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Platonin Mitigates Acute Lung Injury Induced by Bilateral Lower Limb Ischemia-Reperfusion in Rats
Journal of Surgical Research 167, e255–e262 (2011) doi:10.1016/j.jss.2010.03.075
Platonin Mitigates Acute Lung Injury Induced by Bilateral Lower Limb Ischemia-Reperfusion in Rats Kuei-Yao Hsu, M.D.,*,† Pei-Shan Tsai, Ph.D.,‡ Jie-Jen Lee, M.D., Ph.D.,§,k Tao-Yeuan Wang, M.D.,{ and Chun-Jen Huang, M.D., Ph.D.*,†,1 *Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan; †School of Medicine, Tzu Chi University, Hualien, Taiwan; ‡College of Nursing; §Department of Pharmacology, Taipei Medical University, Taipei, Taiwan; kDepartment of Surgery; and {Department of Pathology, Mackay Memorial Hospital, Taipei, Taiwan Submitted for publication February 19, 2010
Background. Oxidative stress and inflammatory response are crucial in mediating the development of acute lung injury induced by bilateral lower limb ischemia-reperfusion (I/R). Platonin, a potent antioxidant, possesses anti-inflammation capacity. We sought to elucidate whether platonin could mitigate acute lung injury induced by lower limb I/R. Materials and Methods. Forty-eight adult male rats were allocated to receive I/R, I/R plus platonin (100 mg/kg intravenous injection immediately after reperfusion), sham instrumentation, or sham instrumentation plus platonin (denoted as the I/R, I/R-platonin, Sham, or Sham-platonin group, respectively; n [ 12 in each group). Bilateral hind limb I/R was induced by applying rubber band tourniquets high around each thigh for 3 h followed by reperfusion for 3 h. After sacrifice, the degree of lung injury was determined. Results. Histologic findings revealed moderate inflammation in lung tissues of the I/R group and mild inflammation in those of the I/R-platonin group. Total cell number and protein concentration in bronchoalveolar lavage fluid as well as the leukocyte infiltration and myeloperoxidase activity in lung tissues of the I/R group were significantly higher than those of the I/Rplatonin group. The pulmonary concentrations of macrophage inflammatory protein-2, interleukin-6, and prostaglandin E2 of the I/R group were significantly higher than those of the I/R-platonin group. Moreover, the plasma nitric oxide concentration as well as the nitric oxide and malondialdehyde concentrations in
1 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, No. 289, Jianguo Rd., Sindian City, Taipei County 231, Taiwan. E-mail: [email protected].
lung tissues of the I/R group were significantly higher than those of the I/R-platonin group. Conclusions. Platonin mitigates acute lung injury induced by bilateral lower limb I/R in rats. Ó 2011 Elsevier Inc. All rights reserved.
Key Words: chemokine; cytokine; nitric oxide; prostaglandin E2.
INTRODUCTION
Acute lower limb ischemia is a common condition that can be observed in clinical situations such as embolism, atherosclerotic thrombosis, traumatic arterial injury, aortic clamping during abdominal aortic aneurysm repair, etc. [1–5]. However, reperfusion of the acutely ischemic lower limb can, in turn, induce reperfusion injury and cause remote vital organ dysfunctions [1, 6, 7]. Oxidative stress and inflammatory response have been shown to play an essential role in mediating the remote organ injury induced by lower limb ischemia-reperfusion [1, 6, 8]. Among the vital organs, lung is one of the most sensitive organs that is subject to the injuries induced by lower limb ischemia-reperfusion [1, 6, 8]. Platonin (4,40 ,400 -thrimethyl-3,30 ,300 -triheptyl-7-[200 trimethinethiazolocyanine-3-300 thia-zolyl]-2,20 diiodide), a cyanine photosensitizing dye and a potent antioxidant, possesses potent reactive oxygen and nitrogen species (ROS/RNS) scavenging capacity [9]. Platonin also has potent immunomodulating action and is currently used for the treatment of immune diseases, especially rheumatoid arthritis, in clinical settings [10]. In addition, platonin has been shown to possess significant anti-inflammation capacity [9, 11, 12]. For
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instance, platonin was reported to improve circulatory failure and mortality in septic rats [9]. Previous data also indicated that endotoxin-induced up-regulation of inflammatory molecules could be inhibited by platonin [11, 12]. However, the question of whether platonin could exert protective effects and attenuate acute lung injury induced by lower limb ischemia-reperfusion remains unstudied. This study was conducted to test the following hypotheses: (1) platonin could mitigate acute lung injury induced by lower limb ischemia-reperfusion in rats, and (2) the mechanisms may involve attenuating oxidative stress and decreasing inflammatory response. METHODS AND MATERIALS All animal studies were approved by the Institutional Animal Use and Care Committee of Buddhist Tzu Chi General Hospital, Taipei Branch, and the care and handling of the animals were in accordance with National Institutes of Health guidelines. All rats were fed a standard laboratory chow and were provided water ad libitum until the day of experiment. A total of 48 adult male Sprague-Dawley rats (200 to 250 g; BioLASCO Taiwan Co., Ltd, Taipei, Taiwan) were used for the experiments.
Blood Sample Collection and Arterial Blood Gas (ABG) Analysis At the end of each experiment, arterial blood (0.5 mL) was drawn. ABG levels were immediately measured with a blood gas analyzer (Rapidlab 348; Bayer Healthcare LLC, East Walpole, MA). In addition, another 5 mL of blood was withdrawn and centrifuged to separate the plasma. Plasma samples were stored at –80 C for subsequent analysis of nitric oxide (NO, the indicator of oxidative stress).
Lung Tissues Collection and Bronchoalveolar Lavage (BAL) At the end of each experiment, the left main bronchus was tied and left lung was removed. The upper and lower lobes of left lung were divided and the left lower lobe lung tissues were snap-frozen in liquid nitrogen and stored at –80 C for subsequent analysis. The right lung tissues of six rats from each group were infused with 4% formaldehyde through the tracheostomy tube and then removed. For the other six rats of each group, the right lung was lavaged with 6-mL aliquots of sterile saline for five times, as we have previously reported [13]. After the BAL fluid (BALF) was collected and recorded, an aliquot of the BALF was diluted 1:1 with trypan blue dye (Life Technologies, Grand Island, NY) and the total cell number was counted using a standard hemocytometer. The remaining BALF was centrifuged and the protein concentration of the supernatant was determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL).
Histologic Analysis Animal Preparation Under anesthesia (ketamine/xylazine: 110/10 mg/kg, intraperitoneal), two polyethylene (PE-50) catheters were placed in the right carotid artery for continuous blood pressure monitoring and the right external jugular vein for intravenous injection, respectively. After tracheostomy, a 14-gauge angiocatheter was inserted as a tracheostomy tube. Rats were mechanically ventilated (tidal volume: 4 mL of room air; rate: 35 breaths/min) with a small animal ventilator (Harvard Apparatus, South Natick, MA). Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored (BIOPAC System, Santa Barbara, CA) throughout the experiments. Supplemental dose of ketamine/xylazine mixture (30/3 mg/kg, intravenous) was administered every 1 h until the end of the experiment.
Lower Limb Ischemia-Reperfusion Protocols The lower limb ischemia-reperfusion injury protocol was adapted from a previous report [6]. In brief, bilateral hind limb ischemiareperfusion was induced by applying rubber band tourniquets high around each thigh for 3 h followed by reperfusion for 3 h.
Experimental Protocols Rats were randomly allocated to one of the four groups (n ¼ 12 in each group), i.e., the sham instrumentation, sham instrumentation plus platonin, ischemia-reperfusion, and ischemia-reperfusion plus platonin groups, and designated as the Sham, Sham-platonin, I/R, and I/R-platonin group, respectively. Rats of the I/R-platonin and Sham-platonin groups received intravenous injection of platonin (100 mg/kg dissolved in 0.5 mL normal saline) immediately after reperfusion or at comparable time point in those that received sham instrumentation. To control for the volume effects of vehicle, rats of the Sham and I/R groups also received 0.5 mL normal saline intravenous injection at comparable time point. The dosage of platonin (i.e., 100 mg/kg) employed in this study was determined accordingly to previous data that platonin at this dosage significantly protected septic rats from circulatory failure and mortality [9]. After reperfusion for 3 ho, all rats were sacrificed with high dose pentobarbital injection.
The formaldehyde-infused right lungs were embedded in paraffin wax, serial sectioned, and stained with hematoxylin and eosin. Histologic characteristics including edematous changes of the alveolar wall, hemorrhage, vascular congestion, and polymorphonuclear leukocytes (PMN) infiltration, were evaluated under a light microscope to evaluate pulmonary inflammation, as we have previously reported [13]. Each histologic characteristic was scored on a scale of 0-normal to 5-severe by a pathologist who was blinded to the experiment. The overall pulmonary inflammation was categorized according to the sum of the score (0–5: normal to minimal inflammation; 6–10: mild inflammation; 11–15: moderate inflammation; 16–20: severe inflammation). The degree of leukocyte infiltration can be quantified by PMN/alveoli ratio [13]. We thus counted PMNs and alveoli per high-power field (HPF, 4003) in 10 randomly selected areas of each sample. The PMNs/alveoli ratio was then calculated by dividing the sum of the PMNs in 10 HPFs by that of the alveoli.
Wet/Dry Weight Ratio and Myeloperoxidase (MPO) Activity Assay We measured the wet/dry weight ratio, i.e., the index of lung water, of the freshly harvested rat lung samples (i.e., left upper lobe) to evaluate pulmonary inflammation. In brief, the whole lobe was weighed and then placed in the oven for 24 h at 60 C, and weighed again when it was dry, as we have previously reported [13]. The wet/dry weight ratio was then calculated. We also quantified pulmonary inflammation by measuring pulmonary MPO activity. The protocol was modified from our previous report.[13] In brief, snap-frozen tissue samples (–80 C) were homogenized, re-suspended, sonicated, and centrifuged. The supernatant was collected and incubated in a water bath for 2 h at 60 C. MPO activity was then measured.
Inflammatory Molecules Snap-frozen lung tissues were processed, as we have previously reported [13]. The pulmonary concentrations of the inflammatory molecules, including chemokine (e.g., macrophage inflammatory
HSU ET AL.: PLATONIN VERSUS LOWER LIMB I/R INJURY
RESULTS
protein-2, MIP-2), cytokine (e.g., interleukin-6, IL-6), and prostaglandin E2 (PGE2), were measured using enzyme-linked immunosorbent assay (ELISA) (MIP-2 ELISA kit; R and D Systems, Inc., Minneapolis, MN; ELISA Kits for IL-6 and PGE2; Pierce).
Reverse Transcription and Polymerase Chain Reaction (RT-PCR) Production of PGE2 is mediated by cyclooxygenase 2 (COX-2) [14]. Transcriptional expression of COX-2 of the harvested lung tissues was measured using RT-PCR. The primer sequences and amplification protocols for COX-2 and b-Actin (as an internal standard) were adapted from those previously published ones [15, 16]. After separation, PCR-amplified cDNA band densities were quantified using densitometric techniques (Scion Image for Windows; Scion Corp, Frederic, MD).
NO Assay for Oxidative Status Evaluation Process of the lung tissues samples were performed as we have previously reported [13]. Then the plasma and pulmonary concentrations of stable NO metabolites, nitrite and nitrate, were measured using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).
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Hemodynamics
The baseline HR and MAP values of these four groups were comparable (data not shown). HR and MAP of the Sham and Sham-platonin groups remained stable throughout the experiment. The HR and MAP values measured at the end of the experiment (i.e., the end HR and MAP) of the Sham and Sham-platonin groups were also comparable. In contrast, the end HR values of the I/R and I/R-platonin groups were significantly higher than that of the Sham group (P ¼ 0.035 and 0.031, respectively; Table 1) whereas the end MAP values of the I/R and I/R-platonin groups were significantly lower than that of the Sham group (P ¼ 0.029 and 0.033, respectively; Table 1). In addition, the end HR and MAP values of the I/R and I/R-platonin groups were comparable (Table 1). ABG Data
Malondialdehyde (MDA) Assay for Lipid Peroxidation Status Evaluation For MDA assay, snap-frozen lung tissues were thawed immediately before assaying. The protocol of MDA assay was modified from our previous report [17]. In brief, tissues homogenates were collected. Then, phosphoric acid and thiobarbituric acid solution were added to 0.5 mL of homogenates. The mixture was heated in boiling water for 45 min. After cooling, the absorbance was measured and the amounts of lipid peroxides were calculated.
Statistical Analysis One-way analysis of variance with the Bonferroni test was used for multiple comparisons. Data were presented as means 6 standard deviations. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL) was used for data analysis.
The baseline ABG values, including pH, PaO2, PaCO2, and base excess (BE), of these four groups were comparable (data not shown). The end ABG values of the Sham and Sham-platonin groups were also comparable (Table 1). In contrast, the end pH, PaO2, and BE values of the I/R and I/R-platonin groups were significantly lower than those of the Sham group (all P < 0.02; Table 1), whereas the end PaCO2 values of the I/R and I/R-platonin groups were significantly higher than that of the Sham group (both P < 0.015; Table 1). Moreover, the end values of pH, PaO2, and BE of the I/R-platonin group were significantly higher than those of the I/R group (P ¼ 0.028, 0.015, and 0.033, respectively; Table 1). The end PaCO2 value of the I/R-platonin group, in contrast, was significantly lower than that of the I/R group (P ¼ 0.031; Table 1).
TABLE 1 Hemodynamics and Arterial Blood Gas Data at the End of Experiment Hemodynamics
Arterial blood gas
Group (n ¼ 12)
HR (beats/min)
MAP (mm Hg)
pH
PaO2 (mm Hg)
PaCO2 (mm Hg)
Sham Sham-platonin I/R I/R-platonin
317 6 21 309 6 18 346 6 22* 338 6 17*
105 6 12 112 6 10 84 6 13* 87 6 12*
7.43 6 0.08 7.40 6 0.05 7.26 6 0.04* 7.35 6 0.04*,y
96 6 5 99 6 7 70 6 4* 86 6 5*,y
41 6 4 38 6 6 56 6 4* 48 6 3*,y
Sham ¼ the sham instrumentation group. Sham-platonin ¼ the sham instrumentation plus platonin group. I/R ¼ the lower limb ischemia-reperfusion group. I/R-platonin ¼ the lower limb ischemia-reperfusion plus platonin group. HR ¼ heart rate. MAP: mean arterial pressure. Data are means 6 standard deviations. * P < 0.05 versus Sham group. y P < 0.05 I/R-platonin group versus I/R group.
Base Excess (mM) 1.8 6 2.9 2.3 6 1.5 7.6 6 1.6* 4.9 6 1.7*,y
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BALF Data
Pulmonary Concentrations of Inflammatory Molecules
The total cell number and total protein concentration in BALF of the Sham and Sham-platonin groups were low (Fig. 1A and B). The total cell number and total protein concentration of the I/R group were significantly higher than those of the Sham group (P ¼ 0.020 and 0.017, respectively; Fig. 1A and B). In contrast, the total cell number and total protein concentration of the I/Rplatonin group were significantly lower than those of the I/R group (P ¼ 0.029 and 0.025, respectively; Fig. 1A and B).
The pulmonary MIP-2, IL-6, PGE2, and COX-2 mRNA concentrations of the Sham and Shamplatonin groups were low (Fig. 4A, B, and C). The MIP-2, IL-6, PGE2, and COX-2 mRNA concentrations of the I/R group were significantly higher than those of the Sham group (all P < 0.001; Fig. 4A, B, and C). In contrast, the MIP-2, IL-6, PGE2, and COX-2 mRNA concentrations of the I/R-platonin group were significantly lower than those of the I/R group (P ¼ 0.028, 0.021, 0.036, and 0.025, respectively; Fig. 4A, B, and C). NO and MDA Assays
Lung Histology and Pulmonary Inflammation Score
Histologic analysis revealed normal to minimal inflammation in the lung tissues of the Sham and Sham-platonin groups (Fig. 2A and B). The lung tissues of the I/R group revealed moderate inflammation (Fig. 2C), whereas those of the I/R-platonin group revealed mild inflammation (Fig. 2D). Findings of the pulmonary inflammation score paralleled the findings of the histological analysis (Fig. 3A).
Wet/Dry Weight Ratio, PMNs/Alveoli Ratio, and MPO Activity
The wet/dry weight ratio, PMNs/alveoli ratio, and MPO activity in lung tissues of the Sham and Shamplatonin groups were low (Fig. 3B, C, and D). The wet/dry weight ratio, PMNs/alveoli ratio, and MPO activity of the I/R group were significantly higher than those of the Sham group (P ¼ 0.028, 0.015, and 0.011, respectively; Fig. 3B, C, and D). In contrast, the PMNs/alveoli ratio and MPO activity of the I/Rplatonin group were significantly lower than those of the I/R group (P ¼ 0.026 and 0.032, respectively; Fig. 3C and D), whereas the wet/dry weight ratio of these two groups were comparable (Fig. 3)B.
The plasma NO concentrations of the Sham and Sham-platonin groups were low (Fig. 5A). The pulmonary NO and MDA concentrations of the Sham and Sham-platonin groups were also low (Fig. 5B and C). The plasma NO concentration of the I/R group was significantly higher than that of the Sham group (P < 0.001; Fig. 5A). Similarly, the pulmonary NO and MDA concentrations of the I/R group were significantly higher than those of the Sham group (P ¼ 0.017 and 0.021, respectively; Fig. 5B and C). In contrast, the plasma NO concentration of the I/R-platonin group was significantly lower than that of the I/R group (P ¼ 0.021; Fig. 5A). The pulmonary NO and MDA concentrations of the I/R-platonin group were also significantly lower than those of the I/R group (P ¼ 0.030 and 0.026, respectively; Fig. 5B and C). DISCUSSION
Data from this study, in concert with those previous ones [1, 6, 8], confirmed that lower limb ischemiareperfusion could induce acute lung injury in rats. In this study, we demonstrated that the acute lung injury induced by lower limb ischemia-reperfusion could be mitigated by platonin. As platonin is currently used in clinical settings [10], data from this study could have
FIG. 1. (A)The total cell number and (B) the total protein concentration in bronchoalveolar lavage fluid (BALF) collected from rats of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
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FIG. 2. Microscopic findings of the lung tissues stained with hematoxylin and eosin (2003). (A) Representative microscopic findings of the sham instrumentation (Sham) group. (B) Representative microscopic findings of the Sham plus platonin (Sham-platonin) group. (C) Representative microscopic findings of the lower limb ischemia-reperfusion (I/R) group. (D) Representative microscopic findings of the I/R plus platonin (I/R-platonin) group.
direct clinical implications. Judging from our data, we believe that incorporation of platonin as part of the therapies should be beneficial in clinical situations associated with lower limb ischemia-reperfusion.
The notion that lower limb-ischemia could exert oxidative stress in rats [6, 8] was confirmed by this study, as our data revealed that the systemic NO concentrations as well as the pulmonary NO and
FIG. 3. (A) The pulmonary inflammation scores, (B) the wet/dry weight ratio, (C) the polymorphonuclear leukocytes (PMN)/alveoli ratio, and (D) the myeloperoxidase (MPO) activity in lung tissues harvested from rats of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
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FIG. 4. The concentrations of (A) macrophage inflammatory protein-2 (MIP-2), (B) interleukin-6 (IL-6), and (C) prostaglandin E2 (PGE2), and cyclooxygenase 2 (COX-2) mRNA in lung tissues harvested from rats of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
MDA concentrations were significantly increased in rats experiencing lower limb ischemia-reperfusion. It is well established that bursts of ROS/RNS could imbalance the cellular redox condition and lead to damages of the vital pathways, including energy metabolism, survival/stress responses, apoptosis, etc. [18]. Moreover, bursts of ROS/RNS may induce inflammatory response, including neutrophil activation and cytokine produc-
tion [19]. In line with this notion, it has been shown that lower limb ischemia-reperfusion could induce inflammatory response [6, 8]. This notion was also confirmed by this study, as our data revealed significant increases in pulmonary leukocyte infiltration (evidenced by histology and MPO assay) and up-regulation of pulmonary inflammatory molecules (e.g., chemokine, cytokine, and PGE2) in rats
FIG. 5. (A) The plasma nitric oxide (NO) concentration, (B) the pulmonary NO concentration, and (C) the pulmonary malondialdehyde (MDA) concentration of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
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experiencing lower limb ischemia-reperfusion. It has been shown that oxidants (especially NO), infiltrated leukocytes, and inflammatory molecules can work together to injure pulmonary microvascular endothelial cell and result in the loss of microvascular integrity and eventually lead to gas exchange impairment and pulmonary dysfunction during ischemia-reperfusion injury [20, 21]. This notion was supported by our data, as we observed a significant decrease in PaO2 and a significant increase in PaCO2 in rats experiencing lower limb ischemia-reperfusion. Platonin possesses potent ROS/RNS scavenging capacity and anti-inflammation capacity [9]. Our data revealed that rats of the I/R-platonin group had higher PaO2 and lower PaCO2 levels compared with those of the I/ R group, indicating that the gas exchange impairment induced by lower limb ischemia-reperfusion could be mitigated by platonin. Moreover, data from this study revealed that rats of the I/R-platonin group had lower systemic NO concentration, pulmonary NO and MDA concentrations, pulmonary leukocyte infiltration, and pulmonary inflammatory molecules concentrations compared with those rats of the I/R group. These data thus provided clear evidence to confirm our hypothesis that platonin could act through decreasing the oxidative stress and ameliorating the inflammatory response to exert its protective effects against acute lung injury induced by lower limb ischemia-reperfusion. It is well established that expression of inflammatory molecules is regulated by the crucial transcription factors, including nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) [22, 23]. In addition, expression of AP1 is under the regulation of mitogen-activated protein kinases (MAPKs) [24]. The question of whether lower limb ischemia-reperfusion induces significant upregulation of NF-kB, MAPKs, and AP-1 remains unstudied. NF-kB, MAPKs, and AP-1 are oxidative stress-responsive transcription factors and their expression can be up-regulated by oxidative stress [25, 26]. Moreover, previous data also confirmed the crucial roles of NF-kB, MAPKs, and AP-1 in regulating the expression of inflammatory molecules in other forms of ischemia-reperfusion [27–29]. Judging from these data, we speculate that lower limb ischemiareperfusion may very likely induce the up-regulation of NF-kB, MAPKs, and AP-1. As aforementioned, our data confirmed that the oxidative stress and pulmonary inflammatory response induced by lower limb ischemia-reperfusion could be attenuated by platonin. The question of whether NF-kB, MAPKs, and AP-1 are involved in mediating the observed effects of platonin also remains unstudied. Previous data indicated that platonin posted significant effects on regulating the expression of NF-kB [30]. Our recent data also confirmed that platonin posted significant effects on
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regulating the expression of MAPKs and AP-1 [31]. Judging from these data, we further speculate that platonin may very likely act through inhibiting the upregulation of NF-kB, MAPKs, and/or AP-1 to exert its protective effects in this regard. Previous data, reported by Hsiao et al., indicated that platonin could significantly attenuate hemodynamic changes in septic rats [9]. Judging from those previous data, one would expect that platonin should exert similar effects in rats experiencing lower limb ischemiareperfusion. However, to our surprise, our data revealed that the end MAP and HR of the I/R and I/R-platonin groups were comparable. It has been postulated that it takes approximately 1 h before the anti-inflammation effects of platonin to come to effect [32]. Therefore, it is likely that the discrepancy between our data and those reported by Hsiao et al. is related to the difference in the timing of platonin administration, as Hsiao et al. chose to administer platonin before endotoxin installation (i.e., pretreatment) and we chose to administer platonin immediately after reperfusion (i.e., cotreatment), instead. Moreover, data from this study confirmed the protective effects of platonin against acute lung injury induced by lower limb ischemia-reperfusion. However, the protective effects of platonin observed in this study were not potent enough to totally reverse the detrimental effects of lower limb ischemia-reperfusion. As the therapeutic efficacy of platonin is affected by the timing of administration [32], it is likely that platonin can exert stronger therapeutic effects in this regard if platonin is administered before reperfusion and/or ischemia. The therapeutic efficacy of platonin is also dose-dependent [11]. The preliminary data observed in this study revealed that platonin at the dosages of 25 and 50 mg/kg exerted no significant effects on ameliorating the upregulation of pulmonary inflammatory molecules as well as the increases in total cell number and protein concentration in BALF induced by lower limb ischemia-reperfusion (data not shown). Judging from these data, we thus speculate that larger dosages of platonin (e.g., >100 mg/kg) may very likely exert stronger therapeutic effects in this regard. More studies are needed before further conclusions can be drawn. Certain study limitations do exist. First, this study focused on the early phase of reperfusion, and the long-term effects of platonin on protecting lung tissues against lower limb ischemia-reperfusion injury remain to be elucidated. Second, interspecies differences should be taken into consideration if further data interpretation is intended. In summary, platonin mitigates acute lung injury induced by bilateral lower limb ischemia-reperfusion in rats. The mechanisms may involve decreasing oxidative stress and attenuating inflammatory response induced by lower limb ischemia-reperfusion.
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ACKNOWLEDGMENTS This work was supported by grants from the Buddhist Tzu Chi General Hospital, Taipei Branch (TCRD-TPE-99-28) awarded to K-Y H and the National Science Council, Taiwan (NSC 98-2314-B-303-012MY3) awarded to C-J H.
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transcription in activated murine macrophages may involve voltage-sensitive naþ channel. Anesth Analg 2006;102:1739. Futaki N, Takahashi S, Kitagawa T, et al. Selective inhibition of cyclooxygenase-2 by NS-398 in endotoxin shock rats in vivo. Inflamm Res 1997;46:496. Chang KY, Tsai PS, Huang TY, et al. HO-1 mediates the effects of HBO pretreatment against sepsis. J Surg Res 2006;136:143. Becker LB. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res 2004;61:461. Kaminski KA, Bonda TA, Korecki J, et al. Oxidative stress and neutrophil activation–the two keystones of ischemia/reperfusion injury. Int J Cardiol 2002;86:41. Wang LF, Patel M, Razavi HM, et al. Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 2002;165:1634. Turnage RH, Guice KS, Oldham KT. Pulmonary microvascular injury following intestinal reperfusion. New Horiz 1994;2:463. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997; 17:3. Adcock IM. Transcription factors as activators of gene transcription: AP-1 and NF-kappa B. Monaldi Arch Chest Dis 1997; 52:178. Kristof AS, Marks-Konczalik J, Moss J. Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation. J Biol Chem 2001;276:8445. Schreck R, Albermann K, Baeuerle PA. Nuclear factor kappa B: An oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun 1992;17:221. Cindrova-Davies T, Spasic-Boskovic O, Jauniaux E, et al. Nuclear factor-kappa B, p38, and stress-activated protein kinase mitogen-activated protein kinase signaling pathways regulate proinflammatory cytokines and apoptosis in human placental explants in response to oxidative stress: Effects of antioxidant vitamins. Am J Pathol 2007;170:1511. Valen G. Signal transduction through nuclear factor kB in ischemia-reperfusion and heart failure. Basic Res Cardiol 2004;99:1. Lysiak JJ, Bang HJ, Nguyen QA, et al. Activation of the nuclear factor kappa B pathway following ischemia-reperfusion of the murine testis. J Androl 2005;26:129. Omura T, Yoshiyama M, Shimada T, et al. Activation of mitogenactivated protein kinases in in vivo ischemia/reperfused myocardium in rats. J Mol Cell Cardiol 1999;31:1269. Shao DZ, Lin MT. Platonin inhibits LPS-induced NF-kappaB by preventing activation of Akt and IKKbeta in human PBMC. Inflamm Res 2008;57:601. Lee JJ, Liu CL, Tsai PS, et al. Platonin inhibits endotoxininduced MAPK and AP-1 up-regulation. J Surg Res 2011; 167:e299. Oyanagui Y. Platonin, a photosensitizing cyanine dye, suppresses acute inflammation. Arch Int Pharmacodyn Ther 1983; 266:162.
Platonin Mitigates Acute Lung Injury Induced by Bilateral Lower Limb Ischemia-Reperfusion in Rats Kuei-Yao Hsu, M.D.,*,† Pei-Shan Tsai, Ph.D.,‡ Jie-Jen Lee, M.D., Ph.D.,§,k Tao-Yeuan Wang, M.D.,{ and Chun-Jen Huang, M.D., Ph.D.*,†,1 *Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan; †School of Medicine, Tzu Chi University, Hualien, Taiwan; ‡College of Nursing; §Department of Pharmacology, Taipei Medical University, Taipei, Taiwan; kDepartment of Surgery; and {Department of Pathology, Mackay Memorial Hospital, Taipei, Taiwan Submitted for publication February 19, 2010
Background. Oxidative stress and inflammatory response are crucial in mediating the development of acute lung injury induced by bilateral lower limb ischemia-reperfusion (I/R). Platonin, a potent antioxidant, possesses anti-inflammation capacity. We sought to elucidate whether platonin could mitigate acute lung injury induced by lower limb I/R. Materials and Methods. Forty-eight adult male rats were allocated to receive I/R, I/R plus platonin (100 mg/kg intravenous injection immediately after reperfusion), sham instrumentation, or sham instrumentation plus platonin (denoted as the I/R, I/R-platonin, Sham, or Sham-platonin group, respectively; n [ 12 in each group). Bilateral hind limb I/R was induced by applying rubber band tourniquets high around each thigh for 3 h followed by reperfusion for 3 h. After sacrifice, the degree of lung injury was determined. Results. Histologic findings revealed moderate inflammation in lung tissues of the I/R group and mild inflammation in those of the I/R-platonin group. Total cell number and protein concentration in bronchoalveolar lavage fluid as well as the leukocyte infiltration and myeloperoxidase activity in lung tissues of the I/R group were significantly higher than those of the I/Rplatonin group. The pulmonary concentrations of macrophage inflammatory protein-2, interleukin-6, and prostaglandin E2 of the I/R group were significantly higher than those of the I/R-platonin group. Moreover, the plasma nitric oxide concentration as well as the nitric oxide and malondialdehyde concentrations in
1 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, No. 289, Jianguo Rd., Sindian City, Taipei County 231, Taiwan. E-mail: [email protected].
lung tissues of the I/R group were significantly higher than those of the I/R-platonin group. Conclusions. Platonin mitigates acute lung injury induced by bilateral lower limb I/R in rats. Ó 2011 Elsevier Inc. All rights reserved.
Key Words: chemokine; cytokine; nitric oxide; prostaglandin E2.
INTRODUCTION
Acute lower limb ischemia is a common condition that can be observed in clinical situations such as embolism, atherosclerotic thrombosis, traumatic arterial injury, aortic clamping during abdominal aortic aneurysm repair, etc. [1–5]. However, reperfusion of the acutely ischemic lower limb can, in turn, induce reperfusion injury and cause remote vital organ dysfunctions [1, 6, 7]. Oxidative stress and inflammatory response have been shown to play an essential role in mediating the remote organ injury induced by lower limb ischemia-reperfusion [1, 6, 8]. Among the vital organs, lung is one of the most sensitive organs that is subject to the injuries induced by lower limb ischemia-reperfusion [1, 6, 8]. Platonin (4,40 ,400 -thrimethyl-3,30 ,300 -triheptyl-7-[200 trimethinethiazolocyanine-3-300 thia-zolyl]-2,20 diiodide), a cyanine photosensitizing dye and a potent antioxidant, possesses potent reactive oxygen and nitrogen species (ROS/RNS) scavenging capacity [9]. Platonin also has potent immunomodulating action and is currently used for the treatment of immune diseases, especially rheumatoid arthritis, in clinical settings [10]. In addition, platonin has been shown to possess significant anti-inflammation capacity [9, 11, 12]. For
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instance, platonin was reported to improve circulatory failure and mortality in septic rats [9]. Previous data also indicated that endotoxin-induced up-regulation of inflammatory molecules could be inhibited by platonin [11, 12]. However, the question of whether platonin could exert protective effects and attenuate acute lung injury induced by lower limb ischemia-reperfusion remains unstudied. This study was conducted to test the following hypotheses: (1) platonin could mitigate acute lung injury induced by lower limb ischemia-reperfusion in rats, and (2) the mechanisms may involve attenuating oxidative stress and decreasing inflammatory response. METHODS AND MATERIALS All animal studies were approved by the Institutional Animal Use and Care Committee of Buddhist Tzu Chi General Hospital, Taipei Branch, and the care and handling of the animals were in accordance with National Institutes of Health guidelines. All rats were fed a standard laboratory chow and were provided water ad libitum until the day of experiment. A total of 48 adult male Sprague-Dawley rats (200 to 250 g; BioLASCO Taiwan Co., Ltd, Taipei, Taiwan) were used for the experiments.
Blood Sample Collection and Arterial Blood Gas (ABG) Analysis At the end of each experiment, arterial blood (0.5 mL) was drawn. ABG levels were immediately measured with a blood gas analyzer (Rapidlab 348; Bayer Healthcare LLC, East Walpole, MA). In addition, another 5 mL of blood was withdrawn and centrifuged to separate the plasma. Plasma samples were stored at –80 C for subsequent analysis of nitric oxide (NO, the indicator of oxidative stress).
Lung Tissues Collection and Bronchoalveolar Lavage (BAL) At the end of each experiment, the left main bronchus was tied and left lung was removed. The upper and lower lobes of left lung were divided and the left lower lobe lung tissues were snap-frozen in liquid nitrogen and stored at –80 C for subsequent analysis. The right lung tissues of six rats from each group were infused with 4% formaldehyde through the tracheostomy tube and then removed. For the other six rats of each group, the right lung was lavaged with 6-mL aliquots of sterile saline for five times, as we have previously reported [13]. After the BAL fluid (BALF) was collected and recorded, an aliquot of the BALF was diluted 1:1 with trypan blue dye (Life Technologies, Grand Island, NY) and the total cell number was counted using a standard hemocytometer. The remaining BALF was centrifuged and the protein concentration of the supernatant was determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL).
Histologic Analysis Animal Preparation Under anesthesia (ketamine/xylazine: 110/10 mg/kg, intraperitoneal), two polyethylene (PE-50) catheters were placed in the right carotid artery for continuous blood pressure monitoring and the right external jugular vein for intravenous injection, respectively. After tracheostomy, a 14-gauge angiocatheter was inserted as a tracheostomy tube. Rats were mechanically ventilated (tidal volume: 4 mL of room air; rate: 35 breaths/min) with a small animal ventilator (Harvard Apparatus, South Natick, MA). Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored (BIOPAC System, Santa Barbara, CA) throughout the experiments. Supplemental dose of ketamine/xylazine mixture (30/3 mg/kg, intravenous) was administered every 1 h until the end of the experiment.
Lower Limb Ischemia-Reperfusion Protocols The lower limb ischemia-reperfusion injury protocol was adapted from a previous report [6]. In brief, bilateral hind limb ischemiareperfusion was induced by applying rubber band tourniquets high around each thigh for 3 h followed by reperfusion for 3 h.
Experimental Protocols Rats were randomly allocated to one of the four groups (n ¼ 12 in each group), i.e., the sham instrumentation, sham instrumentation plus platonin, ischemia-reperfusion, and ischemia-reperfusion plus platonin groups, and designated as the Sham, Sham-platonin, I/R, and I/R-platonin group, respectively. Rats of the I/R-platonin and Sham-platonin groups received intravenous injection of platonin (100 mg/kg dissolved in 0.5 mL normal saline) immediately after reperfusion or at comparable time point in those that received sham instrumentation. To control for the volume effects of vehicle, rats of the Sham and I/R groups also received 0.5 mL normal saline intravenous injection at comparable time point. The dosage of platonin (i.e., 100 mg/kg) employed in this study was determined accordingly to previous data that platonin at this dosage significantly protected septic rats from circulatory failure and mortality [9]. After reperfusion for 3 ho, all rats were sacrificed with high dose pentobarbital injection.
The formaldehyde-infused right lungs were embedded in paraffin wax, serial sectioned, and stained with hematoxylin and eosin. Histologic characteristics including edematous changes of the alveolar wall, hemorrhage, vascular congestion, and polymorphonuclear leukocytes (PMN) infiltration, were evaluated under a light microscope to evaluate pulmonary inflammation, as we have previously reported [13]. Each histologic characteristic was scored on a scale of 0-normal to 5-severe by a pathologist who was blinded to the experiment. The overall pulmonary inflammation was categorized according to the sum of the score (0–5: normal to minimal inflammation; 6–10: mild inflammation; 11–15: moderate inflammation; 16–20: severe inflammation). The degree of leukocyte infiltration can be quantified by PMN/alveoli ratio [13]. We thus counted PMNs and alveoli per high-power field (HPF, 4003) in 10 randomly selected areas of each sample. The PMNs/alveoli ratio was then calculated by dividing the sum of the PMNs in 10 HPFs by that of the alveoli.
Wet/Dry Weight Ratio and Myeloperoxidase (MPO) Activity Assay We measured the wet/dry weight ratio, i.e., the index of lung water, of the freshly harvested rat lung samples (i.e., left upper lobe) to evaluate pulmonary inflammation. In brief, the whole lobe was weighed and then placed in the oven for 24 h at 60 C, and weighed again when it was dry, as we have previously reported [13]. The wet/dry weight ratio was then calculated. We also quantified pulmonary inflammation by measuring pulmonary MPO activity. The protocol was modified from our previous report.[13] In brief, snap-frozen tissue samples (–80 C) were homogenized, re-suspended, sonicated, and centrifuged. The supernatant was collected and incubated in a water bath for 2 h at 60 C. MPO activity was then measured.
Inflammatory Molecules Snap-frozen lung tissues were processed, as we have previously reported [13]. The pulmonary concentrations of the inflammatory molecules, including chemokine (e.g., macrophage inflammatory
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RESULTS
protein-2, MIP-2), cytokine (e.g., interleukin-6, IL-6), and prostaglandin E2 (PGE2), were measured using enzyme-linked immunosorbent assay (ELISA) (MIP-2 ELISA kit; R and D Systems, Inc., Minneapolis, MN; ELISA Kits for IL-6 and PGE2; Pierce).
Reverse Transcription and Polymerase Chain Reaction (RT-PCR) Production of PGE2 is mediated by cyclooxygenase 2 (COX-2) [14]. Transcriptional expression of COX-2 of the harvested lung tissues was measured using RT-PCR. The primer sequences and amplification protocols for COX-2 and b-Actin (as an internal standard) were adapted from those previously published ones [15, 16]. After separation, PCR-amplified cDNA band densities were quantified using densitometric techniques (Scion Image for Windows; Scion Corp, Frederic, MD).
NO Assay for Oxidative Status Evaluation Process of the lung tissues samples were performed as we have previously reported [13]. Then the plasma and pulmonary concentrations of stable NO metabolites, nitrite and nitrate, were measured using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).
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Hemodynamics
The baseline HR and MAP values of these four groups were comparable (data not shown). HR and MAP of the Sham and Sham-platonin groups remained stable throughout the experiment. The HR and MAP values measured at the end of the experiment (i.e., the end HR and MAP) of the Sham and Sham-platonin groups were also comparable. In contrast, the end HR values of the I/R and I/R-platonin groups were significantly higher than that of the Sham group (P ¼ 0.035 and 0.031, respectively; Table 1) whereas the end MAP values of the I/R and I/R-platonin groups were significantly lower than that of the Sham group (P ¼ 0.029 and 0.033, respectively; Table 1). In addition, the end HR and MAP values of the I/R and I/R-platonin groups were comparable (Table 1). ABG Data
Malondialdehyde (MDA) Assay for Lipid Peroxidation Status Evaluation For MDA assay, snap-frozen lung tissues were thawed immediately before assaying. The protocol of MDA assay was modified from our previous report [17]. In brief, tissues homogenates were collected. Then, phosphoric acid and thiobarbituric acid solution were added to 0.5 mL of homogenates. The mixture was heated in boiling water for 45 min. After cooling, the absorbance was measured and the amounts of lipid peroxides were calculated.
Statistical Analysis One-way analysis of variance with the Bonferroni test was used for multiple comparisons. Data were presented as means 6 standard deviations. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL) was used for data analysis.
The baseline ABG values, including pH, PaO2, PaCO2, and base excess (BE), of these four groups were comparable (data not shown). The end ABG values of the Sham and Sham-platonin groups were also comparable (Table 1). In contrast, the end pH, PaO2, and BE values of the I/R and I/R-platonin groups were significantly lower than those of the Sham group (all P < 0.02; Table 1), whereas the end PaCO2 values of the I/R and I/R-platonin groups were significantly higher than that of the Sham group (both P < 0.015; Table 1). Moreover, the end values of pH, PaO2, and BE of the I/R-platonin group were significantly higher than those of the I/R group (P ¼ 0.028, 0.015, and 0.033, respectively; Table 1). The end PaCO2 value of the I/R-platonin group, in contrast, was significantly lower than that of the I/R group (P ¼ 0.031; Table 1).
TABLE 1 Hemodynamics and Arterial Blood Gas Data at the End of Experiment Hemodynamics
Arterial blood gas
Group (n ¼ 12)
HR (beats/min)
MAP (mm Hg)
pH
PaO2 (mm Hg)
PaCO2 (mm Hg)
Sham Sham-platonin I/R I/R-platonin
317 6 21 309 6 18 346 6 22* 338 6 17*
105 6 12 112 6 10 84 6 13* 87 6 12*
7.43 6 0.08 7.40 6 0.05 7.26 6 0.04* 7.35 6 0.04*,y
96 6 5 99 6 7 70 6 4* 86 6 5*,y
41 6 4 38 6 6 56 6 4* 48 6 3*,y
Sham ¼ the sham instrumentation group. Sham-platonin ¼ the sham instrumentation plus platonin group. I/R ¼ the lower limb ischemia-reperfusion group. I/R-platonin ¼ the lower limb ischemia-reperfusion plus platonin group. HR ¼ heart rate. MAP: mean arterial pressure. Data are means 6 standard deviations. * P < 0.05 versus Sham group. y P < 0.05 I/R-platonin group versus I/R group.
Base Excess (mM) 1.8 6 2.9 2.3 6 1.5 7.6 6 1.6* 4.9 6 1.7*,y
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BALF Data
Pulmonary Concentrations of Inflammatory Molecules
The total cell number and total protein concentration in BALF of the Sham and Sham-platonin groups were low (Fig. 1A and B). The total cell number and total protein concentration of the I/R group were significantly higher than those of the Sham group (P ¼ 0.020 and 0.017, respectively; Fig. 1A and B). In contrast, the total cell number and total protein concentration of the I/Rplatonin group were significantly lower than those of the I/R group (P ¼ 0.029 and 0.025, respectively; Fig. 1A and B).
The pulmonary MIP-2, IL-6, PGE2, and COX-2 mRNA concentrations of the Sham and Shamplatonin groups were low (Fig. 4A, B, and C). The MIP-2, IL-6, PGE2, and COX-2 mRNA concentrations of the I/R group were significantly higher than those of the Sham group (all P < 0.001; Fig. 4A, B, and C). In contrast, the MIP-2, IL-6, PGE2, and COX-2 mRNA concentrations of the I/R-platonin group were significantly lower than those of the I/R group (P ¼ 0.028, 0.021, 0.036, and 0.025, respectively; Fig. 4A, B, and C). NO and MDA Assays
Lung Histology and Pulmonary Inflammation Score
Histologic analysis revealed normal to minimal inflammation in the lung tissues of the Sham and Sham-platonin groups (Fig. 2A and B). The lung tissues of the I/R group revealed moderate inflammation (Fig. 2C), whereas those of the I/R-platonin group revealed mild inflammation (Fig. 2D). Findings of the pulmonary inflammation score paralleled the findings of the histological analysis (Fig. 3A).
Wet/Dry Weight Ratio, PMNs/Alveoli Ratio, and MPO Activity
The wet/dry weight ratio, PMNs/alveoli ratio, and MPO activity in lung tissues of the Sham and Shamplatonin groups were low (Fig. 3B, C, and D). The wet/dry weight ratio, PMNs/alveoli ratio, and MPO activity of the I/R group were significantly higher than those of the Sham group (P ¼ 0.028, 0.015, and 0.011, respectively; Fig. 3B, C, and D). In contrast, the PMNs/alveoli ratio and MPO activity of the I/Rplatonin group were significantly lower than those of the I/R group (P ¼ 0.026 and 0.032, respectively; Fig. 3C and D), whereas the wet/dry weight ratio of these two groups were comparable (Fig. 3)B.
The plasma NO concentrations of the Sham and Sham-platonin groups were low (Fig. 5A). The pulmonary NO and MDA concentrations of the Sham and Sham-platonin groups were also low (Fig. 5B and C). The plasma NO concentration of the I/R group was significantly higher than that of the Sham group (P < 0.001; Fig. 5A). Similarly, the pulmonary NO and MDA concentrations of the I/R group were significantly higher than those of the Sham group (P ¼ 0.017 and 0.021, respectively; Fig. 5B and C). In contrast, the plasma NO concentration of the I/R-platonin group was significantly lower than that of the I/R group (P ¼ 0.021; Fig. 5A). The pulmonary NO and MDA concentrations of the I/R-platonin group were also significantly lower than those of the I/R group (P ¼ 0.030 and 0.026, respectively; Fig. 5B and C). DISCUSSION
Data from this study, in concert with those previous ones [1, 6, 8], confirmed that lower limb ischemiareperfusion could induce acute lung injury in rats. In this study, we demonstrated that the acute lung injury induced by lower limb ischemia-reperfusion could be mitigated by platonin. As platonin is currently used in clinical settings [10], data from this study could have
FIG. 1. (A)The total cell number and (B) the total protein concentration in bronchoalveolar lavage fluid (BALF) collected from rats of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
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FIG. 2. Microscopic findings of the lung tissues stained with hematoxylin and eosin (2003). (A) Representative microscopic findings of the sham instrumentation (Sham) group. (B) Representative microscopic findings of the Sham plus platonin (Sham-platonin) group. (C) Representative microscopic findings of the lower limb ischemia-reperfusion (I/R) group. (D) Representative microscopic findings of the I/R plus platonin (I/R-platonin) group.
direct clinical implications. Judging from our data, we believe that incorporation of platonin as part of the therapies should be beneficial in clinical situations associated with lower limb ischemia-reperfusion.
The notion that lower limb-ischemia could exert oxidative stress in rats [6, 8] was confirmed by this study, as our data revealed that the systemic NO concentrations as well as the pulmonary NO and
FIG. 3. (A) The pulmonary inflammation scores, (B) the wet/dry weight ratio, (C) the polymorphonuclear leukocytes (PMN)/alveoli ratio, and (D) the myeloperoxidase (MPO) activity in lung tissues harvested from rats of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
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FIG. 4. The concentrations of (A) macrophage inflammatory protein-2 (MIP-2), (B) interleukin-6 (IL-6), and (C) prostaglandin E2 (PGE2), and cyclooxygenase 2 (COX-2) mRNA in lung tissues harvested from rats of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
MDA concentrations were significantly increased in rats experiencing lower limb ischemia-reperfusion. It is well established that bursts of ROS/RNS could imbalance the cellular redox condition and lead to damages of the vital pathways, including energy metabolism, survival/stress responses, apoptosis, etc. [18]. Moreover, bursts of ROS/RNS may induce inflammatory response, including neutrophil activation and cytokine produc-
tion [19]. In line with this notion, it has been shown that lower limb ischemia-reperfusion could induce inflammatory response [6, 8]. This notion was also confirmed by this study, as our data revealed significant increases in pulmonary leukocyte infiltration (evidenced by histology and MPO assay) and up-regulation of pulmonary inflammatory molecules (e.g., chemokine, cytokine, and PGE2) in rats
FIG. 5. (A) The plasma nitric oxide (NO) concentration, (B) the pulmonary NO concentration, and (C) the pulmonary malondialdehyde (MDA) concentration of the sham instrumentation (Sham), Sham plus platonin (Sham-platonin), lower limb ischemia-reperfusion (I/R), and I/R plus platonin (I/R-platonin) groups. Data are means 6 standard deviations. *P < 0.05 versus the Sham group. yP < 0.05 the I/R-platonin group versus the I/R group.
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experiencing lower limb ischemia-reperfusion. It has been shown that oxidants (especially NO), infiltrated leukocytes, and inflammatory molecules can work together to injure pulmonary microvascular endothelial cell and result in the loss of microvascular integrity and eventually lead to gas exchange impairment and pulmonary dysfunction during ischemia-reperfusion injury [20, 21]. This notion was supported by our data, as we observed a significant decrease in PaO2 and a significant increase in PaCO2 in rats experiencing lower limb ischemia-reperfusion. Platonin possesses potent ROS/RNS scavenging capacity and anti-inflammation capacity [9]. Our data revealed that rats of the I/R-platonin group had higher PaO2 and lower PaCO2 levels compared with those of the I/ R group, indicating that the gas exchange impairment induced by lower limb ischemia-reperfusion could be mitigated by platonin. Moreover, data from this study revealed that rats of the I/R-platonin group had lower systemic NO concentration, pulmonary NO and MDA concentrations, pulmonary leukocyte infiltration, and pulmonary inflammatory molecules concentrations compared with those rats of the I/R group. These data thus provided clear evidence to confirm our hypothesis that platonin could act through decreasing the oxidative stress and ameliorating the inflammatory response to exert its protective effects against acute lung injury induced by lower limb ischemia-reperfusion. It is well established that expression of inflammatory molecules is regulated by the crucial transcription factors, including nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) [22, 23]. In addition, expression of AP1 is under the regulation of mitogen-activated protein kinases (MAPKs) [24]. The question of whether lower limb ischemia-reperfusion induces significant upregulation of NF-kB, MAPKs, and AP-1 remains unstudied. NF-kB, MAPKs, and AP-1 are oxidative stress-responsive transcription factors and their expression can be up-regulated by oxidative stress [25, 26]. Moreover, previous data also confirmed the crucial roles of NF-kB, MAPKs, and AP-1 in regulating the expression of inflammatory molecules in other forms of ischemia-reperfusion [27–29]. Judging from these data, we speculate that lower limb ischemiareperfusion may very likely induce the up-regulation of NF-kB, MAPKs, and AP-1. As aforementioned, our data confirmed that the oxidative stress and pulmonary inflammatory response induced by lower limb ischemia-reperfusion could be attenuated by platonin. The question of whether NF-kB, MAPKs, and AP-1 are involved in mediating the observed effects of platonin also remains unstudied. Previous data indicated that platonin posted significant effects on regulating the expression of NF-kB [30]. Our recent data also confirmed that platonin posted significant effects on
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regulating the expression of MAPKs and AP-1 [31]. Judging from these data, we further speculate that platonin may very likely act through inhibiting the upregulation of NF-kB, MAPKs, and/or AP-1 to exert its protective effects in this regard. Previous data, reported by Hsiao et al., indicated that platonin could significantly attenuate hemodynamic changes in septic rats [9]. Judging from those previous data, one would expect that platonin should exert similar effects in rats experiencing lower limb ischemiareperfusion. However, to our surprise, our data revealed that the end MAP and HR of the I/R and I/R-platonin groups were comparable. It has been postulated that it takes approximately 1 h before the anti-inflammation effects of platonin to come to effect [32]. Therefore, it is likely that the discrepancy between our data and those reported by Hsiao et al. is related to the difference in the timing of platonin administration, as Hsiao et al. chose to administer platonin before endotoxin installation (i.e., pretreatment) and we chose to administer platonin immediately after reperfusion (i.e., cotreatment), instead. Moreover, data from this study confirmed the protective effects of platonin against acute lung injury induced by lower limb ischemia-reperfusion. However, the protective effects of platonin observed in this study were not potent enough to totally reverse the detrimental effects of lower limb ischemia-reperfusion. As the therapeutic efficacy of platonin is affected by the timing of administration [32], it is likely that platonin can exert stronger therapeutic effects in this regard if platonin is administered before reperfusion and/or ischemia. The therapeutic efficacy of platonin is also dose-dependent [11]. The preliminary data observed in this study revealed that platonin at the dosages of 25 and 50 mg/kg exerted no significant effects on ameliorating the upregulation of pulmonary inflammatory molecules as well as the increases in total cell number and protein concentration in BALF induced by lower limb ischemia-reperfusion (data not shown). Judging from these data, we thus speculate that larger dosages of platonin (e.g., >100 mg/kg) may very likely exert stronger therapeutic effects in this regard. More studies are needed before further conclusions can be drawn. Certain study limitations do exist. First, this study focused on the early phase of reperfusion, and the long-term effects of platonin on protecting lung tissues against lower limb ischemia-reperfusion injury remain to be elucidated. Second, interspecies differences should be taken into consideration if further data interpretation is intended. In summary, platonin mitigates acute lung injury induced by bilateral lower limb ischemia-reperfusion in rats. The mechanisms may involve decreasing oxidative stress and attenuating inflammatory response induced by lower limb ischemia-reperfusion.
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JOURNAL OF SURGICAL RESEARCH: VOL. 167, NO. 2, MAY 15, 2011
ACKNOWLEDGMENTS This work was supported by grants from the Buddhist Tzu Chi General Hospital, Taipei Branch (TCRD-TPE-99-28) awarded to K-Y H and the National Science Council, Taiwan (NSC 98-2314-B-303-012MY3) awarded to C-J H.
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