SHR than controls by both tail-cuff and invasive BP measurements. In ex vivo rings, aortic and mesenteric vessels from SHR treated with DHI exhibited significantly greater acetylcholine-mediated relaxation. Among the 282 genes that are differentially expressed in microarray analysis, DHI treatment up-regulated the expression of kallikrein and plasma kallikrein B genes. DHI also significantly increased serum kallikrein content in SHR. Treatment with DHI significantly increased the ratio of aortic lumen to outer diameter. Therefore, the reduction of vascular remodeling and the up-regulation of Kallikrein-kinin system contribute, at least in part, to the antihypertensive effect of DHI in SHR.

Figure 1

Effects of DHI on noninvasive blood pressure in SHR. Panel A–C are representative data plots from (DBP, SBP, and MBP measurements, respectively) using noninvasive tail-cuff method in conscious rats. Panel D represent body weight in (gram) measured with the small animal weighing scale. Compared with saline control, DHI-treated SHR showed no significant difference (p > 0.05 vs. saline group) in weight over the 4 weeks of routine treatment. Data are expressed as mean ± SEM, n = 4.

Figure 2

Effects of DHI on invasive blood pressure in SHR. Invasive BP were measured in urethane anaesthetised rats after treatment with DHI. A heparinized saline, 100 IU/mL was filled in the transducer in clean polyethylene catheter cannulated to the ventricle through the left common carotid artery. DHI decreased BP in SHR (p < 0.01, Fig. 2A [SHR (DHI)] and 2B, n = 3) but did not change the heart rate (p > 0.05, Fig. 2C). DHI did not change BP in normotensive WKY rats (p > 0.05, Fig. 2A [WKY (Saline)]).

Figure 3

Ach and SNP-mediated vasorelaxation in KCl- precontracted thoracic aortas isolated from DHI-treated SHR. (A,B) Raw trace of Ach and SNP-treated aorta and (C,D) Quantification of the data in (A and B) respectively. The rate of Ach and SNP-induced relaxation was calculated with KCl (60 mM)-induced contraction set to 0. (C) DHI treatment significantly (p < 0.01) relaxed aorta in response to Ach stimulation compared with control group in SHR. (D) There was no significant difference in response to SNP stimulation between DHI treatment group and control group in SHRs’ aorta. All data are expressed as mean ± SEM, n = 5.

Figure 4

In vivo and ex vivo effects of DHI on mesenteric arteries from SHR and wild-type rats. (A,B) Raw traces of relaxation of NE-precontracted MAs from SHR in response to ACh (DHI vs. Saline treatment). (C) Quantitation of data in A and B expressed as % relaxation in response to increased Ach concentrations. (D,E) Raw traces of relaxation in response to ACh (DHI vs. Saline treatment) on AD pre-contracted mesenteric arteries. (F) Quantitation of data in (D and E) expressed as % relaxation in response to increased DHI concentrations. The rate of Ach or DHI-induced relaxation was calculated with NE (10 mM) or AD (10 mM) contraction set as 100%. (A and B) Mesenteric arteries from DHI-treated SHR animals for three days significantly relaxed more than that of saline control in response to increasing doses of ACh, p < 0.01. (D and E) DHI treatment also caused a direct endothelial-dependent vasorelaxation in AD pre-contracted mesenteric arteries from normal (WKY) rats, p < 0.01. All data are expressed as mean ± SEM, n = 3.

Figure 5

Effect of DHI on vascular remodeling in SHR. Histology of H&E stain of thoracic aorta at x40 and x400 (A–D) are shown. Summarized ratio of the inner to outer diameter of the thoracic aortas from the 4 groups of rats are quantified and presented in (E and F). (A) Saline treated WKY rats; B: Saline treated SHR rats; (C) SHR rats treated DHI; (D) SHR treated Losartan. Values represent mean ± SEM (n = 4 per group; p < 0.01 vs. SHR treated DHI).

Figure 6

Gene expression changes by Microarray analysis and subsequent confirmation in SHR mesenteric vessels. (A) Microarray confirmation of down-regulation of RAAS genes by Losartan. (B and C) DHI did not alter RAAS and NOS genes in microarray profiling, respectively. (D) Serum renin, angiotensin and aldosterone contents were determined and no significant difference (N.S.) was detected between DHI treatment and the control groups. (E) Effect of DHI on serum kallikrein content determined by ELISA assay (*p < 0.05). (F) Other hypertension-associated genes that are differentially regulated by DHI in microarray and their confirmation by RT-PCR. RT-PCR gene expressions of Kcnj2, Klkb1, and kallikrein were in accordance (I.A) with microarray data whereas Htr6 gene expression was not in accordance (N.I.A.) with that obtained by microarrays. All data are expressed as mean ± SEM, n = 4.