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PROTECTIVE EFFECTS OF DIHYDROXYLPHENYL LACTIC ACID AND SALVIANOLIC ACID B ON LPS-INDUCED MESENTERIC MICROCIRCULATORY DISTURBANCE IN RATS

Guo, Jun*‡; Sun, Kai*; Wang, Chuan-She*†; Fang, Su-Ping*; Horie, Yoshinori; Yang, Ji-Ying*; Liu, Yu-Ying*; Wang, Fang*; Liu, Lian-Yi*; Fan, Jing-Yu*; Hibi, Toshifumi; Han, Jing-Yan*†‡

doi: 10.1097/shk.0b013e318070c61a
Basic Science Aspects
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Salvia miltiorrhiza is a Chinese medicine widely used for treatment of various cardiovascular diseases. However, little is known about the role of dihydroxylphenyl lactic acid (DLA) and salvianolic acid B (SAB), the main ingredients of S. miltiorrhiza, in the microcirculation. This study aimed to investigate the effect of DLA and SAB on LPS-elicited microcirculatory disturbance, focusing especially on leukocyte adhesion and its potential mechanism. Mesenteric venular diameter, velocity of red blood cells in venules, shear rate of the venular wall, numbers of leukocytes adherent to and emigrated across the venular wall, and mast cell degranulation were determined by an inverted microscope in rats after LPS infusion with or without DLA or SAB. Expression of CD11b and CD18 and production of superoxide anion (O2) and hydrogen peroxide (H2O2) by neutrophils were evaluated in vitro by flow cytometry. LPS exposure induced a significant increase in the number of adherent and emigrated leukocytes and mast cell degranulation, and a prominent decrease in the velocity of red blood cells in venules and shear rate of the venular wall. Additionally, in vitro experiments revealed an apparent enhancement in expression of CD11b and CD18 and production of O2 and H2O2 by rat neutrophils by LPS stimulation. Treatment with DLA or SAB significantly ameliorated LPS-induced microcirculatory disturbance in rat mesentery and inhibited both the expression of CD11b and CD18 and the production of O2 and H2O2 by neutrophils caused by LPS.

Abbreviations - O2-superoxide anion; DCFH-DA-2′, 7′-dichlorofluorescein diacetate; DLA-dihydroxylphenyl lactic acid; EB-ethidium bromide; H2O2-hydrogen peroxide; ICAM-1-intercellular adhesion molecule 1; SAB-salvianolic acid B; SM-Salvia miltiorrhiza

*Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China; Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China; and Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

Received 20 Mar 2007; first review completed 2 Apr 2007; accepted in final form 10 Apr 2007

Address reprint requests to Jing-Yan Han, MD, PhD, Director of Tasly Microcirculation Research Center, Peking University Health Science Center, No. 38 Xueyuan Road Haidian District, Beijing 100083, China. E-mail: kan@chuigaku.co.jp.

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INTRODUCTION

Salvia miltiorrhiza (SM) has been popularly used for treatment of a variety of cardiovascular disorders such as coronary heart disease, hyperlipidemia, and cerebrovascular disease, and the rationale for such treatment has attracted much attention in past decades. The major water-soluble components of SM are dihydroxylphenyl lactic acid (DLA; Fig. 1A) and salvianolic acid B (SAB; Fig. 1B). We have previously reported that cardiotonic pills, a major ingredient of which is SM, can attenuate the adhesion of leukocytes to hepatic sinusoids after I/R in rats chronically fed ethanol (1). Moreover, the water-soluble extract of SM and SAB is found to suppress neutrophil-endothelial adhesion and inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells induced by TNF-α (2). It was also reported that water-soluble extract of SM and SAB attenuated the expression of endothelial cell ICAM-1 and vascular cell adhesion molecule 1 induced by TNF-α by inhibiting the activation of nuclear factor-κB (3, 4). On the other hand, it was demonstrated that DLA can effectively scavenge superoxide anion generated from the xanthin-xanthin oxidase system (5). Similarly, antioxidant activity was also reported for SAB (6, 7). However, despite this previous work, little is known regarding the in vivo effect of DLA and SAB on the microcirculatory disturbance occurring in inflammatory conditions and the influence of these compounds on the associated reactions such as adhesion of leukocytes to venular wall, emigration of leukocytes across the venular wall, and degranulation of mast cells.

Fig. 1

Fig. 1

LPS is a component of the cell wall of gram-negative bacteria that gives rise to septic shock in human beings (8), typically with sequential multiple organ injury (9-11). Leukocyte infiltration into tissues is implicated in this process. Leukocyte recruitment and extravasation are controlled by adhesion molecules present on leukocytes and endothelial cells with involvement of reactive oxygen species (12, 13) and degranulation of mast cells (14). Activation and up-regulation of endothelial selectins (E and P selectin) and leukocyte selectin (L selectin) permit transient leukocyte attachment to the endothelial surface followed by rolling of these cells along the vessel wall, whereas the activation of CD11b/CD18 in neutrophils mediates firm neutrophil adhesion to the ICAM-1 on the endothelium (15, 16). Thus, inhibition of adhesion between leukocytes and endothelial cells is one of the crucial steps in the improvement of microcirculatory disturbance induced by LPS (17). Experimentally, i.v. infusion of low concentrations of LPS can evoke the microcirculatory disturbance without interference with systematic circulation and is therefore widely used as a model in the field of microcirculatory disturbance research (18). Using this model, the present study investigated the in vivo effects of DLA and SAB on microcirculatory disturbance in rat mesentery by evaluating the venular diameter, velocity of red blood cells (RBCs) in venules, mesenteric venular shear rate, adhesion of leukocytes to the venular wall, emigration of leukocytes across venular wall, and degranulation of mast cells. Furthermore, the influence of DLA and SAB on the expression of CD11b and CD18 and the production of superoxide anion (O2) and hydrogen peroxide (H2O2) by neutrophils were studied in an in vitro experiment using flow cytometry (fluorescence-activated cell sorter [FACS]).

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MATERIALS AND METHODS

Reagents

Dihydroxylphenyl lactic acid and SAB were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). LPS derived from Escherichia coli serotype O55:B5, toluidine blue, 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), and hydroethidine were obtained from Sigma (St. Louis, Mo). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD11b monoclonal antibody; FITC-conjugated mouse anti-rat CD18 monoclonal antibody; FITC-conjugated mouse immunoglobulin (Ig)A, κ and FITC-conjugated mouse IgG1, κ were purchased from BD Biosciences Pharmingen (San Diego, Calif). Hemolysin was purchased from BD Biosciences Immunocytometer Systems (San Jose, Calif). Mono-Poly resolving medium was purchased from Dainippon Pharmaceutical Corporation (Osaka, Japan). RPMI 1640 and fetal bovine serum were purchased from Hyclone (Logan, Utah). All other chemicals used were of the highest grade available commercially.

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Animals

Male Sprague-Dawley rats weighing 200 to 250 g were provided by the Animal Center of Peking University Health Science Center. All studies were approved by and all animals were handled according to the guidelines of the Peking University Animal Research Committee.

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Observation of mesenteric microcirculation

Sprague-Dawley rats were fasted for 12 h before the experiment, while allowing free access to water. The animals were anesthetized with urethane (1.25 mg kg−1 body weight, i.m.). The left jugular and left femoral veins were cannulated for injection of various drugs. The abdomen was opened via a midline incision 20 to 30 mm long, and the ileocecal portion of the mesentery 20-cm caudal was gently drawn out, exteriorized, and mounted on a transparent plastic stage designed for rats. The mesentery was kept at 37°C by a constant temperature device and moistened by continuous superfusion with physiologic saline. Microcirculatory hemodynamics in the mesentery were observed by a transillumination method using an inverted microscope (DM-IRB; Leica, Wetzlar, Germany). A video camera (Jk-TU53H; TOSHIBA, Tokyo, Japan) mounted on the microscope projected the image onto a color monitor (J2118A; TCL, Huizhou, China), and the images were recorded with a DVD (DVR-R25; Malata, Xiamen, China). A single unbranched venule without an obvious bend and with diameter ranging between 30 and 50 μm and length of about 200 μm was selected for determination of all the parameters in the present study (19).

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LPS-induced microcirculatory disturbance and drug infusion

The rats were randomly divided into four groups, each with 6 animals. After 10 min of observation of the basal hemodynamics in the rat mesenteric microvasculature, the vehicle (saline solution) or experimental drugs were administrated. In the control group, the animals were infused with saline solution from the start of experiment (0 min) to the end of experiment (60 min) via the cannulated left jugular vein (8 mL kg−1 body weight h−1), whereas in the LPS group, animals received LPS (2 mg kg−1 body weight h−1 in saline solution) infused continuously from the start of experiment (0 min) until the end of experiment (60 min) from the left femoral vein (20). In the LPS + DLA group, in addition to LPS infusion (2 mg kg body weight h−1) into the left femoral vein, the animals were subject to DLA infusion (5 mg kg−1 body weight h−1 in saline solution) into the left jugular vein, which started 20 min before the experiment and continued until the end of the observation. The animals in the LPS + SAB group received the same treatment as those in LPS + DLA group, but SAB (5 mg kg−1 body weight h−1 in saline solution) was substituted for DLA. The total infusion amount was the same in the four groups.

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Determination of venular diameter

Venular diameter was determined by Image-Pro Plus 5.0 software (19) at 0, 10, 30, and 50 min of observation by evaluating the dynamic images of venules recorded with this system.

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Measurement of velocity of RBCs in the venules

The velocity of RBCs in the venules was recorded at a rate of 2,000 frames per second by changing the monitor from charge-coupled device to a high-speed video camera system (FASTCAM-ultima APX; Photron, San Diego, Calif), and the recordings were replayed from the high-speed stored images at a rate of 25 frames per second. Red blood cell velocity in the venule was measured with Image-Pro Plus 5.0 software (19) at 0, 10, 30, and 50 min of observation.

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Measurement of shear rate

The shear rate of venules (γ) was calculated according to the formula γ = 8 (mean velocity of RBCs in venules/venular diameter) (21).

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Determination of adherent leukocytes

The leukocytes that were adherent to the venular wall were identified by reviewing the dynamic images of leukocytes recorded with this system. Adherent leukocytes were defined as those that attached to the same site for more than 10 s as determined from replay of the DVD images. The number of adherent leukocytes was counted along 200-μm venules randomly selected from these images at 0, 10, 30, and 50 min of observation (19).

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Determination of emigrated leukocytes

The number of emigrated leukocytes was determined by examining the recorded images. Leukocyte emigration was expressed as the number of leukocytes per 200-μm length of venule surrounding the venule randomly selected from the images at 0, 10, 30, and 50 min of observation (22).

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Determination of mast cell degranulation

Sixty minutes after the infusion of LPS or saline solution, the tissue was stained with 0.1% toluidine blue for 1 min by topical application followed by rinsing with physiologic saline. The number of both nondegranulated mast cells and degranulated mast cells in each field was scored at a magnification of ×20, and a total of five fields were evaluated for each animal. The ratio of the number of degranulated mast cells to the total number of mast cells was presented as the percentage of degranulated mast cells (19).

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Determination of expression of CD11b and CD18 in neutrophils

To determine the expression of CD11b and CD18 in neutrophils, blood was collected from another group of rats (n = 6) via the abdominal aorta and anticoagulated with heparin. The samples were divided into four groups: control group, LPS group (LPS, 2 μg mL−1), LPS + DLA group (2 μg mL−1 LPS plus 0.2, 0.5, or 1.0 mg mL−1 DLA) and LPS + SAB group (2 μg mL−1 LPS plus 0.2, 0.5, or 1.0 mg mL−1 SAB). The samples were incubated at 37°C in a water bath for 2 h and then labeled with either 1 μg mL−1 FITC-conjugated mouse anti-rat CD11b antibody or 1 μg mL−1 FITC-conjugated mouse anti-rat CD18 anti-body, or the FITC-conjugated corresponding isotypes (mouse IgA, κ for CD11b; mouse IgG1, κ for CD18) for 20 min at room temperature. Afterwards, erythrocytes were lysed using hemolysin as described by the manufacturer (BD Biosciences), and the remaining cells were washed twice with phosphate-buffered saline. Neutrophils were then sorted by FACS Calibur Flow cytometry (BD Biosciences) based on characteristic forward-/side-scatter expression, and 5,000 neutrophils were evaluated from each sample for determination of mean fluorescence intensity (23).

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Determination of intracellular H2O2 and O2 production by neutrophils

Blood was taken from a separate group of rats and anticoagulated with heparin. Neutrophils were isolated using Mono-Poly resolving medium as described by Ting and Morris (24) and suspended in 0.1 M phosphate-buffered saline. Intracellular production of O2 and H2O2 was analyzed by FACS Calibur Flow cytometry (BD Biosciences) (25). Briefly, collected neutrophils were incubated at 37°C for 5 min with 20 mM DCFH-DA and then for an additional 15 min with 10 mM of hydroethidine. The acetate moieties of DCFH-DA are cleaved off intracellularly by esterases, liberating the membrane-impermeable 2′, 7′-dichlorofluorescein, which fluoresces when oxidized to DCF by H2O2; hydroethidium, on the other hand, can be directly oxidized by O2 to ethidium bromide, which fluoresces after intercalating with nucleic acids. The cells were next treated with DLA (0.5 mg mL−1) or SAB (0.5 mg mL−1) and stimulated with LPS (2 μg mL−1). After 20 min incubation on ice, the production of H2O2 and O2 was quantified by flow cytometry by measuring emission at 525 (FL1, for DCF) and 590 nm (FL2, for ethidium bromide).

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Statistical analysis

Statistical significance was calculated using ANOVA and Fisher post hoc test. A level of P < 0.05 was considered statistically significant. Data were expressed as mean ± SEM.

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RESULTS

Effects of DLA and SAB on LPS-induced changes in mesenteric venular diameter

The venular diameters under baseline conditions were 39.29 ± 3.79 μm in the control group, 39.83 ± 1.72 μm in the LPS group, 34.96 ± 2.22 μm in the LPS + DLA group, and 39.44 ± 3.29 μm in the LPS + SAB group. There was no significant difference among these four groups, and there was no significant difference among them at any time in the observation (data not shown).

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Effect of DLA and SAB on LPS-induced changes in velocity of RBCs in venules

The velocities of RBCs in venules in the four groups were determined at different time points (Table 1). Thirty minutes after LPS infusion, a decrease in velocity of RBCs in venules to 74% of the baseline was elicited, and this effect remained almost constant until the end of observation. This LPS-induced decrease in the velocity of RBCs in venules was obviously attenuated by treatment with either DLA or SAB.

Table 1

Table 1

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Effect of DLA and SAB on LPS-induced changes in mesenteric venular shear rate

Table 2 tabulates mesenteric venular shear rates in the four groups observed at different time points. No significant difference was found in baseline venular shear rate among the control, LPS, LPS + DLA, and LPS + SAB groups. LPS exposure led to an apparent decrease in mesenteric venular shear rate, which became significant after 30 min, and this decrease was sustained until 50 min after the LPS infusion. Dihydroxylphenyl lactic acid or SAB treatment attenuated the decrease in mesenteric venular shear rate induced by LPS, although a slight, but not significant, decrease in the shear rate in either the LPS + DLA or LPS + SAB group could be detected 50 min after LPS infusion.

Table 2

Table 2

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Effect of DLA and SAB on LPS-induced changes in the number of leukocytes adherent to venule wall

The time course of changes in the number of leukocytes adherent to venule wall under various conditions was evaluated (Fig. 2). The number of adherent leukocytes in the control group increased only slightly with time over the period of observation. However, LPS stimulation elicited a significant and progressive increase in the number of adherent leukocytes, starting from 10 min, and this tendency to increase was maintained until 50 min after LPS infusion. Treatment with either DLA or SAB resulted in marked inhibition of the LPS-induced leukocyte adhesion.

Fig. 2

Fig. 2

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Effect of DLA and SAB on LPS-induced changes in the number of leukocytes emigrated across venule wall

The time course of changes in the number of leukocytes emigrated across venule wall is shown in Figure 3. In the control group, few or no emigrated leukocytes were observed in the areas examined at all time points. Administration of LPS caused a significant increase in the number of emigrated leukocytes at 50 min, and this increase was almost completely abolished by treatment with DLA or SAB.

Fig. 3

Fig. 3

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Effect of DLA and SAB on LPS-induced mast cell degranulation

Figure 4 shows the percentage of degranulated mast cells observed along venules in each of the four groups. A small population of degranulated mast cells can be detected even in the control group. The number of degranulated mast cells increased at 60 min after LPS infusion, whereas DLA or SAB treatment significantly inhibited LPS-induced degranulation of mast cells.

Fig. 4

Fig. 4

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Effect of DLA and SAB on expression of CD11b and CD18 in neutrophils in vitro

Flow cytometry was used to analyze the expression of adhesion molecules CD11b and CD18 in neutrophils subject to various treatments. Figure 5A shows that the fluorescence intensity of CD11b of neutrophils was significantly enhanced after exposure to LPS in comparison with control, and this LPS-enhanced CD11b fluorescence intensity was profoundly diminished by addition of either SAB or DLA, with effective concentrations of the former as low as 0.2 mg mL−1 and the latter 0.5 mg mL−1. Figure 5B demonstrates that a similar tendency was observed for adhesion molecule CD18: the fluorescence intensity of neutrophil CD18 increased significantly in response to LPS, and treatment with DLA or SAB resulted in a dramatic inhibition of this response when the concentrations that were used reached 0.2 mg mL−1.

Fig. 5

Fig. 5

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Effect of DLA and SAB on production of intracellular O2 and H2O2 by neutrophils in vitro

Antioxidative effects of DLA and SAB were accessed by flow cytometry. Production of intracellular O2- in neutrophils was elevated significantly when the cells were subjected to LPS stimulation, and addition of DLA or SAB at a concentration of 0.5 mg mL−1 depressed the production of O2- remarkably (Fig. 6A). LPS stimulation evoked an almost 2-fold increase in the fluorescence intensity of neutrophil H2O2 as compared with the control, and this effect was significantly attenuated by treatment with either DLA or SAB at a concentration of 0.5 mg mL−1 (Fig. 6B).

Fig. 6

Fig. 6

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DISCUSSION

The present study demonstrated the protective effects of DLA and SAB on the microcirculatory disturbance induced by LPS in rat mesentery, including the inhibition of leukocyte adhesion and emigration, attenuation of the decrease in velocity of RBCs and venular shear rate, and suppression of mast cell degranulation. Furthermore, DLA and SAB were found to suppress the expression of adhesion molecules CD11b and CD18 and the production of O2 and H2O2 by neutrophils after LPS stimulation.

In inflammatory tissue reactions in response to LPS, leukocyte infiltration has been well documented to mediate local tissue damage (15). Leukocyte adhesion and extravasation are controlled by adhesion molecules on leukocytes and endothelial cells, including CD11b and CD18 in neutrophils. Hence, immunoneutralization of one or more adhesion molecules would be a potential strategy for interruption of the LPS-induced inflammatory process. On the other hand, accumulating evidence suggests that free radicals are involved directly and indirectly in the pathophysiology of sepsis or endotoxemia. Free radicals cause cytotoxicity in endothelial cells and induce immediate proinflammatory cytokine and chemokine production by macrophages, which subsequently attract inflammatory cells into the tissue. Based on these facts, some efforts have been made to scavenge free radicals after LPS exposure by antioxidants to prevent tissue injury. However, such approaches have proven to be quite challenging due to the complexity of these inflammatory reactions. Therefore, it seems appropriate to suggest use of medicine that exert effect on LPS-induced microcirculatory disorders not only by inhibition of adhesion between leukocytes and endothelial cells but also by scavenging free radicals. The findings in the present study suggest that both DLA and SAB, the two water-soluble components of SM, have such therapeutic capacity and may account for the recognized effectiveness of SM in clinical use for improvement of microcirculatory disturbance in various vascular disorders.

It is well known that several families of cell adhesion molecules are implicated in LPS-induced leukocyte accumulation at the site of inflammation. Initial tethering of leukocytes in postcapillary venules requires the expression of selectins on endothelial cells and expression of their counter-receptors on leukocytes. Subsequent activation of CD11b and CD18 on leukocytes by chemotactic factors and up-regulation of ICAM-1 on endothelial cells leads to the firm adhesion of leukocytes on the endothelial surface followed by extravasation and emigration to the inflammatory site (26). Obviously, interruption of any of these steps will prevent leukocyte accumulation at the site of inflammation. The present in vitro study demonstrated that DLA and SAB attenuate the LPS-induced enhancement of the CD11b and CD18 expression in neutrophils. It is thus proposed that the inhibitory effect of these two agents on the expression of adhesion molecules CD11b and CD18 in neutrophils is most likely one of the reactions that ultimately leads to blockage of the adhesion of leukocytes to venules. The effect of DLA and SAB on the expression of other adhesion molecules remains to be explored.

Several possibilities exist regarding the pathway by which DLA and SAB exert their inhibitory effect on the LPS-induced expression of CD11b and CD18 in neutrophils. First, activation of Toll-like receptor 4 signals in the process of CD11b/CD18 expression in neutrophils involves the activation of at least the transcription factors NF-κB and c-Jun/PU.1 (27). It was reported that water-soluble extract of SM and SAB attenuated the expression of endothelial cell ICAM-1 and vascular cell adhesion molecule 1 induced by TNF-α by inhibiting the activation of NF-κB (3, 4). But whether DLA and SAB have the effect on NF-κB and c-Jun/PU.1 of neutrophils needs further study. It is also recognized that preformed CD11b/CD18 in neutrophils is present inside secretary granules under resting conditions, and is translocated to the cell surface upon stimulation. Furthermore, it has been demonstrated that both O2 and H2O2 induce CD11b/CD18 up-regulation and CD11b-/CD18-mediated neutrophil adhesion (12, 13) through increased translocation of CD11b/CD18 from secretary vesicles to the plasma membrane (28). Finally, the integrins on the cell surface assume such a dynamic structure arrangement that in the low-affinity conformation, the headpiece of the molecule folds over its legs and face down toward the membrane and extends upward upon activation (29). Taking into account findings in the present study that DLA and SAB are able to inhibit both the expression of adhesion molecules CD11b and CD18 and the production of O2 and H2O2 in neutrophils, it is most likely that the ability of DLA and SAB to inhibit the expression of CD11b and CD18 is correlated with their antioxidant potential, although more work is needed to understand the exact detail in the mechanism by which DLA and SAB exert inhibitory action on the expression of CD11b and CD18 in neutrophils.

It is well recognized that mast cell degranulation induced by continuous LPS infusion leads to the release of cytokines such as histamine, IL-5, IL-6, IL-10, and TNF-α through the Toll-like receptor 4 (14, 30-32). These vasoactive substances attack vessels from the outside, promote the expression of VCAM and E selectin on endothelial cells (33), increase the rolling and adhesion of leukocytes, and enhance vascular albumin leakage (22). In the present study, the degranulation of mast cells induced by LPS infusion was found to be inhibited markedly by treatment with DLA and SAB as measured by the percent of degranulated mast cells, suggesting that DLA and SAB were also capable of protecting the microvasculature by blockage of vasoactive substances from outside. However, the mechanism by which DLA and SAB inhibit mast cell degranulation remains unknown.

In summary, our in vivo study provides definitive evidences that DLA and SAB are both able to improve mesenteric microcirculatory disturbance induced by LPS in rats, and this effect of improvement is correlated with the suppression of expression of adhesion molecules CD11b and CD18, and suppression of the production of free radicals in neutrophils, as is suggested by the results of our in vitro experiments.

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ACKNOWLEDGMENTS

The authors thank Dr. Michael Allen McNutt for this valuable discussion.

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REFERENCES

1. Horie Y, Han JY, Mori S, Konishi M, Kajihara M, Kaneko T, Yamagishi Y, Kato S, Ishii H, Hibi T: Herbal cardiotonic pills prevent gut ischemia/reperfusion-induced hepatic microvascular dysfunction in rats fed ethanol chronically. World J Gastroenterol 11:511-515, 2005.
2. Ren de C, Du GH, Zhang JT: Inhibitory effect of the water-soluble extract of Salvia miltiorrhiza on neutrophil-endothelial adhesion. Jpn J Pharmacol 90:276-280, 2002.
3. Chen YH, Lin SJ, Ku HH, Shiao MS, Lin FY, Chen JW, Chen YL: Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-alpha-treated human aortic endothelial cells. J Cell Biochem 82:512-521, 2001.
4. Ding M, Zhao GR, Yuan YJ, Guo ZX: Aqueous extract of Salvia miltiorrhiza regulates adhesion molecule expression of tumor necrosis factor alpha-induced endothelial cells by blocking activation of nuclear factor kappaB. J Cardiovasc Pharmacol 45:516-524, 2005.
5. Zhao BL, Jiang W, Zhao Y, Hou JW, Xin WJ: Scavenging effects of Salvia miltiorrhiza on free radicals and its protection for myocardial mitochondrial membranes from ischemia-reperfusion injury. Biochem Mol Biol Int 38:1171-1182, 1996.
6. Lin YH, Liu AH, Wu HL, Westenbroek C, Song QL, Yu HM, Ter Horst GJ, Li XJ: Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta (25-35)-induced reduction in BPRP in PC12 cells. Biochem Biophys Res Commun 348:593-599, 2006.
7. Zhang HS, Wang SQ: Salvianolic acid B from Salvia miltiorrhiza inhibits tumor necrosis factor-alpha (TNF-alpha)-induced MMP-2 upregulation in human aortic smooth muscle cells via suppression of NAD(P)H oxidase-derived reactive oxygen species. J Mol Cell Cardiol 41:138-148, 2006.
8. Pernerstorfer T, Stohlawetz P, Hollenstein U, Dzirlo L, Eichler HG, Kapiotis S, Jilma B, Speiser W: Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen. Arterioscler Thromb Vasc Biol 19:2517-2523, 1999.
9. Coimbra R, Melbostad H, Loomis W, Tobar M, Hoyt DB: Phosphodiesterase inhibition decreases nuclear factor-kappaB activation and shifts the cytokine response toward anti-inflammatory activity in acute endotoxemia. J Trauma 59:575-582, 2005.
10. Shimada H, Hasegawa N, Koh H, Tasaka S, Shimizu M, Yamada W, Nishimura T, Amakawa K, Kohno M, Sawafuji M, et al.et al: Effects of initial passage of endotoxin through the liver on the extent of acute lung injury in a rat model. Shock 26:311-315, 2006.
11. Hickson-Bick DL, Jones C, Buja LM: The response of neonatal rat ventricular myocytes to lipopolysaccharide-induced stress. Shock 25:546-552, 2006.
12. Serrano CV Jr., Mikhail EA, Wang P, Noble B, Kuppusamy P, Zweier JL: Superoxide and hydrogen peroxide induce CD18-mediated adhesion in the postischemic heart. Biochim Biophys Acta 1316:191-202, 1996.
13. Fraticelli A, Serrano CV Jr, Bochner BS, Capogrossi MC, Zweier JL: Hydrogen peroxide and superoxide modulate leukocyte adhesion molecule expression and leukocyte endothelial adhesion. Biochim Biophys Acta 1310:251-259, 1996.
14. Varadaradjalou S, Feger F, Thieblemont N, Hamouda NB, Pleau JM, Dy M, Arock M: Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur J Immunol 33:899-906, 2003.
15. Butcher EC: Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033-1036, 1991.
16. Lynam EB, Simon SI, Rochon YP, Sklar LA: Lipopolysaccharide enhances CD11b/CD18 function but inhibits neutrophil aggregation. Blood 83:3303-3311, 1994.
17. Davenpeck KL, Zagorski J, Schleimer RP, Bochner BS: Lipopolysaccharide-induced leukocyte rolling and adhesion in the rat mesenteric microcirculation: regulation by glucocorticoids and role of cytokines. J Immunol 161:6861-6870, 1998.
18. Egan BM, Chen G, Kelly CJ, Bouchier-Hayes DJ: Taurine attenuates LPS-induced rolling and adhesion in rat microcirculation. J Surg Res 95:85-91, 2001.
19. Han JY, Miura S, Akiba Y, Higuchi H, Kato S, Suzuki H, Yokoyama H, Ishii H: Chronic ethanol consumption exacerbates microcirculatory damage in rat mesentery after reperfusion. Am J Physiol Gastrointest Liver Physiol 280:G939-G948, 2001.
20. Kurose I, Suematsu M, Miura S, Fukumura D, Sekizuka E, Nagata H, Oshio C, Tsuchiya M: Oxyradical generation from leukocytes during endotoxin-induced microcirculatory disturbance in rat mesentery-attenuating effect of cetraxate. Toxicol Appl Pharmacol 120:37-44, 1993.
21. Dunne JL, Ballantyne CM, Beaudet AL, Ley K: Control of leukocyte rolling velocity in TNF-alpha-induced inflammation by LFA-1 and Mac-1. Blood 99:336-341, 2002.
22. Kurose I, Argenbright LW, Wolf R, Lianxi L, Granger DN: Ischemia/reperfusion-induced microvascular dysfunction: role of oxidants and lipid mediators. Am J Physiol 272:H2976-H2982, 1997.
23. Sun K, Wang CS, Guo J, Liu YY, Wang F, Liu LY, He JG, Fan JY, Han JY: Effect of Panax notoginseng saponins on lipopolysaccharide-induced adhesion of leukocytes in rat mesenteric venules. Clin Hemorheol Microcirc 34:103-108, 2006.
24. Ting A, Morris PJ: A technique for lymphocyte preparation from stored heparinized blood. Vox Sang 20:561-563, 1971.
25. Shen YC, Sung YJ, Chen CF: Magnolol inhibits Mac-1 (CD11b/CD18)-dependent neutrophil adhesion: relationship with its antioxidant effect. Eur J Pharmacol 343:79-86, 1998.
26. Darley-Usmar V, Halliwell B: Blood radicals: reactive nitrogen species, reactive oxygen species, transition metal ions, and the vascular system. Pharm Res 13:649-662, 1996.
27. Zhou X, Gao XP, Fan J, Liu Q, Anwar KN, Frey RS, Malik AB: LPS activation of Toll-like receptor 4 signals CD11b/CD18 expression in neutrophils. Am J Physiol Lung Cell Mol Physiol 288:L655-L662, 2005.
28. Simms HH, D'Amico R: Subcellular location of neutrophil opsonic receptors is altered by exogenous reactive oxygen species. Cell Immunol 166:71-82, 1995.
29. Xie C, Shimaoka M, Xiao T, Schwab P, Klickstein LB, Springer TA: The integrin alpha-subunit leg extends at a Ca2+-dependent epitope in the thigh/genu interface upon activation. Proc Natl Acad Sci U S A 101:15422-15427, 2004.
30. McCurdy JD, Lin TJ, Marshall JS: Toll-like receptor 4-mediated activation of murine mast cells. J Leukoc Biol 70:977-984, 2001.
31. Vannier E, Miller LC, Dinarello CA: Histamine suppresses gene expression and synthesis of tumor necrosis factor alpha via histamine H2 receptors. J Exp Med 174:281-284, 1991.
32. Ikeda T, Funaba M: Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan. Immunol Lett 88:21-26, 2003.
33. Van Haaster CM, Derhaag JG, Engels W, Lemmens PJ, Gijsen AP, Hornstra G, Van der Vusse GJ, Duijvestijn AM: Mast cell-mediated induction of ICAM-1, VCAM-1 and E-selectin in endothelial cells in vitro: constitutive release of inducing mediators but no effect of degranulation. Pflugers Arch 435:137-144, 1997.
Keywords:

Salvia miltiorrhiza; antioxidants; microcirculation; leukocyte adhesion; mast cell degranulation

©2008The Shock Society