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Original article

CB1 cannabinoid receptor participates in the vascular hyporeactivity resulting from hemorrhagic shock in rats

HOU, Li-chao; LI, Nan; ZHENG, Li-na; LU, Yan; XIE, Ke-liang; WANG, Yue-min; JI, Gen-lin; XIONG, Li-ze

Editor(s): CHEN, Li-min

Author Information
doi: 10.3760/cma.j.issn.0366-6999.2009.08.014
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Abstract

Vascular hyporeactivity, the decreased response of arterial blood pressure to vasoexcitors such as norepinephrine, is mainly developed in the terminal stage of hemorrhagic shock.1 Hemorrhagic shock accompanied with vascular hyporeactivity is one of the causes of multiple organ failure. The vascular hyporeactivity represents a major therapeutic challenge since medical therapies for it generally are ineffective.2 Vascular hyporeactivity is well documented and is associated with adrenoceptor dysfunction,3 inflammatory factors,4 NO,5 endothelin,6 calcium and potassium channels desensitization7-9 and Rho kinase regulation,10 but the underlying cellular and molecular mechanisms are not well understood.

It is reported that the activation of cannabinoid receptors, which are expressed in blood vessel and immunocytes, can influence immune function and reduce blood pressure.11,12 Blocking a subtype of CB1 cannabinoid receptor (CB1R) improves hypotension in vivo13 and vascular contractile function in vitro.14 Whether CB1R is involved in the development of shock-induced vascular hyporeactivity is not clear. The present study was designed to examine whether CB1R is involved in the development of vascular hyporeactivity in rats suffering from hemorrhagic shock.

METHODS

Animals

Male Sprague-Dawley rats (280-320 g, 3-6 months) were obtained from the Laboratory Animal Center of the Fourth Military Medical University. The animals were housed in plastic boxes at 20-22°C with a constant 12 hour light/dark cycle. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. The Institutional Animal Care and Use Committee of the Fourth Military Medical University approved the experimental protocols.

Chemicals and solutions

Heparin sodium was purchased from Sigma Chemical Co. (MO, USA). N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1 H-pyrazole -3-carboxamide, SR141716A (Sciphar Biotechnology Co., China) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1 -(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-e-3-carboxamide, AM251 (Tocris Cookson Inc., USA) were freshly dissolved in the vehicle (DMSO:Tween80:PBS=1:1:18). The CB1R primary antibody was purchased from Chemicon International (Temecula, USA), the secondary antibody was purchased from Vector Co. (Burlingame, USA). The RNeasy Protect kit for isolating RNA was purchased from Qiagen (Netherlands). TRIzol Reagent was purchased from Invitrogen (Carlsbad, USA). Norepinephrine was purchased from Serva (Heidelberg, Germany).

Hemorrhagic shock model

The animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). A polyethylene cannula (Intramedic PE-50, Becton-Dickinson, USA) was inserted into the femoral vein for administrating drugs. Two additional polyethylene cannula were inserted into the femoral arteries on both sides for drawing blood samples and measuring mean arterial blood pressure (MAP). The MAP was measured by a polygraph recorder (RM-6200C, Nihon Kohden, Japan) after heparinization with heparin sodium (800 IU/kg i.v., Sigma Chemical Co., USA). Hemorrhagic shock was induced by stepwise bleeding for 20 minutes. The MAP was reduced to and stabilized at (25±5) mmHg for 2 hours according to the method described by Cainazzo et al.13 The vascular reactivity was determined by the responses of MAP to norepinephrine at 6 μg/kg (3 ml·kg-1·h-1).15,16

RT-PCR

The tissues of aorta and 2-3 branches of superior mesenteric artery (SMA) were fixed in TRIzol reagent (Invitrogen Corp, USA) and stored at -80°C. RNA was isolated with the RNeasy Protect kit (Qiagen). The 2 μg of total RNA were reverse-transcribed using oligo (dT) primers. PCR amplification of the first-strand cDNA product was carried out according to the manufacturer's protocol (Invitrogen) in the presence of either two CB1R cDNA specific primers (sense primer, 5′-ATCCTGGTGGTGTTGATCATCTGC-3′; antisense primer, 5′-GGTACAGACACAGGTGTCTGTGCA-3′) or two GAPDH cDNA-specific primers. The following temperature profile was used for PCR amplification: an initial denaturing step at 94°C for 5 minutes, 35 cycles consisting of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute, followed by a final extension step at 72°C for 5 minutes. The films were scanned and analyzed by using the Quantity One (Ver.4.6.2) 1-D Analysis Software (Bio-Rad Co., USA) to obtain the integrated densitometric values. GAPDH was amplified as the internal control.

Western blotting analysis

Membrane proteins from the aorta and SMA were extracted and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane. The membranes were blocked for 1 hour at room temperature in 5% milk washing solution (50 mmol/L Tris-HCl, 100 mmol/L NaCl, and 0.1% Tween-20 at pH 7.5). Subsequently, the membranes were incubated with rabbit anti-rat CB1R antibody (1:500; Chemicon International, USA) in blocking solution overnight at 4°C. The membranes were washed, incubated with goat anti-rabbit IgG (1:2000; Vector Co., USA) in blocking solution at 37°C for 1 hour. The blots were developed using an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA) and exposed to film (Hyperfilm-ECL; Amersham Pharmacia Biotech). The films were scanned and analyzed by using the Quantity One 1-D Analysis Software (Bio-Rad Co). β-actin was used to normalize the protein loaded on membranes.

Experimental design

Changes of vascular reactivity in rats with hemorrhagic shock

Sixteen animals were randomly divided into two groups (n=8 in each group): sham-operated (Sham) and hemorrhagic shock (HS) groups. The animals in Sham group had identical procedures as the HS group without hemorrhage. The changes of vascular reactivity were observed after the end of bleeding in the HS group and at the same time points in the Sham group.

Changes of CB1R mRNA and protein

Twelve additional animals were used in the study. The grouping method and experimental protocols were the same as described above. The changes of CB1R mRNA and protein in the aorta and SMA were analyzed by RT-PCR and Western blotting.

Effect of CB1R antagonists on the vascular hyporeactivity

In order to examine the role of CB1R in the development of vascular hyporeactivity, 32 animals were randomly divided into four groups (n=8 in each group): HS+SR141716A, HS+AM251, HS+Vehicle and HS+Saline groups. Hemorrhagic shock was induced in all animals as described above. The animals in the HS+SR141716A and HS+AM251 groups were injected with the CB1R antagonist, SR141716A 3 mg/kg13 and AM251 3 mg/kg17 after vascular hyporeactivity was determined. The animals in HS+Vehicle and HS+Saline groups were given the same volume of vehicle and saline at the same time point. MAP was monitored all the time during the experiment. Vascular reactivity was determined again 30 minutes after administration of the CB1R antagonist, vehicle and saline.

Effects of CB1R antagonists on the survival rates in rats with hemorrhagic shock

One hundred animals were randomly divided into five groups (n=20 in each group): Sham-operated (Sham), hemorrhagic shock (HS), HS+SR141716A, HS+AM251 and HS+vehicle groups. The survival rates in each group were observed 4 hours after the end of bleeding.

Statistical analysis

All measurements are given as mean±standard error (SE). Survival rates were analyzed by Fisher's exact probability test. MAP response was analyzed by one-way analysis of variance (ANOVA). A P <0.05 was considered statistically significant. The statistical analysis was performed with SPSS 13.0 software.

RESULTS

Vascular hyporeactivity developed in all animals suffering from hemorrhagic shock

The MAP response to norepinephrine (NE) (6 μg/kg, i.v.) in the Sham group was (9.63±0.25) mmHg 1 hour after the end of bleeding. The MAP response to NE in the HS group, however, was reduced to (8.56±0.49) mmHg (P <0.01 vs the Sham group). At 1.5 hours after the end of bleeding the MAP response to NE in HS group was (2.99 ± 0.37) mmHg and it was still significantly lower than that in Sham group (29.75±0.21) mmHg (P <0.01). These results suggest that vascular hyporeactivity is developed in all animals with hemorrhagic shock (Figure 1).

Figure 1.
Figure 1.:
Changes of vascular reactivity in rats with hemorrhagic shock. HS group: Hemorrhagic shock was induced by stepwise bleeding over a period of 20 minutes. MAP was reduced to and stabilized at (25±5) mmHg. Sham group: The animals in this group had identical procedures as the HS group without hemorrhage. Vascular reactivity was measured by the MAP response to NE, (6 μg/kg, 3 ml·kg-1·h-1, i.v.). All measurements are given as mean±SE. *P <0.01 vs Sham group.

Expression of CB1R mRNA and protein increased after the development of vascular hyporeactivity

To determine the expression of CB1R mRNA and protein in the development of vascular hyporeactivity the tissues of the aorta and SMA were collected for analysis of CB1R mRNA and protein. The levels of CB1R mRNA expression in the aorta and SMA were significantly increased in the animals that developed vascular hyporeactivity compared to the Sham group (P <0.01, Figure 2a and 2b). The Western blotting analysis demonstrated similar results regarding the changes of CB1R protein HS vs Sham (P <0.01, Figure 2c and 2d). These results suggest that CB1R is associated with vascular hyporeactivity induced by hemorrhagic shock.

Figure 2.
Figure 2.:
The expression of CB1R mRNA and protein increased after the development of vascular hyporeactivity. a: Representative RT-PCR analysis. b: bar graph shows the results of quantification analysis of CB1R mRNA expression. c: Representative Western blot analysis. d: bar graph shows the results of quantification analysis of CB1R protein expression. *P <0.01 vs Sham group.

CB1R antagonists improved the vascular hyporeactivity and hypotension

To determine the role of CB1R in the development of vascular hyporeactivity, the effects of CB1R antagonists on vascular hyporeactivity were observed at 1.5 hours after the end of bleeding. As seen in Figure 3, the vascular hyporeactivity was significantly potentiated by CB1R antagonists, SR141716A and AM251. The MAP response to NE (6 μg/kg, i.v.) in the SR141716A-treated or AM251-treated groups was (41.75±4.08) or (44.78±1.80) mmHg respectively, which had a significant difference when compared to that in the saline group respectively with (4.22±1.12) mmHg (P <0.01). The above results indicate that CB1R is involved in the development of vascular hyporeactivity resulting from hemorrhagic shock. And no statistically significant difference existed in the change of vascular hyporeactivity between the HS+Saline and HS+Vehicle groups.

Figure 3.
Figure 3.:
CB1R antagonist, SR141716A and AM251, improved the vascular hyporeactivity and hypotension. *P <0.05 vs before NE injection in the same group; †P <0.01 vs before NE injection in HS+Vehicle group; ‡P <0.01 vs after NE injection in HS+Vehicle group. HS: hemorrhagic shock.

CB1R antagonists increased the survival rates in rats suffering from hemorrhagic shock

We further observed the effects of CB1R antagonists, SR141716A and AM251, on the survival rates in the animals that developed vascular hyporeactivity. The survival rates at 2 and 3 hours after the end of bleeding in the HS+SR141716A group were 100% and 90% respectively, which were increased when compared to those in the HS groups respectively with 20% and 0 (P <0.05). The survival rate at 4 hours after the end of bleeding in the HS+SR141716A and HS groups was 20% and 0 respectively with no significant difference between the two groups (Figure 4). In the HS+AM251 group, the survival rates at 2, 3 and 4 hours after the end of bleeding were 100%, 90% and 30% respectively, which were significantly increased when compared with those in HS group respectively (P <0.05). And no statistically significant difference existed in the survival rates at 2, 3 and 4 hours after the end of bleeding between the HS and HS+Vehicle groups. These results demonstrate that both SR141716A and AM251 increase the survival rates of rats that developed vascular hyporeactivity during the observation period (up to 4 hours).

Figure 4.
Figure 4.:
CB1R antagonist, SR141716A and AM251, increased the survival rates in rats suffering from hemorrhagic shock. The animals in HS+SR141716A and HS+AM251 groups received i.v. bolus injections of SR141716A (3 mg/kg) and AM251 (3 mg/kg) at 1.5 hours after the end of bleeding. The animals in the HS+Vehicle and HS, Sham groups were given the same volume of vehicle (DMSO: Tween80:PBS=1:1:18) and saline at the same time points. *P <0.05 vs Sham group, †P <0.05 vs HS+Vehicle group.

DISCUSSION

Hemorrhagic shock is common in the patients with traumatic or surgical shock. A volume-controlled hemorrhagic shock rat model,13 characterized by low blood pressure, hypovolemia and low organ perfusion, was used in the present study. Hemorrhagic shock accompanied with vascular hyporeactivity is one of the causes of multiple organ failure. The change of vascular reactivity induced by hemorrhagic shock has two distinct phases, an initial stage of compensatory adjustment and a terminal stage of decompensatory adjustment that leads to vascular hyporeactivity.1 The present results showed that the vascular hyporeactivity was developed in all animals with hemorrhagic shock.

Previous studies reported that CB1R is expressed in vascular smooth muscle cells of cat cerebral artery, rat mesenteric artery, rabbit and rat aorta, and human coronary artery, as well as vascular endothelial cells of rat mesenteric artery and renal artery.11,18-22 It is believed that the altered behavior of the resistance vessels is the main reason of the development of vascular reactivity.1 In the present study, we examined the changes of CB1R mRNA and protein expression in 2-3 branches of the superior mesenteric artery by RT-PCR and Western blotting analysis. We found that the levels of CB1R mRNA and protein expression increased significantly in 2-3 branches of the superior mesenteric artery and aorta during the phase of vascular hyporeactivity, suggesting that CB1R is associated with vascular hyporeactivity induced by hemorrhagic shock.

We further demonstrated that the CB1R antagonists, SR141716A and AM251, improved vascular hyporeactivity and hypotension when applied during the terminal stage of hemorrhagic shock. It has been reported that SR141716A and AM251 could improve hypotension associated with hemorrhagic, endotoxic, and cardiogenic shock in vivo,23 and improve vascular contractile function in vitro.14 We also showed that the CB1R antagonist could improve the survival rates when used during the terminal stage of hemorrhagic shock accompanied by vascular hyporeactivity. The present study suggests that the CB1R antagonists, AM251 or SR141716A, are capable of preventing mortality by indirect inhibition of inflammation factors for a number of diseases in vivo,24 which may be explained by some reasons such as the release of endothelium-derived hyperpolarizing factor,25 activation of potassium channels, inhibition of calcium channels,26 direct effects of cannabinoid receptors on vascular smooth muscle,20 release of mediators from sensory nerves, and presynaptic inhibition of norepinephrine release in vitro and in vivo.27,28 We supposed that CB1R participate in development of vascular hyporeactivity by the above mechanism. The detailed mechanisms, however, remain to be evaluated.

In summary, we provide evidence that CB1R is involved in the development of vascular hyporeactivity resulting from hemorrhagic shock. CB1R antagonists could improve vascular hyporeactivity and hypotension, and thus increase the survival rates in rats. The present results suggest that CB1R antagonists may be useful in treating patients with traumatic, hemorrhagic shock who need field-rescue or initial treatment.

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Keywords:

hemorrhagic shock; vascular hyporeactivity; CB1 cannabinoid receptor

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