内源性一氧化碳在大鼠败血症性休克时
低血压发生机制中的作用

欧和生　杨军　董林旺　庞永政　苏静怡　唐朝枢　刘乃奎

　　摘要　应用盲肠结扎法制备大鼠败血症休克模型,研究内源性一氧化碳(CO)在败血症休克时低血压发病中的作用。用血红素加氧酶(heme oxygenase,HO)抑制剂2,4-二甘油次卟啉锌(zinc deuteroporphyrin 2,4-bisglycol,ZnDPBG)处理大鼠后,观察动物动脉血压,同时测定主动脉平滑肌组织中HO活性和CO生成量。结果发现: 败血症大鼠动脉收缩压、 舒张压降低,同时血管平滑肌HO活性和CO生成明显增加。败血症大鼠用ZnDPBG处理后,动脉血压明显回升,同时HO活性和CO生成明显被抑制。实验表明败血症休克时低血压的发生与血管平滑肌细胞HO活性增加和内源性CO生成增多明显相关;应用HO抑制剂阻断HO活性能导致内源性CO生成减少,继而使败血症休克时大鼠血压明显回升。实验提示,内源性CO对血管张力具有重要的调节作用;HO活性和内源性CO生成增加是败血症休克时低血压发生的重要机制之一。
　　关键词: 低血压； 败血症休克； 一氧化碳； 血红素加氧酶
　　学科分类号: R54.4

ROLE OF ENDOGENOUS CARBON MONOXIDE IN THE
PATHOGENESIS OF HYPOTENSION DURING SEPTIC SHOCK^*

OU　HE-SHENG，YANG　JUN,DONG　LIN-WANG,PANG　YONG-ZHENG,
SU　JING-YI,TANG　CHAO-SHU,LIU　NAI-KUI
(Institute of Cardiovascular Research,Beijing Medical University,Bejing 100083)

　　ABSTRACT　A sepsis model induced by cecal ligation and puncture was used to study the role of endogenous carbon monoxide in hypotension pathogenesis of rats during septic shock. After administration of zinc deuteroporphyrin 2,4-bisglycol (ZnDPBG),an inhibitor of heme oxygenase (HO),blood pressure (BP),HO activity and carbon monoxide (CO) release from vascular muscle tissue were measured. The results showed that BP of sepsis rats,including systolic and diastolic arterial BP,decreased significantly while HO activity and CO content were significantly increased. In contrast,after administration of ZnDPBG,BP of sepsis rats was significantly increased while the HO activity and CO production were significantly decreased. These findings suggest that HO activity and CO release within vascular musculature are increased during septic shock;inhibition of HO may elevate BP of rats during septic shock through a decrease of endogenous CO production. It is concluded that endogenous CO derived from vascular muscle cells plays an important role in regulating vascular tone,and the up-regulation of HO activity followed by subsequent CO production contributes to hypotension pathogenesis during septic shock.
　　Key words: hypotension;septic shock;carbon monoxide;heme oxygenase

　　Endotoxic shock is a detrimental consequence of severe Gram-negative bacterial infection characterized by vascular smooth muscle cell relaxation and severe hypotension. The mechanisms of hypotension during septic shock are unclear. Some previous studies suggested that nitric oxide (NO) may play an important role in the pathogenesis of endotoxic shock^［1，2］. However,MacMicking and his colleagues reported that an inducible NOS-independent pathway contributed to hypotension and death in septic shock^［3］,and recent studies expressed an interesting speculation that one potential pathway involves heme oxygenase (HO)^［4，5］.
　　Heme oxygenase (HO)is a mammalian enzyme that converts almost 1% of the heme in blood to bilirubin and carbon monoxide (CO)^［6］,and zinc deuteroporphyrin 2,4-bisglycol (ZnDPBG) is an in～hib～itor of this enzyme.Recent studies revealed that CO may be endogenously produced by arteries^［7，8］. As a gas molecule,CO can activate soluble guanylate cyclase and relaxes vascular smooth muscle,similar to NO.The regulatory functions of endogenous CO on hemodynamics during septic shock are not clear.Some reports showed that hypoxia may induce HO activity and subsequently promote accumulation of cGMP in vascular smooth muscle cells^［7］.Endogenous CO derived from vascular smooth muscle cells also inhibits the production of endothelium-derived vasoactive agents such as endothelin-1 and platelete-derived growth factor-B under hypoxic conditions^［5］.These previous studies suggest that endogenous CO plays an important role in vascular biology.
　　We designed the present study to assess the contribution of heme oxygenase activity of endogenous CO to production of hypotension of rats during septic shock.To accomplish these,we used ZnDPBG as an inhibitor of HO to sepsis rats for investigation of the hemodynamics,HO activity and CO release from vascular smooth muscle in contrast with those rats without administration of ZnDPBG.

　　1　MATERIALS AND METHODS

　　1.1　 Animal model and measurement of hemodynamic parameters　　 Male Sprague-Dawley rats (270～320 g) were used.All animals were fasted overnight with free access to water.Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et al.^［8］ with minor modification.Under halothane anesthesia,a laparotomy was performed,and the cecum was ligated with 3～0 silk ligature and punctured twice with an 18-gauge needle.The cecum was then returned to the peritoneal cavity and the abdomen was closed in two layers.Control rats were sham-operated (a laparotomy was performed and the cecum was manipulated with neither ligation nor puncture).All animals are resuscitated with 4 ml/100　g body weight of normal saline at the completion of surgery and also 7 h after surgery.Animals were fasted but had free access to water after operative procedures.Septic shock animals refers to those sacrificed at 18 h after CLP.The mortality rates were 0% (0/6) for control and 20% (4/20) for septic shock.
　　All animals were divided into four groups (n=6 in each): the sham,the sham plus ZnDPBG,sepsis and sepsis plus ZnDPBG group,which were intraperitoneally injected with the following different drugs at 17 h after CLP,i.e.,1 h before measurement of hemodynamic parameters.The rats in the sham plus ZnDPBG group and the sepsis plus ZnDPBG group were injected with ZnDPBG (45 μmol/kg,ip),and the rats in the sham group and the sepsis group were treated with a drug vehicle (50 mmol/L Na₂CO₃).
　　At 18 h after CLP,the rats were anesthetized with sodium pentobarbital (60 mg/kg,ip) and instrumented with a vascular indwelling polyethylene cannula (PE-50) filled with heparinized saline,which was introduced into the right common carotid artery.The arterial cannula was connected to a pressure transducer (Model P223XL,Gould Inc) for recording arterial blood pressure (ABP) on a polygraph (Model 7D,Grass Instruments Co.).Heart rate (HR) was monitored by means of a cardiotachometer (Model 7P44C,Grass Instruments Co.) triggered by the systolic pressure.
　　After measurement of hemodynamic parameters the animals were killed through exosanguisation.The aorta was immediately removed to assay vascular HO activity and CO release,respectively.
　　1.2　Assay of HO activity　　 The method for assaying HO activity of vascular smooth muscle microsomes has been described in detail previously^［9］.Briefly,the microsome was isolated from vascular musculature and was then added to a reaction mixture containing liver cytosol (0.6 mg),NADP (0.8 mmol/L),glucose-6-phosphate (1 mmol/L),and glucose-6 phosphate dehydrogenase (0.2 U).Finally,10 μl of 2.5 mmol/L hemin was added as the substrate.The mixtures were aerobically incubated for 10 min at 37℃ in the dark.After the reaction was stopped,the amount of bilirubin formed was measured with a double-beam spectrophotometer of 464～530 nm OD.HO activity was expressed as pmol of bilirubin formed per mg of aortic microsome in 60 min.The protein content was determined by a dye-binding assay kit (Bio-Rad).
　　1.3　Measurement of CO release from vascular muscle tissue　　 The method for measuring carboxyhemoglobin (HbCO) formation was described in detail previously^［9］.Briefly,aortic muscle tissues were cut into 3 mm×3 mm sections and incubated in DMEM medium containing 50 μmol/L pure Hb equilibrated with 95%　O₂-5%　CO₂ at 37℃ for 1 h.HbCO gives the maximal absorbance on a CO-oximeter at a wavelength of 569 nm and is calculated as the percentage of total Hb.The added amount of Hb was determined from a standard curve using exogenous CO and varying amounts of Hb over a range of concentrations where the absorbance was linear.
　　1.4　Statistics　　 Results are expressed as (±) and were analyzed by ANOVA.A P<0.05 was considered to indicate significance.

　　2　RESULTS

　　2.1　Changes in mean arterial blood pressure (MABP) and HR of rats after administration of ZnDPBG
　　Table 1 shows the data from the rats of the four groups.As compared with the sham group,the systolic ABP,diastolic ABP and pulse difference of ABP in sepsis rats were significantly decreased (P<0.01) together with the HR (P<0.01).No such change was found between the sham group and the sham plus ZnDPBG group.In contrast,the systolic ABP,diastolic ABP and pulse difference of ABP in sepsis plus ZnDPBG group were significantly elevated (P<0.01),and the heart rate in sepsis plus ZnDPBG group is significantly increased (P<0.05) compared with sepsis group.

Table　1　Changes in MABP and HR of rats after administration of ZnDPBG

Group	Sham	Sham+ZnDPBG	Sepsis	Sepsis+ZnDPBG
Systolic ABP (kPa)	16.32±0.92	15.53±0.79	12.12±1.05^**	13.95±1.05^##
Diastolic ABP (kPa)	10.26±0.53	10.53±0.53	7.90±0.53^**	9.74±0.53^##
Pulse difference of BP (kPa)	6.05±0.66	5.13±0.66	4.21±0.53^**	4.61±0.53
Heart rate (beats/min)	414±22	406±17	302±32^**	364±28^#

^**P<0.01,compared with the sham group;^#P<0.05,^##P<0.01,compared with sepsis group;n=6 in each group.

　　2.2　Changes in HO activity of vascular smooth muscle cell microsomes after administration of ZnDPBG
　　Figure 1 shows the data on the changes of HO activity of vascular smooth muscle cell microsomes in the four group rats.Compared with the sham group,the HO activity in sham plus ZnDPBG group was significantly decreased (54±9 vs 386±28 pmol/(mg^.h),P<0.01);while that in sepsis group was significantly increased (898±44 vs 386±28 pmol/(mg^.h),P<0.01).But the HO activity in sepsis plus ZnDPBG group was significantly decreased compared with sepsis group (96±10 vs 898±44 pmol/(mg.h),P<0.01).

Fig.1　Changes of HO activity in smooth muscle
tissues after administration of ZnDPBG
^**P<0.01,compared with the sham group;^##P<0.01,
compared with sepsis group (n=6 in each group).

　　2.3　Changes in carbon monoxide formation of smooth muscle tissues after administration of ZnDPBG
　　Figure 2 shows the data of the changes of CO release from vascular smooth muscle tissues in the four groups of rats.Compared with the sham group,the CO formation in the sham plus ZnDPBG group was significantly decreased (1.22±0.03 vs 6.02±0.60 μmol/L,P<0.01) while that in sepsis group was significantly increased (16.66±2.21 vs 6.02±0.60 μmol/L,P<0.01).But the CO formation in sepsis plus ZnDPBG was significantly decreased compared with sepsis group (6.88±1.01 vs 16.66±2.21 μmol/L,P<0.01).

Fig.2　Changes of HbCO formation in smooth muscle tissues after administration of ZnDPBG^**P<0.01,compared with the sham group;^##P<0.01,compared with sepsis group,n=6 in each group.

　　3　DISCUSSION

　　Endotoxemia leading to septic shock is a severe consequence of Gram-negative bacterial infection.One of the pathological characters of the case is vascular smooth muscle cell relaxation and severe hypotension.The mechanism of severe hypotension in this pathological condition is unclear.Althrough the physiologic importance of NO and NO synthase (NOS) have been studied in detail during septic shock,some investigators have found a potential NOS-independent pathway contributing to regulating vascular tone^［3］,which appears to involve endogenous CO derived from vascular smooth muscle cells according to our experiments.
　　Heme oxygenase (HO) is an enzyme that converts heme to bilirubin and CO.As a byproduct of heme degradation,CO is an activator of guanylyl cyclase relaxing vascular smooth muscle via cGMP-dependent mechanism.This gas molecule shares many biological functions of NO,for example,both of them are capable of binding and activating guanylyl cyclase.Recent studies demonstrate that CO may be endogenously produced by arteries^［5,10］,and administration of HO inhibitor,such as ZnDPBG,can inhibit HO activity with subsequent CO formation in vascular smooth muscle tissue^［11］.
　　In the present study,the arterial blood pressure of sepsis rats was significantly decreased,while the HO activity and CO formation in vascular smooth muscle were significantly increased compared with the sham rats.It is suggested that the pressure in sepsis is associated with increased HO activity and subsequent endogenous CO production.To further verify this hypothesis,we treated the sepsis rats with ZnDPBG which can completely inhibit HO activity and CO formation in vascular smooth muscle.Our results suggest that administration of ZnDPBG to sepsis rats can increase arterial blood pressure and decrease HO activity and CO formation.No such effects were found when the sham rats were treated with ZnDPBG,implying that the effects of HO activity inhibitor on hemodynamic measurement in this study were associated with those of bacterial infection.Recent reports revealed that some cytokines,such as IL-1β,may involve the relaxation of vascular smooth muscle cells during endotoxemia.IL-1β is an important mediator during septic shock which stimulates an increase of NO production in vascular smooth muscle cells^［1］,and induces HO mRNA^［12］.But a recent report by Durante et al.revealed that NO may directly induce HO gene expression and CO production in vascular smooth muscle cells^［13］.These studies suggest that the increase of HO activity and subsequent CO production may involve both NO-dependent and NO-independent pathway.Data of this study suggest that ZnDPBG may elevate the blood pressure through some inflammatory mediators,such as IL-1β and TNF-α,because no such effect was found in the sham plus ZnDPBG rats.
　　Our study demonstrate that (1) HO activity can be up-regulated within vascular smooth muscle cells by some pathological factors such as infection of Gram-negative bacteria;（2) the increasing HO activity and subsequent CO production in vascular smooth muscle cells contribute to the relaxation of smooth muscle and hypotension during septic shock;and （3) inhibitor of HO ZnDPBG can elevate arterial blood pressure of sepsis rats during septic shock,implying that inhibition of HO and endogenous CO formation may restore hypotension during severe endotoxemia.We conclude that endogenous CO plays an important role in regulating vascular tone,and ZnDPBG may be applied clinically for preventing relaxation of vascular smooth muscle tone and hypotension during severe endotoxemia.

^*国家自然科学基金资助项目(No.39730220)
This work was supported by research grants from National Natural Science Foundation of China (No.39730220).

作者单位：北京医科大学心血管研究所，北京 100083

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Received 1998-03-15　Revised 1998-05-29