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Effect of Ligustrazine on liver injury after burn trauma
Burns 32 (2006) 328–334 www.elsevier.com/locate/burns
Effect of Ligustrazine on liver injury after burn trauma Hong Zheng a,1, Xu-Lin Chen b,1, Zhi-Xun Han b, Si-Ying Wang a, Zhi-Wu Chen c,* a
Department of Pathophysiology, Anhui Medical University, Hefei, Anhui 230032, People’s Republic of China b Department of Burns, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, People’s Republic of China c Department of Pharmacology, Anhui Medical University, Hefei, Anhui 230032, People’s Republic of China
Abstract This study was designed to investigate the effect of Ligustrazine on burn-induced liver injury as well as the activation of nuclear factor kB (NF-kB) in severely burned rats. Sprague–Dawley rats were divided into three groups: (1) sham group, rats who underwent sham burn; (2) control group, rats given third-degree burns over 30% total body surface area (TBSA) and lactated Ringer solution for resuscitation; (3) Ligustrazine group, rats given burn and lactated Ringer’s solution with Ligustrazine inside for resuscitation. Liver injury was assessed at 24 h post-burn by serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as liver wet/dry weight ratio. Liver myeloperoxidase (MPO) activity was also analyzed. Hepatic NF-kB activity was examined by electrophoretic mobility shift assay (EMSA). Burn results in hepatic dysfunction and increased hepatic NF-kB activity, elevated liver wet/dry ratio and hepatic MPO activity. Ligustrazine inhibited these changes and alleviated burn-mediated hepatic dysfunction. The data indicated that Ligustrazine has a protective effect on burn-induced liver injury and possible mechanism may be attributed to its inhibitory action on the activation of NF-kB following burn trauma. # 2005 Elsevier Ltd and ISBI. All rights reserved. Keywords: Burns; Liver injury; NF-kappB; Ligustrazine
Severe burn causes damage to multiple organs distant from the original burn wound, leading to multiorgan failure, a serious clinical problem. Because of its important role in metabolism, homeostasis, and host defense mechanisms, the liver has been investigated extensively, and is thought to be a major organ responsible for initiating MODS [1,2]. Recent studies suggest that a severe burn can change liver morphology in rats and decreases concentrations of protein and DNA [3]. Earlier studies indicated that a cutaneous wound can signal changes in gene expression by the liver suggesting that this organ plays a pivotal role in immune function, inflammatory processes, and the acute phase response [4–7] to burn. Endotoxin and other bacterial by* Corresponding author. Tel.: +86 551 3413638. E-mail address: [email protected] (Z.-W. Chen). 1 They contributed equally to this work and are therefore both first authors. 0305-4179/$30.00 # 2005 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2005.10.007
products are potent activators of the macrophage and neutrophil. This leads to the release of massive amounts of oxidants, arachidonic acid metabolites, proteases, etc. which cause further local and systemic inflammation-induced tissue damage [8]. The synthesis of constitutive hepatic proteins, acute phase proteins, cytokines, and other mediators by the livers makes it a determining factor in burn survival. Although the pathophysiological basis of such tissue injury remain unclear, based on current research findings, oxidative stress is believed to be the major causative agent to damage the organs distant from the original burn wound. The role of free oxygen radicals, neutrophils and endothelial cells in ischemic insult or organ failure has well been documented. A previous study showed that oxidative stress in the liver of rats was associated with the original burn wound [9]. It has also become clear that oxidative stress initiates a chemokine and mediator cascade, and these
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mediators, in turn, act on additional target cells to produce an array of pro-inflammatory cytokines leading to cardiovascular shock, MODS, or even death. The NF-kB system is another major signaling pathway responsible for oxidative stress and pro-inflammatory cytokine release after burn trauma [10]. A previous study showed that nuclear factor kB (NF-kB) was crucial in liver regeneration [11]. NF-kB is a ubiquitous, rapidly acting transcription factor involved in immune and inflammatory reactions [11–13]. It exerts its immune and inflammatory response by regulating expression of cytokines, chemokines, cell adhesion molecules, and growth factors [11–13]. Though a role for NF-kB in the regulation of cell proliferation and apoptosis in human hepatocellular has also emerged [14,15], the function of NF-kB during liver injury after burn is not entirely well defined. If this transcription factor plays a pivotal role in hepatocellular secretion of inflammatory cytokines and burn-related hepatic dysfunction, then strategies that inhibit this transcription factor would be expected to improve postburn hepatic performance. Ligustrazine (tetramethy pyrazine), a component contained in Chuanxiong (Ligusticum chuanxiong Hort, one of the ABCRBS herbs), is widely applied in the treatment of vascular diseases in China [16]. It blocks calcium channels, reduces the bioactivity of platelets and platelet aggregation, and inhibits free radicals [17]. Ligustrazine has been demonstrated to have protective effects against lung injury after burn [18] and kidney ischemia-reperfusion injury in rats and prevent acute myocardial infarction and cerebrovascular accidents in humans [19,20]. However, there are few reports regarding the effect of Ligustrazine on liver injury after severe burn. In the present report, we examined the effect of Ligustrazine on burn-induced liver injury as well as the activation of NF-kB in severely burned rats so as to define its role in neutrophil-mediated injury to the liver after thermal trauma.
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1.2. Burn procedure Two days before receiving burns, external jugular catheters were placed in all rats under general anesthesia and analgesia (pentobarbital 30 mg/kg i.p.). The catheters were tunneled subcutaneously to the posterior neck and secured with a spring wire. The day before experiment the animals were anesthetized with pentobarbital again; then, the dorsal surfaces were shaved and rats were secured in a constructed template device. The surface area of the skin exposed through the template device was immersed in 98 8C water for 12 s on the dorsal surface. All were quickly dried after each exposure to avoid additional injury. With the use of this technique, full-thickness dermal burns comprising 30% TBSA were obtained [21]. 1.3. Experimental design Animals were randomly divided into three groups as follows: (1) sham group (n = 24); (2) burns group (n = 24); (3) burns plus Ligustrazine group (n = 24). The burned rats were resuscitated with lactated Ringer’s solution by a continuous-infusion pump (Zhejiang Medical Apparatus and Instruments Factory, Zhejiang, China) at a rat of 2 ml/ kg/h. Ligustrazine (The Seventh Pharmaceutical Factory, WuXi, China) was administered intravenously at a dose of 10 mg/kg in animals of burn plus Ligustrazine group immediately after burn, while rats in burn group was given identical saline solution in same manner. The sham burn rats were subjected to an identical preparation except that they were immersed in room temperature water and were not given any fluid resuscitation. In a pilot experiment, main parameters determined in the current study in normal animals were found to be highly constant, without marked change over time. Thus, time course control was not included in this study. Under anesthesia, blood samples were obtained. Then tissue specimens were taken from livers. All of these tests were performed at 24 h after thermal injury.
1. Materials and methods
1.4. Transaminase analysis
1.1. Animals
Evaluation of hepatic injury was performed by determinations of aspartate transaminase (AST) and alanine transaminase (ALT) in blood plasma by using a commercial kit from Boehringer Manheim (Munich, Germany), according to the supplier’s specifications and the results were expressed in international units per liter.
This study was approved by the Animal Welfare and Ethics Committee of Anhui Medical University and followed their guidelines for animal experimentation. Healthy adult male Sprague–Dawley rats (weight range 225–250 g), purchased from the Laboratory Animal Center, Anhui Medical University, were used for the study. The animals were housed in separate cages in a temperaturecontrolled room with alternating 12-h light:12-h dark cycles, and were allowed 1 week to acclimate to their surroundings. Rats were fed a standard animal diet; food and tap water would be available at will throughout the experimental protocol.
1.5. Water content in liver tissue The water content of the livers was determined by calculating the wet/dry weight ratio of liver tissues at 24 h after thermal injury. Post-experimental liver tissue samples were weighed and then desiccated at 85 8C for 48 h, and the wet/dry weight ratio was calculated. In this determination,
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liver edema was represented by an increase in the wet-to-dry weight ratios.
determined with the use of a protein assay (Bio-Rad; Hercules, CA). Nuclear and cytosolic extracts were stored at 80 8C.
1.6. Myeloperoxidase (MPO) activity As a biochemical quantitative marker of pulmonary and hepatic neutrophil infiltration, MPO activity was measured at 24 h after burn tramua. The method described by Mullane et al. [22] was used to measure MPO activity in the livers. Liver tissue was removed and immediately frozen and stored at 80 8C until the MPO assay could be performed. Tissue specimens were homogenized in a 50-mM potassium phosphate buffer with 0.5% hexadecyl-trimethyl-ammonium bromide (HTAB, pH 6.0; Sigma), and centrifuged at 40,000 g for 15 min at 4 8C. The supernatant was assayed for MPO activity spectrophotometrically. Then 20 ml of supernatant were combined with 12 ml of 25 mM H2O2, 30 ml of 40 mM O-dianisidine hydrochloride, and 1.938 ml of 50 mM phosphate buffer (pH 6.0). Color development was stopped by the addition of 0.1 ml of 1% NaN3 at room temperature after 5 and 20 min, respectively. The optical density was measured at 460 nm with a spectrophotometer (U-2001, Hitachi, Tokyo, Japan). Color development from 5 to 20 min was linear. One unit of MPO activity is defined as the activity degrading 1 mmol of H2O2/min at 25 8C. The results are expressed as units per gram of tissue (U/g) [23].
1.7.2. Electrophoretic mobility shift assays The EMSA method was used to characterize the binding activities of NF-kB in nuclear extracts. Double-stranded oligonucleotides were end-labeled with T4 polynucleotide kinase in the presence of [g-32P] ATP (3000 Ci/mmol). Five to 20 mg of nuclear protein was incubated in binding buffer containing g-32P-labeled double-stranded probe for NF-kB (Promega) and polydeoxyinosinic-deoxycytidylic acid (DidC) for 30 min at 4 8C, loaded onto a 6% polyacrylamide gel and electrophoresed for 1 h at 20 8C. Finally, the gels were dried and exposed to X-ray film. 1.8. Statistical analysis Data were expressed as mean S.E.M. Statistical evaluation of the continuous data was performed by oneway analysis of variance, followed by Dunnett’s t-test for between-group comparisons. The level of significance was considered to be P < 0.05.
2. Results
1.7. NF-kappaB activation
2.1. Liver function tests
1.7.1. Nuclear protein extraction A modified procedure based on the method of Grabellus et al. [24] was used. All steps were performed on ice. Livers were thawed in the presence of buffer A (10 mM HEPES, pH 7.9; 10 mM KCl, 1.5 mM MgCl2, 0.1% Nonidet P-40, and protease inhibitors plus 0.5 mM DTT); the proteases included a protease inhibitor cocktail containing the following: 20 mM 4-(2-aminoethyl)-benzenesulfaonylflouride, 14 mm trans-epoxysuccinyl-L-leucylamido-(4-guandidino) butane, 1300 mM bestatin, 10 mM leupeptin, 3 mM aprotinin, and 10mM sodium EDTA. The livers were allowed to thaw on ice for 20 min and were then suspended in 500 ml of buffer A per liver. The tissue was then homogenized with the use of a dounce homogenizer by hand. The cells were then allowed to incubate for 20 min on ice and then centrifuged at 1200 g for 5 min at 4 8C. The supernatants (cytosolic fraction) were removed and frozen at 80 8C. The remaining pellet was then resuspended in 200 ml of ice-cold buffer C (20 mM HEPES, pH 7.9; 420 mM NaCl, 1.8 mM MgCl2, 0.2 mM EDTA, pH 8; 25% glycerol) and protease inhibitor as described above plus 0.5 mM DTT. The pellets were completely resuspended by being pipetted and were incubated for 20 min on ice with gentle resuspension of the pellets every 2 min. After incubation, the pellets were centrifuged at full speed for 5 min at 4 8C. The supernatant was collected (nuclear fraction) and frozen at 80 8C. Protein concentrations were
Hepatocyte injury was evaluated by determining the plasma concentration of AST and ALT. At 24 h following a 30% TBSA full-thickness burn, plasma hepatic transaminases (AST and ALT) increased three- to four-fold in rats compared with sham animals (P < 0.05 versus sham). In vivo administration of Ligustrazine decreased plasma AST and ALT in burn rats significantly (P < 0.05 versus vehicle) (Fig. 1).
Fig. 1. Serum AST, ALT activities in rats. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control.
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Fig. 2. Liver edema was quantitated by liver wet-to-dry weight ratios at 24 h post-burn. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control. N = 8 animals per group.
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Fig. 3. Liver sequestration of neutrophils after thermal wounds as assessed by liver myeloperoxidase activity in sham, control, Ligustrazine groups. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control. N = 8 animals per group.
2.2. Liver wet/dry weight ratio Liver wet/dry weight ratio was significantly higher at 24 h after burn (2.9 0.12 versus 3.9 0.23; P < 0.01) in the control group compared with the sham group. In the Ligustrazine group the ratio was 3.3 0.16, which was significantly (P < 0.01) lower compared with the control group (Fig. 2). 2.3. MPO activity The activity of MPO, an enzyme in azurophilic granules of neutrophils, was used as a well-established marker to quantitate tissue neutrophil sequestration. Extracts of the liver samples were examined for content of MPO at 24 h after thermal injury of skin. As depicted in Fig. 4, the mean MPO level of the sham group was 10.8 1.25 U/g compared to 28.18 2.30 U/g (P < 0.01) in livers obtained from burned rats. In animals treated with Ligustrazine group, the MPO activity significantly decreased to levels of 15.42 1.56 U/g (P < 0.01 versus control; Fig. 3). 2.4. Hepatic NF-kB activation Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NF-kB were assessed by EMSA in liver of rats after burn trauma. At 24 h after a 30%TBSA full-thickness burn, the NF-kB DNA binding activity incresed significantly in liver harvested from burn rats than that from shams and this activity was inhibited by treatment with Ligustrazine (Fig. 4).
3. Discussion Severe burn, one of the most common problems faced in the emergency room, may cause damage to multiple organs
Fig. 4. Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NF-kB was assessed by EMSA in liver of rats after burn trauma. (A) Representative autoradiograms demonstrated that the burn-mediated increase in NF-kB activation was inhibited by treatment with Ligustrazine. (B) Densitometric analysis of EMSA confirmed that Ligustrazine can prevent the activation of NF-kB in liver harvested from burn rats. The values were expressed as fold change from the sham rats in each comparison. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control. N = 8 animals per group.
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distant from the original burn wound and may lead to multiorgan failure. The local and systemic inflammatory response to severe cutaneous thermal trauma is extremely complex, resulting in both local burn tissue damage and subsequent edema as well as marked systemic effects, which demand urgent medical interventions to ensure the survival of the patient. Despite many advanced medical treatments, multiorgan failure management is still a great problem. The data from this study showed that burn traumainduced hepatocellular injury as evidenced by increased plasma AST and ALT levels, increases liver wet/dry ratio, and upregulated NF-kB activity. The in vivo administration of Ligustrazine attenuated post-burn hepatocelluar injury, decreased burn-mediated MPO and NF-kB activity and alleviate histologic edema. It is well-known that circulating endotoxins and other bacterial by-products produced as a result of burn wound colonization are potent activators of the macrophages and neutrophils [25,26]. The activation of these inflammatory cells contributes to distant tissue damage through the release of reactive oxygen metabolites and various enzymes, such as proteases and myeloperoxidase. In the present study, we observed plasma ALT and AST activities-the most commonly used indicators of hepatocellular injury were markedly high in the burn group compared to controls and a significant increase in myeloperoxidase activity in liver samples of animals parallel with elevated liver wet/dry weight ratio, indicating that burn results in hepatic injury. This finding that plasma AST and ALT levels go up dramatically in rats after burn is consistent with previous reports [27,28], however, similar changes appeared slowly or indistinctively in human studies. Chiarelli et al. [29] had studied 43 burned patients, both during and after recovery, and found a 67.44% increase in transaminases in patients. The exact mechanism remains unclear and it seems more likely that human livers have stronger compensatory ability than animals. But in severely burned patients we observed the immediate increase of the enzymes. It has been shown that post-burn intravascular hemolysis may be largely prevented by neutrophil depletion of experimental animals, suggesting that neutrophils play an important role in the development of remote organ injury [30]. Our results showed that hepatic MPO activity (as an index of tissue polymorphonuclear neutrophil uptake) was markedly elevated after burn injury and Ligustrazine led to the downregulation of MPO concentration. MPO is one of the special oxidase in PMN, and MPO activity in tissue reflects the PMN chemotaxis and infiltration. PMN infiltration and activation could produce large amount of oxidants and cause tissue oxidative injury, which contributes to the organ functional damage in MODS. It is well known that accumulated PMNs in the grafted liver sinusoids might be important effector cells in the pathogenosis of liver injury after burn. The reactive oxygen and proteases released by activated PMNs might promote progressive hepatocellular damage.
Although it is well accepted that activated neutrophils play a key role in the development of microvascular injury at both the burn site and remote organs, it remains a matter of speculation as to which mediators might be responsible for activation of blood neutrophils. NF-kB system is another major signaling pathway responsible for cytokine expression. NF-kB plays a key role in the transcriptional regulation of various chemokines, including IL-8 [31]. The IL-8 promoter region contains binding sites for a number of important transcriptional factors including NF-kB [32]. In response to pro-inflammatory stimuli, IL-8 production is dependent on mitogen-activated protein kinases (MAPKS) and NF-kB pathways [33]. In patients with major trauma or burns, cytokines mediate neutrophil dysfunction contributes to ARDS, to immunological alterations involved in the increased susceptibility to infection, and to the systemic inflammatory responses that lead to shock and multiple organ dysfunction [34–36]. In our previous study [18], we found that the serum IL-8 levels of rats were markedly elevated after a 30% TBSA full-thickness burn. Furthermore, we also demonstrated that IL-8 is associated with the activation of neutrophils. Wang et al. [37] have reported that nuclear translocation of NF-kB was upregulated post-burn, in the current study we confirmed that burn trauma upregulated NF-kB activity in the liver of rats, From these findings we speculated that NF-kB system mediates the production of IL-8 and contributes to burn-induced liver injury. Chinese herbal medicine contributes a great deal to the health of the Chinese nation. Ligustrazine, a compound purified from Ligustium Wollichii Francha, is a widely used active ingredient in Chinese herbal medicine for the treatment of cardiovascular diseases due to its vasodilatory actions and antiplatelet activity [20,38,39]. It has been shown in animal models and clinical investigations that Ligustrazine is effective on ischemic diseases, such as heart, brain, and lung. However, the roles and mechanisms of Ligustrazine in treatment of digestive diseases have not been extensively studied. Ligustrazine has been proposed to act as an inhibitor of phosphodiesterase (PDE) and thereby it increases intracellular cAMP [39]. Hemorheological studies show that parameters such as hematocrit, fibrinogen level, and plasma viscosity, have a tendency to increase after large burns. Hemorheological parameters are modified by ligustrazini hydrochloride [16,40,41]. Ligustrazine could affect hemorheological events, such as blood flow, erythrocyte deformation, leukocyte adhesion, platelet aggregation, and thrombolysis [20]. These results suggest that Ligustrazine may play a role in tissue repair and blood hemeostasis. Furthermore, based on the current findings, the protective effect of Ligustrazine, in part, may be attributed to its inhibitory action on tissue neutrophil infiltration. We reported first that Ligustrazine attenuates acute lung injury after burn trauma, and inhibits serum IL-8 concentrations [18].
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In the present study, we examined the effect of Ligustrazine on the liver injury of severely burned rats. Our results showed that Ligustrazine decreased serum levels of aspartate aminotransferase and alanine aminotransferase, attenuated the wet/dry ratio and MPO activity, and downregulated the activity of NF-kB, suggesting that the protective effects of Ligustrazine against burn-induced liver injury may be attributable to influencing NF-kB activation and further preventing post-burn pro-inflammatory cytokines release and accumulation of neutrophils. In conclusion, the early use of Ligustrazine seems to be effective in ameliorating liver injury after major thermal injury. We conclude that Ligustrazine may act through inhibiting NF-kB activity. Although the final molecular mechanism remains to be further studied, this will likely provide further evidence for Ligustrazine as a therapeutic strategy in burn-induced liver injury.
References [1] Wang P, Chaudry IH. Mechanism of hepatocellular dysfunction during hyperdynamic sepsis. Am J Physiol 1996;270:R927–38. [2] Matuschak GM. Lung-liver interactions in sepsis and multiple organ failure syndrome. Clin Chest Med 1996;17:83–98. [3] Baron P, Traber LD, Traber DL, Nguyen T, Hollyoak M, Heggers JP, et al. Gut failure and translocation following burn and sepsis. J Surg Res 1994;57:197–204. [4] Gilpin DA, Hsieh CC, Kuninger DT, Herndon DN, Papaconstantinou J. Effect of thermal injury on the expression of transcription factors that regulate acute phase response genes: the response of C/EBP alpha, C/EBP beta, and C/EBP delta to thermal injury. Surgery 1996;119: 674–83. [5] Blom WM, De Bont HJ, Meijerman I, Kuppen PJ, Mulder GJ, Nagelkerke JF. Interleukin-2-activated natural killer cells can induce both apoptosis and necrosis in rat hepatocytes. Hepatology 1999;29: 785–92. [6] Fey GH, Gauldie J. The acute phase response of the liver in inflammation. Prog Liver Dis 1990;9:89–116. [7] Jeschke MG, Herndon DN, Wolf SE, DebRoy MA, Rai J, Lichtenbelt BJ, et al. Recombinant human growth hormone alters acute phase reactant proteins, cytokine expression, and liver morphology in burned rats. J Surg Res 1999;83:122–9. [8] Youn YK, LaLonde C, Demling R. The role of mediators in the response to thermal injury. World J Surg 1992;16:30–6. [9] Ding HQ, Zhou BJ, Liu L, Cheng S. Oxidative stress and metallothionein expression in the liver of rats with severe thermal injury. Burns 2002;28:215–21. [10] Wang M, Tai G, Zhang Y, Xiao G. The modulating effects of transfected IkappaBalpha gene on the NF-kappaB activity of hepatic tissue in scalded rats. Zhonghua Shao Shang Za Zhi 2002;18: 42–4. [11] Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, et al. NF-kappaB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 1998;101:802–11. [12] Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 1994;10:405–55. [13] Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I (kappa) B kinase-beta. Nature 1998;396:77–80. [14] Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE. Inhibition of NFkappa B activity induces apoptosis in murine hepatocytes. Am J Pathol 1997;151:891–6.
333
[15] Wang CY, Mayo MW, Baldwin Jr AS.. TNF- and cancer therapyinduced apoptosis: potentiation by inhibition of NF-kappaB. Science 1996;274:784–7. [16] Liao F. Treatment of blood stasis syndrome and hemorheology. Clin Hemorheol 1994;14:463–71. [17] Zou LY, Hao XM, Zhang GQ, Zhang M, Guo JH, Liu TF. Effect of tetramethyl pyrazine on L-type calcium channel in rat ventricular myocytes. Can J Physiol Pharmacol 2001;79:621–6. [18] Zheng H, Chen XL, Han ZX, Zhang Z, Wang SY, Xu QL. Ligustrazine attenuates acute lung injury after burn trauma. Burns 2005;39:635–40. [19] Sun L, Li Y, Shi J, Wang X, Wang X. Protective effects of ligustrazine on ischemia-reperfusion injury in rat kidneys. Microsurgery 2002;22: 343–6. [20] Liao F. Herbs of activating blood circulation to remove blood stasis. Clin Hemorheol Microcirc 2000;23:127–31. [21] Chen XL, Xia ZF, Ben DF, Wang GQ, Wei D. Role of p38 mitogenactivated protein kinase in lung injury after burn trauma. Shock 2003;19:475–9. [22] Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Methods 1985;14:157–67. [23] Cutrin JC, Boveris A, Zingaro B, Corvetti G, Poli G. In situ determination by surface chemiluminescence of temporal relationships between evolving warm ischemia-reperfusion injury in rat liver and phagocyte activation and recruitment. Hepatology 2000;31:622–32. [24] Grabellus F, Levkau B, Sokoll A, Welp H, Schmid C, Deng MC, et al. Reversible activation of nuclear factor-kappaB in human end-stage heart failure after left ventricular mechanical support. Cardiovasc Res 2002;53:124–30. [25] Jones 2nd WG, Minei JP, Barber AE, Fahey 3rd TJ, Shires 3rd GT, Shires GT. Splanchnic vasoconstriction and bacterial translocation after thermal injury. Am J Physiol 1991;261:H1190–6. [26] Morris SE, Navaratnam N, Herndon DN. A comparison of effect of thermal injury and smoke inhalation on bacterial translocation. J Trauma 1990;30:639–43. [27] Jeschke MG, Jeschke MG, Aili Low JF, Spies M, Vita R, Hawkins HK, et al. Barrow cell proliferation, apoptosis, NF-kB expression, enzyme, protein, and weight changes in livers of burned rats. Am J Physiol Gastrointest Liver Physiol 2001;280:G1314–20. [28] Chen XL, Xia ZF, Yu YX, Wei D, Wang CR, Ben DF. P38 mitogenactivated protein kinase inhibition attenuates burn-induced liver injury in rats. Burns 2005;31:320–30. [29] Chiarelli A, Casadei A, Pornaro E, Siliprandi L, Mazzoleni F. Alanine and aspartate aminotransferase serum levels in burned patients: a longterm study. J Trauma 1987;27(7):790–4. [30] Hansbrough JF, Wikstrom T, Braide M, Tenenhaus M, Rennekampff OH, Kiessig V, et al. Neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury. J Surg Res 1996;61: 17–22. [31] Holtmann HR, Winzen P, Holland S, Eichemeier E, Hoffmann D, Wallach N. Induction of interleukin-8 synthesis integrates effects of transcription and Mrna degradation from at least three different cytokine-or stress-activated signal transduction pathways. Mol Cell Biol 1999;19:6742–53. [32] Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, et al. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 1993;90: 10193–7. [33] Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol 2000;18: 621–63. [34] Beal AL, Cerra FB. Multiple organ failure syndrome in the 1990s. Systemic inflammatory response and organ dysfunction. JAMA 1994; 271:226–33. [35] Bellomo R. The cytokine network in the critically ill. Anaesth Intensive Care 1992;20:288–302.
334
H. Zheng et al. / Burns 32 (2006) 328–334
[36] Drost AC, Burleson DG, Cioffi Jr WG, Jordan BS, Mason AD, Pruitt Jr BA. Plasma cytokines following thermal injury and their relationship with patient mortality, burn size, and time postburn. J Trauma 1993;35:335–9. [37] Wang GQ, Xia ZF, Yu BJ, Xiao SC, Chen YL. Cardiac apoptosis in burned rats with delayed fluid resuscitation. Burns 2001;27:250–3. [38] Liu SY, Sylvester DM. Antiplatelet activity of tetranmethylpyrazine. Thromb Res 1994;75:51–62.
[39] Kwan CY. Plant-derived drugs acting on celluar Ca2+ mobilization in vascular smooth muscle: tetramethylpyrazine and tetrandrine. Stem Cells 1994;12:64–7. [40] Luo G. Effects of ligustrazine and anisodamine on whole blood viscosity and erythrocyte deformability in rabbits. Zhonghua Yi Xue Za Zhi 1987;67:607–9. [41] Sutara SP. Effects of three Chinese medicinal herbs on erythrocyte deformability. Clin Hemorheol 1987;7:773–9.
Effect of Ligustrazine on liver injury after burn trauma Hong Zheng a,1, Xu-Lin Chen b,1, Zhi-Xun Han b, Si-Ying Wang a, Zhi-Wu Chen c,* a
Department of Pathophysiology, Anhui Medical University, Hefei, Anhui 230032, People’s Republic of China b Department of Burns, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, People’s Republic of China c Department of Pharmacology, Anhui Medical University, Hefei, Anhui 230032, People’s Republic of China
Abstract This study was designed to investigate the effect of Ligustrazine on burn-induced liver injury as well as the activation of nuclear factor kB (NF-kB) in severely burned rats. Sprague–Dawley rats were divided into three groups: (1) sham group, rats who underwent sham burn; (2) control group, rats given third-degree burns over 30% total body surface area (TBSA) and lactated Ringer solution for resuscitation; (3) Ligustrazine group, rats given burn and lactated Ringer’s solution with Ligustrazine inside for resuscitation. Liver injury was assessed at 24 h post-burn by serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as liver wet/dry weight ratio. Liver myeloperoxidase (MPO) activity was also analyzed. Hepatic NF-kB activity was examined by electrophoretic mobility shift assay (EMSA). Burn results in hepatic dysfunction and increased hepatic NF-kB activity, elevated liver wet/dry ratio and hepatic MPO activity. Ligustrazine inhibited these changes and alleviated burn-mediated hepatic dysfunction. The data indicated that Ligustrazine has a protective effect on burn-induced liver injury and possible mechanism may be attributed to its inhibitory action on the activation of NF-kB following burn trauma. # 2005 Elsevier Ltd and ISBI. All rights reserved. Keywords: Burns; Liver injury; NF-kappB; Ligustrazine
Severe burn causes damage to multiple organs distant from the original burn wound, leading to multiorgan failure, a serious clinical problem. Because of its important role in metabolism, homeostasis, and host defense mechanisms, the liver has been investigated extensively, and is thought to be a major organ responsible for initiating MODS [1,2]. Recent studies suggest that a severe burn can change liver morphology in rats and decreases concentrations of protein and DNA [3]. Earlier studies indicated that a cutaneous wound can signal changes in gene expression by the liver suggesting that this organ plays a pivotal role in immune function, inflammatory processes, and the acute phase response [4–7] to burn. Endotoxin and other bacterial by* Corresponding author. Tel.: +86 551 3413638. E-mail address: [email protected] (Z.-W. Chen). 1 They contributed equally to this work and are therefore both first authors. 0305-4179/$30.00 # 2005 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2005.10.007
products are potent activators of the macrophage and neutrophil. This leads to the release of massive amounts of oxidants, arachidonic acid metabolites, proteases, etc. which cause further local and systemic inflammation-induced tissue damage [8]. The synthesis of constitutive hepatic proteins, acute phase proteins, cytokines, and other mediators by the livers makes it a determining factor in burn survival. Although the pathophysiological basis of such tissue injury remain unclear, based on current research findings, oxidative stress is believed to be the major causative agent to damage the organs distant from the original burn wound. The role of free oxygen radicals, neutrophils and endothelial cells in ischemic insult or organ failure has well been documented. A previous study showed that oxidative stress in the liver of rats was associated with the original burn wound [9]. It has also become clear that oxidative stress initiates a chemokine and mediator cascade, and these
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mediators, in turn, act on additional target cells to produce an array of pro-inflammatory cytokines leading to cardiovascular shock, MODS, or even death. The NF-kB system is another major signaling pathway responsible for oxidative stress and pro-inflammatory cytokine release after burn trauma [10]. A previous study showed that nuclear factor kB (NF-kB) was crucial in liver regeneration [11]. NF-kB is a ubiquitous, rapidly acting transcription factor involved in immune and inflammatory reactions [11–13]. It exerts its immune and inflammatory response by regulating expression of cytokines, chemokines, cell adhesion molecules, and growth factors [11–13]. Though a role for NF-kB in the regulation of cell proliferation and apoptosis in human hepatocellular has also emerged [14,15], the function of NF-kB during liver injury after burn is not entirely well defined. If this transcription factor plays a pivotal role in hepatocellular secretion of inflammatory cytokines and burn-related hepatic dysfunction, then strategies that inhibit this transcription factor would be expected to improve postburn hepatic performance. Ligustrazine (tetramethy pyrazine), a component contained in Chuanxiong (Ligusticum chuanxiong Hort, one of the ABCRBS herbs), is widely applied in the treatment of vascular diseases in China [16]. It blocks calcium channels, reduces the bioactivity of platelets and platelet aggregation, and inhibits free radicals [17]. Ligustrazine has been demonstrated to have protective effects against lung injury after burn [18] and kidney ischemia-reperfusion injury in rats and prevent acute myocardial infarction and cerebrovascular accidents in humans [19,20]. However, there are few reports regarding the effect of Ligustrazine on liver injury after severe burn. In the present report, we examined the effect of Ligustrazine on burn-induced liver injury as well as the activation of NF-kB in severely burned rats so as to define its role in neutrophil-mediated injury to the liver after thermal trauma.
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1.2. Burn procedure Two days before receiving burns, external jugular catheters were placed in all rats under general anesthesia and analgesia (pentobarbital 30 mg/kg i.p.). The catheters were tunneled subcutaneously to the posterior neck and secured with a spring wire. The day before experiment the animals were anesthetized with pentobarbital again; then, the dorsal surfaces were shaved and rats were secured in a constructed template device. The surface area of the skin exposed through the template device was immersed in 98 8C water for 12 s on the dorsal surface. All were quickly dried after each exposure to avoid additional injury. With the use of this technique, full-thickness dermal burns comprising 30% TBSA were obtained [21]. 1.3. Experimental design Animals were randomly divided into three groups as follows: (1) sham group (n = 24); (2) burns group (n = 24); (3) burns plus Ligustrazine group (n = 24). The burned rats were resuscitated with lactated Ringer’s solution by a continuous-infusion pump (Zhejiang Medical Apparatus and Instruments Factory, Zhejiang, China) at a rat of 2 ml/ kg/h. Ligustrazine (The Seventh Pharmaceutical Factory, WuXi, China) was administered intravenously at a dose of 10 mg/kg in animals of burn plus Ligustrazine group immediately after burn, while rats in burn group was given identical saline solution in same manner. The sham burn rats were subjected to an identical preparation except that they were immersed in room temperature water and were not given any fluid resuscitation. In a pilot experiment, main parameters determined in the current study in normal animals were found to be highly constant, without marked change over time. Thus, time course control was not included in this study. Under anesthesia, blood samples were obtained. Then tissue specimens were taken from livers. All of these tests were performed at 24 h after thermal injury.
1. Materials and methods
1.4. Transaminase analysis
1.1. Animals
Evaluation of hepatic injury was performed by determinations of aspartate transaminase (AST) and alanine transaminase (ALT) in blood plasma by using a commercial kit from Boehringer Manheim (Munich, Germany), according to the supplier’s specifications and the results were expressed in international units per liter.
This study was approved by the Animal Welfare and Ethics Committee of Anhui Medical University and followed their guidelines for animal experimentation. Healthy adult male Sprague–Dawley rats (weight range 225–250 g), purchased from the Laboratory Animal Center, Anhui Medical University, were used for the study. The animals were housed in separate cages in a temperaturecontrolled room with alternating 12-h light:12-h dark cycles, and were allowed 1 week to acclimate to their surroundings. Rats were fed a standard animal diet; food and tap water would be available at will throughout the experimental protocol.
1.5. Water content in liver tissue The water content of the livers was determined by calculating the wet/dry weight ratio of liver tissues at 24 h after thermal injury. Post-experimental liver tissue samples were weighed and then desiccated at 85 8C for 48 h, and the wet/dry weight ratio was calculated. In this determination,
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liver edema was represented by an increase in the wet-to-dry weight ratios.
determined with the use of a protein assay (Bio-Rad; Hercules, CA). Nuclear and cytosolic extracts were stored at 80 8C.
1.6. Myeloperoxidase (MPO) activity As a biochemical quantitative marker of pulmonary and hepatic neutrophil infiltration, MPO activity was measured at 24 h after burn tramua. The method described by Mullane et al. [22] was used to measure MPO activity in the livers. Liver tissue was removed and immediately frozen and stored at 80 8C until the MPO assay could be performed. Tissue specimens were homogenized in a 50-mM potassium phosphate buffer with 0.5% hexadecyl-trimethyl-ammonium bromide (HTAB, pH 6.0; Sigma), and centrifuged at 40,000 g for 15 min at 4 8C. The supernatant was assayed for MPO activity spectrophotometrically. Then 20 ml of supernatant were combined with 12 ml of 25 mM H2O2, 30 ml of 40 mM O-dianisidine hydrochloride, and 1.938 ml of 50 mM phosphate buffer (pH 6.0). Color development was stopped by the addition of 0.1 ml of 1% NaN3 at room temperature after 5 and 20 min, respectively. The optical density was measured at 460 nm with a spectrophotometer (U-2001, Hitachi, Tokyo, Japan). Color development from 5 to 20 min was linear. One unit of MPO activity is defined as the activity degrading 1 mmol of H2O2/min at 25 8C. The results are expressed as units per gram of tissue (U/g) [23].
1.7.2. Electrophoretic mobility shift assays The EMSA method was used to characterize the binding activities of NF-kB in nuclear extracts. Double-stranded oligonucleotides were end-labeled with T4 polynucleotide kinase in the presence of [g-32P] ATP (3000 Ci/mmol). Five to 20 mg of nuclear protein was incubated in binding buffer containing g-32P-labeled double-stranded probe for NF-kB (Promega) and polydeoxyinosinic-deoxycytidylic acid (DidC) for 30 min at 4 8C, loaded onto a 6% polyacrylamide gel and electrophoresed for 1 h at 20 8C. Finally, the gels were dried and exposed to X-ray film. 1.8. Statistical analysis Data were expressed as mean S.E.M. Statistical evaluation of the continuous data was performed by oneway analysis of variance, followed by Dunnett’s t-test for between-group comparisons. The level of significance was considered to be P < 0.05.
2. Results
1.7. NF-kappaB activation
2.1. Liver function tests
1.7.1. Nuclear protein extraction A modified procedure based on the method of Grabellus et al. [24] was used. All steps were performed on ice. Livers were thawed in the presence of buffer A (10 mM HEPES, pH 7.9; 10 mM KCl, 1.5 mM MgCl2, 0.1% Nonidet P-40, and protease inhibitors plus 0.5 mM DTT); the proteases included a protease inhibitor cocktail containing the following: 20 mM 4-(2-aminoethyl)-benzenesulfaonylflouride, 14 mm trans-epoxysuccinyl-L-leucylamido-(4-guandidino) butane, 1300 mM bestatin, 10 mM leupeptin, 3 mM aprotinin, and 10mM sodium EDTA. The livers were allowed to thaw on ice for 20 min and were then suspended in 500 ml of buffer A per liver. The tissue was then homogenized with the use of a dounce homogenizer by hand. The cells were then allowed to incubate for 20 min on ice and then centrifuged at 1200 g for 5 min at 4 8C. The supernatants (cytosolic fraction) were removed and frozen at 80 8C. The remaining pellet was then resuspended in 200 ml of ice-cold buffer C (20 mM HEPES, pH 7.9; 420 mM NaCl, 1.8 mM MgCl2, 0.2 mM EDTA, pH 8; 25% glycerol) and protease inhibitor as described above plus 0.5 mM DTT. The pellets were completely resuspended by being pipetted and were incubated for 20 min on ice with gentle resuspension of the pellets every 2 min. After incubation, the pellets were centrifuged at full speed for 5 min at 4 8C. The supernatant was collected (nuclear fraction) and frozen at 80 8C. Protein concentrations were
Hepatocyte injury was evaluated by determining the plasma concentration of AST and ALT. At 24 h following a 30% TBSA full-thickness burn, plasma hepatic transaminases (AST and ALT) increased three- to four-fold in rats compared with sham animals (P < 0.05 versus sham). In vivo administration of Ligustrazine decreased plasma AST and ALT in burn rats significantly (P < 0.05 versus vehicle) (Fig. 1).
Fig. 1. Serum AST, ALT activities in rats. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control.
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Fig. 2. Liver edema was quantitated by liver wet-to-dry weight ratios at 24 h post-burn. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control. N = 8 animals per group.
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Fig. 3. Liver sequestration of neutrophils after thermal wounds as assessed by liver myeloperoxidase activity in sham, control, Ligustrazine groups. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control. N = 8 animals per group.
2.2. Liver wet/dry weight ratio Liver wet/dry weight ratio was significantly higher at 24 h after burn (2.9 0.12 versus 3.9 0.23; P < 0.01) in the control group compared with the sham group. In the Ligustrazine group the ratio was 3.3 0.16, which was significantly (P < 0.01) lower compared with the control group (Fig. 2). 2.3. MPO activity The activity of MPO, an enzyme in azurophilic granules of neutrophils, was used as a well-established marker to quantitate tissue neutrophil sequestration. Extracts of the liver samples were examined for content of MPO at 24 h after thermal injury of skin. As depicted in Fig. 4, the mean MPO level of the sham group was 10.8 1.25 U/g compared to 28.18 2.30 U/g (P < 0.01) in livers obtained from burned rats. In animals treated with Ligustrazine group, the MPO activity significantly decreased to levels of 15.42 1.56 U/g (P < 0.01 versus control; Fig. 3). 2.4. Hepatic NF-kB activation Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NF-kB were assessed by EMSA in liver of rats after burn trauma. At 24 h after a 30%TBSA full-thickness burn, the NF-kB DNA binding activity incresed significantly in liver harvested from burn rats than that from shams and this activity was inhibited by treatment with Ligustrazine (Fig. 4).
3. Discussion Severe burn, one of the most common problems faced in the emergency room, may cause damage to multiple organs
Fig. 4. Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NF-kB was assessed by EMSA in liver of rats after burn trauma. (A) Representative autoradiograms demonstrated that the burn-mediated increase in NF-kB activation was inhibited by treatment with Ligustrazine. (B) Densitometric analysis of EMSA confirmed that Ligustrazine can prevent the activation of NF-kB in liver harvested from burn rats. The values were expressed as fold change from the sham rats in each comparison. Symbol (*) indicates significance by P < 0.01 with respect to sham. Symbol (#) indicates significance by P < 0.01 with respect to control. N = 8 animals per group.
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distant from the original burn wound and may lead to multiorgan failure. The local and systemic inflammatory response to severe cutaneous thermal trauma is extremely complex, resulting in both local burn tissue damage and subsequent edema as well as marked systemic effects, which demand urgent medical interventions to ensure the survival of the patient. Despite many advanced medical treatments, multiorgan failure management is still a great problem. The data from this study showed that burn traumainduced hepatocellular injury as evidenced by increased plasma AST and ALT levels, increases liver wet/dry ratio, and upregulated NF-kB activity. The in vivo administration of Ligustrazine attenuated post-burn hepatocelluar injury, decreased burn-mediated MPO and NF-kB activity and alleviate histologic edema. It is well-known that circulating endotoxins and other bacterial by-products produced as a result of burn wound colonization are potent activators of the macrophages and neutrophils [25,26]. The activation of these inflammatory cells contributes to distant tissue damage through the release of reactive oxygen metabolites and various enzymes, such as proteases and myeloperoxidase. In the present study, we observed plasma ALT and AST activities-the most commonly used indicators of hepatocellular injury were markedly high in the burn group compared to controls and a significant increase in myeloperoxidase activity in liver samples of animals parallel with elevated liver wet/dry weight ratio, indicating that burn results in hepatic injury. This finding that plasma AST and ALT levels go up dramatically in rats after burn is consistent with previous reports [27,28], however, similar changes appeared slowly or indistinctively in human studies. Chiarelli et al. [29] had studied 43 burned patients, both during and after recovery, and found a 67.44% increase in transaminases in patients. The exact mechanism remains unclear and it seems more likely that human livers have stronger compensatory ability than animals. But in severely burned patients we observed the immediate increase of the enzymes. It has been shown that post-burn intravascular hemolysis may be largely prevented by neutrophil depletion of experimental animals, suggesting that neutrophils play an important role in the development of remote organ injury [30]. Our results showed that hepatic MPO activity (as an index of tissue polymorphonuclear neutrophil uptake) was markedly elevated after burn injury and Ligustrazine led to the downregulation of MPO concentration. MPO is one of the special oxidase in PMN, and MPO activity in tissue reflects the PMN chemotaxis and infiltration. PMN infiltration and activation could produce large amount of oxidants and cause tissue oxidative injury, which contributes to the organ functional damage in MODS. It is well known that accumulated PMNs in the grafted liver sinusoids might be important effector cells in the pathogenosis of liver injury after burn. The reactive oxygen and proteases released by activated PMNs might promote progressive hepatocellular damage.
Although it is well accepted that activated neutrophils play a key role in the development of microvascular injury at both the burn site and remote organs, it remains a matter of speculation as to which mediators might be responsible for activation of blood neutrophils. NF-kB system is another major signaling pathway responsible for cytokine expression. NF-kB plays a key role in the transcriptional regulation of various chemokines, including IL-8 [31]. The IL-8 promoter region contains binding sites for a number of important transcriptional factors including NF-kB [32]. In response to pro-inflammatory stimuli, IL-8 production is dependent on mitogen-activated protein kinases (MAPKS) and NF-kB pathways [33]. In patients with major trauma or burns, cytokines mediate neutrophil dysfunction contributes to ARDS, to immunological alterations involved in the increased susceptibility to infection, and to the systemic inflammatory responses that lead to shock and multiple organ dysfunction [34–36]. In our previous study [18], we found that the serum IL-8 levels of rats were markedly elevated after a 30% TBSA full-thickness burn. Furthermore, we also demonstrated that IL-8 is associated with the activation of neutrophils. Wang et al. [37] have reported that nuclear translocation of NF-kB was upregulated post-burn, in the current study we confirmed that burn trauma upregulated NF-kB activity in the liver of rats, From these findings we speculated that NF-kB system mediates the production of IL-8 and contributes to burn-induced liver injury. Chinese herbal medicine contributes a great deal to the health of the Chinese nation. Ligustrazine, a compound purified from Ligustium Wollichii Francha, is a widely used active ingredient in Chinese herbal medicine for the treatment of cardiovascular diseases due to its vasodilatory actions and antiplatelet activity [20,38,39]. It has been shown in animal models and clinical investigations that Ligustrazine is effective on ischemic diseases, such as heart, brain, and lung. However, the roles and mechanisms of Ligustrazine in treatment of digestive diseases have not been extensively studied. Ligustrazine has been proposed to act as an inhibitor of phosphodiesterase (PDE) and thereby it increases intracellular cAMP [39]. Hemorheological studies show that parameters such as hematocrit, fibrinogen level, and plasma viscosity, have a tendency to increase after large burns. Hemorheological parameters are modified by ligustrazini hydrochloride [16,40,41]. Ligustrazine could affect hemorheological events, such as blood flow, erythrocyte deformation, leukocyte adhesion, platelet aggregation, and thrombolysis [20]. These results suggest that Ligustrazine may play a role in tissue repair and blood hemeostasis. Furthermore, based on the current findings, the protective effect of Ligustrazine, in part, may be attributed to its inhibitory action on tissue neutrophil infiltration. We reported first that Ligustrazine attenuates acute lung injury after burn trauma, and inhibits serum IL-8 concentrations [18].
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In the present study, we examined the effect of Ligustrazine on the liver injury of severely burned rats. Our results showed that Ligustrazine decreased serum levels of aspartate aminotransferase and alanine aminotransferase, attenuated the wet/dry ratio and MPO activity, and downregulated the activity of NF-kB, suggesting that the protective effects of Ligustrazine against burn-induced liver injury may be attributable to influencing NF-kB activation and further preventing post-burn pro-inflammatory cytokines release and accumulation of neutrophils. In conclusion, the early use of Ligustrazine seems to be effective in ameliorating liver injury after major thermal injury. We conclude that Ligustrazine may act through inhibiting NF-kB activity. Although the final molecular mechanism remains to be further studied, this will likely provide further evidence for Ligustrazine as a therapeutic strategy in burn-induced liver injury.
References [1] Wang P, Chaudry IH. Mechanism of hepatocellular dysfunction during hyperdynamic sepsis. Am J Physiol 1996;270:R927–38. [2] Matuschak GM. Lung-liver interactions in sepsis and multiple organ failure syndrome. Clin Chest Med 1996;17:83–98. [3] Baron P, Traber LD, Traber DL, Nguyen T, Hollyoak M, Heggers JP, et al. Gut failure and translocation following burn and sepsis. J Surg Res 1994;57:197–204. [4] Gilpin DA, Hsieh CC, Kuninger DT, Herndon DN, Papaconstantinou J. Effect of thermal injury on the expression of transcription factors that regulate acute phase response genes: the response of C/EBP alpha, C/EBP beta, and C/EBP delta to thermal injury. Surgery 1996;119: 674–83. [5] Blom WM, De Bont HJ, Meijerman I, Kuppen PJ, Mulder GJ, Nagelkerke JF. Interleukin-2-activated natural killer cells can induce both apoptosis and necrosis in rat hepatocytes. Hepatology 1999;29: 785–92. [6] Fey GH, Gauldie J. The acute phase response of the liver in inflammation. Prog Liver Dis 1990;9:89–116. [7] Jeschke MG, Herndon DN, Wolf SE, DebRoy MA, Rai J, Lichtenbelt BJ, et al. Recombinant human growth hormone alters acute phase reactant proteins, cytokine expression, and liver morphology in burned rats. J Surg Res 1999;83:122–9. [8] Youn YK, LaLonde C, Demling R. The role of mediators in the response to thermal injury. World J Surg 1992;16:30–6. [9] Ding HQ, Zhou BJ, Liu L, Cheng S. Oxidative stress and metallothionein expression in the liver of rats with severe thermal injury. Burns 2002;28:215–21. [10] Wang M, Tai G, Zhang Y, Xiao G. The modulating effects of transfected IkappaBalpha gene on the NF-kappaB activity of hepatic tissue in scalded rats. Zhonghua Shao Shang Za Zhi 2002;18: 42–4. [11] Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, et al. NF-kappaB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 1998;101:802–11. [12] Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 1994;10:405–55. [13] Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I (kappa) B kinase-beta. Nature 1998;396:77–80. [14] Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE. Inhibition of NFkappa B activity induces apoptosis in murine hepatocytes. Am J Pathol 1997;151:891–6.
333
[15] Wang CY, Mayo MW, Baldwin Jr AS.. TNF- and cancer therapyinduced apoptosis: potentiation by inhibition of NF-kappaB. Science 1996;274:784–7. [16] Liao F. Treatment of blood stasis syndrome and hemorheology. Clin Hemorheol 1994;14:463–71. [17] Zou LY, Hao XM, Zhang GQ, Zhang M, Guo JH, Liu TF. Effect of tetramethyl pyrazine on L-type calcium channel in rat ventricular myocytes. Can J Physiol Pharmacol 2001;79:621–6. [18] Zheng H, Chen XL, Han ZX, Zhang Z, Wang SY, Xu QL. Ligustrazine attenuates acute lung injury after burn trauma. Burns 2005;39:635–40. [19] Sun L, Li Y, Shi J, Wang X, Wang X. Protective effects of ligustrazine on ischemia-reperfusion injury in rat kidneys. Microsurgery 2002;22: 343–6. [20] Liao F. Herbs of activating blood circulation to remove blood stasis. Clin Hemorheol Microcirc 2000;23:127–31. [21] Chen XL, Xia ZF, Ben DF, Wang GQ, Wei D. Role of p38 mitogenactivated protein kinase in lung injury after burn trauma. Shock 2003;19:475–9. [22] Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Methods 1985;14:157–67. [23] Cutrin JC, Boveris A, Zingaro B, Corvetti G, Poli G. In situ determination by surface chemiluminescence of temporal relationships between evolving warm ischemia-reperfusion injury in rat liver and phagocyte activation and recruitment. Hepatology 2000;31:622–32. [24] Grabellus F, Levkau B, Sokoll A, Welp H, Schmid C, Deng MC, et al. Reversible activation of nuclear factor-kappaB in human end-stage heart failure after left ventricular mechanical support. Cardiovasc Res 2002;53:124–30. [25] Jones 2nd WG, Minei JP, Barber AE, Fahey 3rd TJ, Shires 3rd GT, Shires GT. Splanchnic vasoconstriction and bacterial translocation after thermal injury. Am J Physiol 1991;261:H1190–6. [26] Morris SE, Navaratnam N, Herndon DN. A comparison of effect of thermal injury and smoke inhalation on bacterial translocation. J Trauma 1990;30:639–43. [27] Jeschke MG, Jeschke MG, Aili Low JF, Spies M, Vita R, Hawkins HK, et al. Barrow cell proliferation, apoptosis, NF-kB expression, enzyme, protein, and weight changes in livers of burned rats. Am J Physiol Gastrointest Liver Physiol 2001;280:G1314–20. [28] Chen XL, Xia ZF, Yu YX, Wei D, Wang CR, Ben DF. P38 mitogenactivated protein kinase inhibition attenuates burn-induced liver injury in rats. Burns 2005;31:320–30. [29] Chiarelli A, Casadei A, Pornaro E, Siliprandi L, Mazzoleni F. Alanine and aspartate aminotransferase serum levels in burned patients: a longterm study. J Trauma 1987;27(7):790–4. [30] Hansbrough JF, Wikstrom T, Braide M, Tenenhaus M, Rennekampff OH, Kiessig V, et al. Neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury. J Surg Res 1996;61: 17–22. [31] Holtmann HR, Winzen P, Holland S, Eichemeier E, Hoffmann D, Wallach N. Induction of interleukin-8 synthesis integrates effects of transcription and Mrna degradation from at least three different cytokine-or stress-activated signal transduction pathways. Mol Cell Biol 1999;19:6742–53. [32] Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, et al. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 1993;90: 10193–7. [33] Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol 2000;18: 621–63. [34] Beal AL, Cerra FB. Multiple organ failure syndrome in the 1990s. Systemic inflammatory response and organ dysfunction. JAMA 1994; 271:226–33. [35] Bellomo R. The cytokine network in the critically ill. Anaesth Intensive Care 1992;20:288–302.
334
H. Zheng et al. / Burns 32 (2006) 328–334
[36] Drost AC, Burleson DG, Cioffi Jr WG, Jordan BS, Mason AD, Pruitt Jr BA. Plasma cytokines following thermal injury and their relationship with patient mortality, burn size, and time postburn. J Trauma 1993;35:335–9. [37] Wang GQ, Xia ZF, Yu BJ, Xiao SC, Chen YL. Cardiac apoptosis in burned rats with delayed fluid resuscitation. Burns 2001;27:250–3. [38] Liu SY, Sylvester DM. Antiplatelet activity of tetranmethylpyrazine. Thromb Res 1994;75:51–62.
[39] Kwan CY. Plant-derived drugs acting on celluar Ca2+ mobilization in vascular smooth muscle: tetramethylpyrazine and tetrandrine. Stem Cells 1994;12:64–7. [40] Luo G. Effects of ligustrazine and anisodamine on whole blood viscosity and erythrocyte deformability in rabbits. Zhonghua Yi Xue Za Zhi 1987;67:607–9. [41] Sutara SP. Effects of three Chinese medicinal herbs on erythrocyte deformability. Clin Hemorheol 1987;7:773–9.