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resuscitation in rats
Cytokine xxx (2015) xxx–xxx
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Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats Ming-Chang Kao a,b,c, Chen-Hsien Yang b,c, Joen-Rong Sheu a,⇑, Chun-Jen Huang a,b,c,⇑ a
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Anesthesiology, Taipei Tzu Chi Hospital, Taipei, Taiwan c School of Medicine, Tzu Chi University, Hualien, Taiwan b
a r t i c l e
i n f o
Article history: Received 18 May 2015 Received in revised form 2 September 2015 Accepted 8 September 2015 Available online xxxx Keywords: Anti-inflammatory Tumor necrosis factor-a Interleukin Heme oxygenase-1 Tin protoporphyrin
a b s t r a c t Background: Cepharanthine possesses strong anti-inflammation capacity. We sought to clarify whether cepharanthine could mitigate pro-inflammatory cytokine production in acute lung injury induced by hemorrhagic shock/resuscitation (HS/RES). The involvement of heme oxygenase-1 (HO-1) was also investigated. Methods: Male Sprague Dawley rats were allocated to receive HS/RES, HS/RES plus iv cepharanthine or HS/RES plus cepharanthine plus the HO-1 activity inhibitor tin protoporphyrin (SnPP) and denoted as the HS/RES, HS/RES + CEP, and HS/RES + CEP + SnPP group, respectively. HS/RES was achieved by blood drawing to lower mean arterial pressure (40–45 mmHg for 60 min) followed by shed blood/saline mixtures re-infusion. The rats were monitored for another 5 h before sacrifice. Results: Arterial blood gas, lung permeability and histologic assays (including histopathology, neutrophil infiltration, and lung water content) confirmed that HS/RES induced significant lung injury. Significant increases in pulmonary levels of tumor necrosis factor-a, interleukin-1b, interleukin-6, prostaglandin E2 and cyclooxygenase-2 confirmed that HS/RES induced a significant inflammatory response in the lungs. Cepharanthine significantly attenuated the pulmonary pro-inflammatory cytokine production and lung injury induced by HS/RES. However, the protective effects of cepharanthine were blocked by SnPP, the potent HO-1 activity inhibitor. Conclusion: Cepharanthine significantly mitigates pro-inflammatory cytokine response in acute lung injury induced by HS/RES in rats. The mechanism may involve the HO-1 pathway. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Hemorrhagic shock followed by resuscitation (HS/RES) are two early pathophysiological phases in the sequela of the clinical reversal of hemorrhage [1]. Hemorrhagic shock induces tissue ischemia and oxidative stress. Reperfusion induced by resuscitation causes the release of toxic mediators from ischemic tissues and the subsequent triggering of inflammation cascades [2], which in turn leads to additional local and distal tissue injury. In the process, the free heme released from damaged erythrocytes catalyzes the formation of reactive oxygen species and greatly increases cell dysfunction [3–5]. Under physiological conditions the enzymes of heme ⇑ Corresponding authors at: Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing St., Taipei 110, Taiwan. (J.-R. Sheu). Department of Anesthesiology, Taipei Tzu Chi Hospital, 289, Jianguo Rd., Sindian District, New Taipei City 231, Taiwan (C.-J. Huang). E-mail addresses: [email protected] (M.-C. Kao), [email protected] (C.-H. Yang), [email protected] (J.-R. Sheu), [email protected] (C.-J. Huang).
oxygenase (HO) tightly regulate the catabolism of heme to produce metabolites that possess anti-oxidative and anti-inflammatory properties [6,7]. The lung tissues, being a downstream filter that receives the entire cardiac output, are most vulnerable to the toxic metabolites released from ischemic organs after HS/RES [8]. HS/RES readily upregulates the expression of inflammatory molecules in lung tissues, which leads to overt pulmonary inflammation and the progression of lung injury [9–12]. Therapies aimed at attenuating pulmonary inflammation may have beneficial consequences against lung injury induced by HS/RES [8]. Cepharanthine is a biscoclaurine alkaloid derived from Stephania cepharantha, a plant of the Menispermaceae family [13]. Cepharanthine possesses potent in vivo and in vitro antineoplastic, immunomodulatory and anti-inflammation effects [14–18] and has been used clinically to successfully treat radiation-induced leukopenia, hair loss, bronchial asthma, and certain types of allergic inflammation [19,20]. Moreover, in animal
http://dx.doi.org/10.1016/j.cyto.2015.09.008 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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models of sepsis cepharanthine can exert protective effects and mitigate inflammatory responses [21,22]. To date, the question of whether cepharanthine can mitigate pro-inflammatory cytokine response in acute lung injury induced by HS/RES remains unstudied. To examine this hypothesis, we conducted this study using a rodent model of HS/RES. Moreover, it has been shown that induction of heme oxygenase-1 (HO-1) exerts protective effects against HS/RES [23]. We also examined whether HO-1 was involved in mediating the anti-inflammatory effect of cepharanthine. 2. Materials and methods 2.1. Animal preparation Ninety male Sprague–Dawley rats (BioLASCO Taiwan Co., Ltd, Taipei, Taiwan) weighing 250–300 g were used in the experiments. All animal experiments were approved by Institutional Animal Use and Care Committee, Taipei Tzu Chi Hospital (100-IACUC No. 003). All experiments were done in accordance with the guidelines of the National Institutes of Health. The rats were anesthetized using an intramuscular injection of a ketamine/xylazine mixture (110/10 mg/kg respectively). Additional doses of ketamine/xylazine mixture (30/3 mg/kg respectively) were given hourly until the end of the experiment. The rats were placed supine on a heating pad. A rectal temperature probe was inserted and the body temperature was maintained at 37 °C throughout the experiments using the heating pad and heating lamps. Polyethylene catheters (PE-50, Becton Dickinson, Sparks, MD, USA) were inserted into the right femoral artery for blood pressure monitoring and the left femoral vein for blood withdrawal and intravenous (iv) injection, respectively. A tracheostomy was performed to allow airway clearance. 2.2. HS/RES protocols Protocols of HS/RES were adapted from our previous study [24]. In brief, hemorrhagic shock was achieved by blood drawing over 10 min to reduce mean arterial pressure (MAP; BIOPAC System, Santa Barbara, CA, USA) from the physiologic level to 40– 45 mmHg. The shed blood was stored in a syringe containing 20 units of heparin at room temperature. This lowered MAP was then kept for 60 min by drawing or re-infusing blood as needed. Resuscitation was achieved by re-infusing the shed blood, supplemented with twice the maximum blood volume drawn of normal saline over a 10-min period. All rats were monitored for another 300 min. 2.3. Experimental protocols The rats were randomized to 5 experimental groups (n = 18 in each group). The Sham group received sham operation (i.e., cannulation of vessels and tracheostomy) plus a 30 lL intravenous injection (iv) of dimethylsulfoxide (DMSO) vehicle (Sigma, St Louis, Mo, USA). The Sham + CEP group received sham operation plus cepharanthine (5 mg/kg, iv; LKT Laboratories, Inc. St. Paul, MN, USA). The HS/RES group received HS/RES plus vehicle. The HS/RES + CEP group received HS/RES plus cepharanthine (5 mg/kg, iv). The HS/RES + CEP + SnPP group received HS/RES plus cepharanthine (5 mg/kg, iv) plus the potent HO-1 enzymatic activity inhibitor tin protoporphyrin (SnPP, 30 mg/kg, iv; Enzo Life Sciences, Farmingdale, NY, USA). All rats received intravenous injection of vehicle, cepharanthine, or cepharanthine plus SnPP immediately before resuscitation or at corresponding time points in the Sham groups. At 300 min following resuscitation, arterial blood (0.5 mL) was drawn from 12 randomly selected rats in each group. Arterial blood
gas (ABG) was immediately analyzed with a blood gas analyzer (Gem Premier 3000; Instrumentation Laboratory, Bedford, MA, USA). The lung samples were then harvested immediately after euthanization with pentobarbital (100 mg/kg, iv). The remaining 6 rats of each group were used for lung permeability measurements.
2.4. Lung tissues collection and bronchoalveolar lavage (BAL) For the above-mentioned 12 rats from each group, the left main bronchus was tied and the left lung was excised. The superior and inferior lobes of the left lung were separated, and the inferior lobe was snap frozen in liquid nitrogen and stored at 80 °C. The left superior lobe was used for wet/dry weight ratio measurement. Then, 6 of these 12 rats from each group received right lung perfusion with 4% formaldehyde and then excised. For the remaining 6 rats, the right lung was lavaged 5 times with 3 mL sterile saline and the BAL fluid (BALF) was collected [24]. An aliquot of BALF was diluted 1:1 with trypan blue dye for counting total cell number. The remaining BALF was collected and centrifuged. The protein concentration of the supernatant was measured using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, IL, USA).
2.5. Histologic analysis The formalin-fixed and paraffin-embedded lung tissues were serial sectioned and stained with hematoxylin and eosin. Histologic features including alveolar wall edema, vascular congestion, hemorrhage, and polymorphonuclear (PMN) leukocyte infiltration were examined under a light microscope using our previously published protocol [24]. Each histologic feature was scored on a 5-grade scale: 0 (normal) to 5 (severe). The overall lung injury in each rat was classified as normal to minimal when the sum of the scores was 0–5, mild when 6–10, moderate when 11–15 and severe when 16–20.
2.6. Wet/dry weight ratio and myeloperoxidase (MPO) activity assay Wet/dry weight ratio (i.e., lung water content) and MPO activity (i.e., quantification of tissue PMN accumulation) were analyzed by protocols we have previously described [25]. The freshly harvested left superior lobe was weighed and then dried in the oven at 80 °C for 24 h. The lobe was then weighed again in dry condition. The wet/dry weight ratio was then calculated. For MPO activity assay, the snap-frozen lung tissues were homogenized and centrifuged. The suspension was then sonicated and the supernatant was obtained and incubated in a water bath at 60 °C for 2 h. MPO activity was measured using a MPO fluorometric detection kit (Enzo Life Science) according to the manufacturer’s instructions.
2.7. Lung permeability Lung permeability was determined by the Evans blue dye (EBD) extravasation [26]. In brief, rats received 30 mg/kg (iv) of EBD (Sigma) at 300 min after resuscitation. A blood sample (1 mL) was drawn at 5 min after EBD injection to determine the plasma EBD concentration. The rats were then euthanized at 20 min after EBD injection and BAL was performed, as above-mentioned. The collected BALF was then centrifuged at 1500 rpm at 4 °C for 10 min. The EBD concentrations of the BALF and the plasma were then analyzed by spectrophotometry at 620 nm. The EBD concentration of the BALF was then compared to that of the plasma to determine lung permeability.
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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2.8. Inflammatory molecules For inflammatory molecule assay, the snap-frozen lung tissues were processed by a protocol we have previously described [24]. The concentrations of tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1b), interleukin-6 (IL-6) and prostaglandin E2 (PGE2) were determined using enzyme-linked immunosorbent assay kits (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. 2.9. Reverse transcription-polymerase chain reaction (RT-PCR) Transcriptional expression of cyclooxygenase-2 (COX-2), i.e., the enzyme regulating PGE2 expression, and HO-1 of the lung tissues were determined using RT-PCR. The protocols of primer sequences and amplification for COX-2, HO-1, and b-Actin were performed according to our previous report [27]. After separation, band intensities of PCR-amplified cDNA were quantified by densitometric techniques (Scion Image for Windows, Scion Corp, Frederic, MD, USA). 2.10. HO activity assay HO activity was analyzed from the microsomal fraction by measuring the formation of bilirubin, using a protocol we have previously described [5]. The lung tissues were homogenized and the homogenates were centrifuged. After disposing of the supernatant, the microsomal pellet was re-suspended in a solution of 100 mM potassium phosphate buffer and 2 mM MgCl2 (pH 7.4). The bilirubin concentration was measured as difference in absorbance between 450 and 600 nm and was calculated with an excitation coefficient of 27.3 mM1cm1. HO activity was expressed as pmol of bilirubin formed per mg of tissue protein. Protein concentration was measured using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, IL, USA). 2.11. Statistical analysis Data were expressed as means ± standard deviations. Differences among groups were tested using one-way analysis of variance (ANOVA). If one-way ANOVA revealed significant differences among groups, then the post-hoc tests with the Bonferroni multiple comparison tests were performed. Differences were considered significant at P < 0.05. 3. Results 3.1. Hemodynamic data One-way ANOVA revealed that the baseline MAP and heart rate (HR) were not significantly different among these five groups (data not shown). The MAP and HR of the Sham and the Sham + CEP groups were stable during the whole experiment. One-way ANOVA revealed significant group differences in the end MAP (P < 0.001) and HR (P < 0.001). Multiple comparison tests revealed that the end MAP and HR of the HS/RES group were significantly lower than those of the Sham group (both P < 0.001, Table 1). Moreover, the end MAP and HR of the HS/RES + CEP group were significantly higher than those of both the HS/RES group (both P < 0.001, Table 1) and the HS/RES + CEP + SnPP group (both P < 0.001, Table 1). 3.2. ABG data The ABG data including the values of pH, PaO2, PaCO2 and base excess (BE) were measured. One-way ANOVA revealed significant
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group differences in the pH, PaO2, BE and PaCO2 (all P < 0.001). Multiple comparison tests revealed that the pH, PaO2, and base excess (BE) of the Sham and the Sham + CEP groups were not significantly different (Table 1). The pH, PaO2 and BE of the HS/RES group were significantly lower than those of the Sham group (all P < 0.001, Table 1). The pH, PaO2 and BE values of the HS/RES + CEP group were significantly higher than those of the HS/RES group (all P < 0.001), whereas the PaCO2 values between the two groups were not significantly different (Table 1). In addition, the pH, PaO2, and BE values of the HS/RES + CEP + SnPP group were significantly lower than those of the HS/RES + CEP group (all P < 0.001), whereas the PaCO2 value of the HS/RES + CEP + SnPP group was significantly higher than that of the HS/RES + CEP group (P < 0.001, Table 1). 3.3. Histologic lung injury score Histologic analysis disclosed normal to minimal lung injury in rats of the Sham and the Sham + CEP groups (Fig. 1A and B). Histologic analysis also disclosed moderate to severe lung injury in rats of the HS/RES and the HS/RES + CEP + SnPP groups (Fig. 1C and E) and mild injury in rats of the HS/RES + CEP group (Fig. 1D). Lung injury scores (Fig. 1F) basically paralleled the histologic analysis. 3.4. BALF analysis, wet/dry weight ratio, MPO activity and lung permeability One-way ANOVA revealed a significant group difference in the BALF total cell numbers (P < 0.001). The BALF total cell numbers of the Sham and the Sham + CEP groups were low (Fig. 2A). Multiple comparison tests revealed that the BALF total cell numbers of the HS/RES group was significantly higher than that of the Sham group (P < 0.001, Fig. 2A). In contrast, the BALF total cell numbers of the HS/RES + CEP group was significantly lower than of the HS/RES group (P < 0.001, Fig. 2A). However, the BALF total cell numbers of the HS/RES + CEP + SnPP group was significantly higher than that of the HS/RES + CEP group (P = 0.005, Fig. 2A). Data of the BALF protein concentration (Fig. 2B), wet/dry weight ratio (Fig. 2C), MPO activity (Fig. 2D) and lung permeability (Fig. 2E) essentially matched the data of the BALF total cell numbers (Fig. 2A), except that the wet/dry weight ratio of the HS/RES and the HS/RES + CEP groups were not significantly different (Fig. 2C). 3.5. Inflammatory molecules data One-way ANOVA revealed a significant group difference in the pulmonary TNF-a concentration (P < 0.001).The pulmonary TNF-a concentration of the Sham and the Sham + CEP groups were low (Fig. 3A). As expected, multiple comparison tests revealed that the pulmonary TNF-a concentration of the HS/RES group was significantly higher than that of the Sham group (P < 0.001, Fig. 3A). Moreover, the pulmonary TNF-a concentration of the HS/RES + CEP group was significantly lower than that of the HS/RES group (P < 0.001, Fig. 3A). In contrast, the pulmonary TNF-a concentration of the HS/RES + CEP + SnPP group was significantly higher than that of the HS/RES + CEP group (P < 0.001, Fig. 3A). Data of the pulmonary concentrations of IL-1b (Fig. 3B), IL-6 (Fig. 3C), COX-2 mRNA (Fig. 3D) and PGE2 (Fig. 3E) essentially matched the data of TNF-a (Fig. 3A). 3.6. HO-1 expression One-way ANOVA revealed significant group differences in the pulmonary HO-1 mRNA and HO activity (P < 0.001). The pulmonary concentrations of HO-1 mRNA and HO activity of the Sham group were low. Multiple comparison tests revealed that the
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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Table 1 Hemodynamic and arterial blood gas data at the end of experiment. Group (n = 12)
MAP (mmHg)
HR (beats/min)
pH
PaO2 (mm Hg)
PaCO2 (mm Hg)
Base excess (mM)
Sham Sham + CEP HS/RES HS/RES + CEP HS/RES + CEP + SnPP
113 ± 13 112 ± 15 83 ± 3* 103 ± 10# 81 ± 3*,y
332 ± 14 348 ± 16 266 ± 11* 294 ± 21*,# 250 ± 7*,y
7.26 ± 0.04 7.26 ± 0.03 7.15 ± 0.03* 7.23 ± 0.04# 7.14 ± 0.05*,y
130 ± 11 126 ± 10 99 ± 8* 123 ± 7# 88 ± 7*,#,y
47 ± 3 47 ± 3 45 ± 2 44 ± 2 49 ± 3#,y
8.1 ± 1.4 7.9 ± 0.9 12.6 ± 1.4* 8.8 ± 1.2# 14.5 ± 1.8*,#,y
Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. HR: heart rate. MAP: mean arterial pressure. Data were means ± standard deviations. * P < 0.05 vs. the Sham group. # P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. y P < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
Fig. 1. Representative microscopic findings of the lung tissues stained with hematoxylin-eosin (10) and the lung injury scores (n = 6 in each group). (A) The Sham group. (B) The Sham plus cepharanthine (Sham + CEP) group. (C) The hemorrhagic shock/resuscitation (HS/RES) group. (D) The HS/RES + CEP group. (E) The HS/RES + CEP plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (HS/RES + CEP + SnPP) group. (F) The lung injury scores. DMSO: dimethylsulfoxide. Data were means ± standard deviations. ⁄P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/ RES + CEP group.
pulmonary concentrations of HO-1 mRNA and HO activity of the Sham + CEP group were significantly higher than those of the Sham group (P < 0.001 and =0.013, Fig. 4A and B). The pulmonary concentrations of HO-1 mRNA and HO activity of the HS/RES group were also significantly higher than those of the Sham group (both P < 0.001, Fig. 4A and B). Moreover, the pulmonary concentrations of HO-1 mRNA and HO activity of the HS/RES + CEP group were significantly higher than those of the HS/RES group (P = 0.028 and 0.002, Fig. 4A and B). The pulmonary HO activity of the HS/RES + CEP group was significantly higher than that of the HS/RES + CEP + SnPP group (P = 0.002, Fig. 4B), whereas the pulmonary HO-1 mRNA concentrations of the two groups were not significantly different (P = 0.995, Fig. 4A). 4. Discussion This study replicates the findings of earlier studies [8,9,24], and demonstrates conclusively that this model of HS/RES causes increases in pulmonary pro-inflammatory cytokines and acute lung injury in rats. The situation closely mimics the clinical
situation of the early phase of hemorrhagic shock. The novel finding of our study is that iv cepharanthine administered immediately before resuscitation significantly ameliorated all experimental indices of pulmonary inflammation and lung injury caused by HS/RES. Moreover, our data demonstrates the active involvement of the HO-1 dependent pathway in mediating the protective effects of cepharanthine against HS/RES. The shock event and subsequent post-resuscitation event are two dependent yet distinct patho-physiologies of the early phase of hemorrhagic shock [1]. Tissue ischemia induced by the hemorrhagic event and subsequent reperfusion event, performed as a component procedure of resuscitation, can lead to the release of toxic mediators from ischemic tissues and subsequently trigger the local and distal inflammation cascades [2,28]. The resultant systemic pro-inflammatory response from both cascades can cause persistent hypotension, despite resuscitation [29,30]. Hemodynamics and anatomy combine to determine that the lung tissues are extremely vulnerable to the insults of HS/RES. Synergistic interaction between local pulmonary inflammatory molecules and the infiltrated neutrophils can damage pulmonary microvascular
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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Fig. 2. (A) The total cell number in bronchoalveolar lavage fluid (BALF; n = 6 in each group), (B) the total protein concentration in BALF (n = 6), (C) the wet/dry weight ratio of the lungs (n = 12), (D) the myeloperoxidase (MPO) activity of the lungs (n = 12) and (E) the lung permeability (n = 6). Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. DMSO: dimethylsulfoxide. Data were means ± standard deviations. ⁄ P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
Fig. 3. The concentrations of (A) tumor necrosis factor-a (TNF-a), (B) interleukin-1b (IL-1b), (C) IL-6, (D) cyclooxygenase-2 (COX-2) mRNA and (E) prostaglandin E2 (PGE2) in rat lungs (n = 12 in each group). Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. DMSO: dimethylsulfoxide. Data were means ± standard deviations. ⁄P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/ RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
endothelium and exacerbate acute lung injury that affecting gas exchange [8]. The impaired gas exchange in addition to the postresuscitation hypotension may elicit a vicious cycle of systemic and pulmonary pro-inflammatory response. In agreement with previous reports [2,8,24], our data demonstrated that HS/RES upregulates expression of a range of inflammatory molecules in lung tissues and causes lung injury. Moreover,
the rats in HS/RES group had significant hemodynamic compromise and lower PaO2 after resuscitation. The significantly lower MAP and HR as well as lower PaO2 in rats of HS/RES group could be a consequence of the vicious cycle from greater systemic and pulmonary inflammation. As lung injury impairs gas exchange [31], the lower PaO2 corresponded to the reduced delivery of O2 from the lungs to the bloodstream after HS/RES. Importantly, our
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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molecules upregulation induced by HS/RES. These data provide clear evidence to highlight the involvement of the HO-1 dependent pathway in mediating the protective effects of cepharanthine against pulmonary inflammation and acute lung injury induced by HS/RES. The limitations of the present study should be acknowledged. Firstly, our investigation was limited to the early phase of HS/RES. The question of whether cepharanthine exerts therapeutic effects in the delayed phase of HS/RES remains un-answered. Secondly, this study highlights the effect of cepharanthine on modulating HO-1 expression. However, the underlying mechanisms remain to be elucidated. Since HO-1 induction is tightly controlled by nuclear factor E2-related factor 2 (Nrf2) [7,35] and our results demonstrate that cepharanthine can induce HO-1 expression in sham-operated rat lungs, it is probable that cepharanthine can modulate Nrf2 expression. 5. Conclusions Cepharanthine significantly mitigates pro-inflammatory cytokine response in acute lung injury induced by HS/RES in rats. The mechanism may involve the HO-1 dependent pathway. Conflict of interests The authors declare that there is no conflict of interest regarding the publication of this paper. Fig. 4. (A) Representative gel photography and densitometric analysis data illustrate the transcriptional expression of heme oxygenase-1 (HO-1) and (B) the microsomal bilirubin concentrations in rat lungs (n = 12 in each group). Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. DMSO: dimethylsulfoxide. b-Actin was used as the internal standard. Data were means ± standard deviations. ⁄P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
study revealed that both the biochemical indices of inflammation and the physical lung injury induced by HS/RES were significantly attenuated by iv cepharanthine. These results provide basis for further research to incorporate cepharanthine into the treatment of HS/RES-induced acute lung injury. The inducible HO-1 enzyme has anti-oxidative and antiinflammatory properties, as it tightly regulates the catabolism of heme to produce carbon monoxide, iron, and biliverdin [6,7]. However, endogenous HO-1 induction takes part in a regulatory rather than therapeutic role against hemorrhagic shock [4,23]. Only further enhancement of HO-1 expression can be beneficial in reducing systemic inflammatory response after HS/RES [23,32]. Data from this study, in accordance with the previous report [23], confirmed that HS/RES induces HO-1 expression in rat lungs. Our data also disclosed that, through as yet unknown mechanisms, cepharanthine can induce HO-1 expression in rat lungs (as seen in the Sham + CEP group). Our data further disclosed that cepharanthine can further enhance HO-1 induction in rat lungs after HS/RES. In addition, SnPP is commonly used in experimental models to antagonize the function of HO-1 [33,34]. Our preliminary examination of SnPP at commonly used dose (30 mg/kg) showed that SnPP has no significant effects and side effects in rat lungs of the Sham and HS/RES groups (please see supplemental material). However, the expected findings from the present study disclosed that SnPP can significantly counteract the effects of cepharanthine on mitigating hemodynamic compromise, O2 exchange, lung injury indices and pulmonary inflammatory
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Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats Ming-Chang Kao a,b,c, Chen-Hsien Yang b,c, Joen-Rong Sheu a,⇑, Chun-Jen Huang a,b,c,⇑ a
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Anesthesiology, Taipei Tzu Chi Hospital, Taipei, Taiwan c School of Medicine, Tzu Chi University, Hualien, Taiwan b
a r t i c l e
i n f o
Article history: Received 18 May 2015 Received in revised form 2 September 2015 Accepted 8 September 2015 Available online xxxx Keywords: Anti-inflammatory Tumor necrosis factor-a Interleukin Heme oxygenase-1 Tin protoporphyrin
a b s t r a c t Background: Cepharanthine possesses strong anti-inflammation capacity. We sought to clarify whether cepharanthine could mitigate pro-inflammatory cytokine production in acute lung injury induced by hemorrhagic shock/resuscitation (HS/RES). The involvement of heme oxygenase-1 (HO-1) was also investigated. Methods: Male Sprague Dawley rats were allocated to receive HS/RES, HS/RES plus iv cepharanthine or HS/RES plus cepharanthine plus the HO-1 activity inhibitor tin protoporphyrin (SnPP) and denoted as the HS/RES, HS/RES + CEP, and HS/RES + CEP + SnPP group, respectively. HS/RES was achieved by blood drawing to lower mean arterial pressure (40–45 mmHg for 60 min) followed by shed blood/saline mixtures re-infusion. The rats were monitored for another 5 h before sacrifice. Results: Arterial blood gas, lung permeability and histologic assays (including histopathology, neutrophil infiltration, and lung water content) confirmed that HS/RES induced significant lung injury. Significant increases in pulmonary levels of tumor necrosis factor-a, interleukin-1b, interleukin-6, prostaglandin E2 and cyclooxygenase-2 confirmed that HS/RES induced a significant inflammatory response in the lungs. Cepharanthine significantly attenuated the pulmonary pro-inflammatory cytokine production and lung injury induced by HS/RES. However, the protective effects of cepharanthine were blocked by SnPP, the potent HO-1 activity inhibitor. Conclusion: Cepharanthine significantly mitigates pro-inflammatory cytokine response in acute lung injury induced by HS/RES in rats. The mechanism may involve the HO-1 pathway. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Hemorrhagic shock followed by resuscitation (HS/RES) are two early pathophysiological phases in the sequela of the clinical reversal of hemorrhage [1]. Hemorrhagic shock induces tissue ischemia and oxidative stress. Reperfusion induced by resuscitation causes the release of toxic mediators from ischemic tissues and the subsequent triggering of inflammation cascades [2], which in turn leads to additional local and distal tissue injury. In the process, the free heme released from damaged erythrocytes catalyzes the formation of reactive oxygen species and greatly increases cell dysfunction [3–5]. Under physiological conditions the enzymes of heme ⇑ Corresponding authors at: Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing St., Taipei 110, Taiwan. (J.-R. Sheu). Department of Anesthesiology, Taipei Tzu Chi Hospital, 289, Jianguo Rd., Sindian District, New Taipei City 231, Taiwan (C.-J. Huang). E-mail addresses: [email protected] (M.-C. Kao), [email protected] (C.-H. Yang), [email protected] (J.-R. Sheu), [email protected] (C.-J. Huang).
oxygenase (HO) tightly regulate the catabolism of heme to produce metabolites that possess anti-oxidative and anti-inflammatory properties [6,7]. The lung tissues, being a downstream filter that receives the entire cardiac output, are most vulnerable to the toxic metabolites released from ischemic organs after HS/RES [8]. HS/RES readily upregulates the expression of inflammatory molecules in lung tissues, which leads to overt pulmonary inflammation and the progression of lung injury [9–12]. Therapies aimed at attenuating pulmonary inflammation may have beneficial consequences against lung injury induced by HS/RES [8]. Cepharanthine is a biscoclaurine alkaloid derived from Stephania cepharantha, a plant of the Menispermaceae family [13]. Cepharanthine possesses potent in vivo and in vitro antineoplastic, immunomodulatory and anti-inflammation effects [14–18] and has been used clinically to successfully treat radiation-induced leukopenia, hair loss, bronchial asthma, and certain types of allergic inflammation [19,20]. Moreover, in animal
http://dx.doi.org/10.1016/j.cyto.2015.09.008 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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models of sepsis cepharanthine can exert protective effects and mitigate inflammatory responses [21,22]. To date, the question of whether cepharanthine can mitigate pro-inflammatory cytokine response in acute lung injury induced by HS/RES remains unstudied. To examine this hypothesis, we conducted this study using a rodent model of HS/RES. Moreover, it has been shown that induction of heme oxygenase-1 (HO-1) exerts protective effects against HS/RES [23]. We also examined whether HO-1 was involved in mediating the anti-inflammatory effect of cepharanthine. 2. Materials and methods 2.1. Animal preparation Ninety male Sprague–Dawley rats (BioLASCO Taiwan Co., Ltd, Taipei, Taiwan) weighing 250–300 g were used in the experiments. All animal experiments were approved by Institutional Animal Use and Care Committee, Taipei Tzu Chi Hospital (100-IACUC No. 003). All experiments were done in accordance with the guidelines of the National Institutes of Health. The rats were anesthetized using an intramuscular injection of a ketamine/xylazine mixture (110/10 mg/kg respectively). Additional doses of ketamine/xylazine mixture (30/3 mg/kg respectively) were given hourly until the end of the experiment. The rats were placed supine on a heating pad. A rectal temperature probe was inserted and the body temperature was maintained at 37 °C throughout the experiments using the heating pad and heating lamps. Polyethylene catheters (PE-50, Becton Dickinson, Sparks, MD, USA) were inserted into the right femoral artery for blood pressure monitoring and the left femoral vein for blood withdrawal and intravenous (iv) injection, respectively. A tracheostomy was performed to allow airway clearance. 2.2. HS/RES protocols Protocols of HS/RES were adapted from our previous study [24]. In brief, hemorrhagic shock was achieved by blood drawing over 10 min to reduce mean arterial pressure (MAP; BIOPAC System, Santa Barbara, CA, USA) from the physiologic level to 40– 45 mmHg. The shed blood was stored in a syringe containing 20 units of heparin at room temperature. This lowered MAP was then kept for 60 min by drawing or re-infusing blood as needed. Resuscitation was achieved by re-infusing the shed blood, supplemented with twice the maximum blood volume drawn of normal saline over a 10-min period. All rats were monitored for another 300 min. 2.3. Experimental protocols The rats were randomized to 5 experimental groups (n = 18 in each group). The Sham group received sham operation (i.e., cannulation of vessels and tracheostomy) plus a 30 lL intravenous injection (iv) of dimethylsulfoxide (DMSO) vehicle (Sigma, St Louis, Mo, USA). The Sham + CEP group received sham operation plus cepharanthine (5 mg/kg, iv; LKT Laboratories, Inc. St. Paul, MN, USA). The HS/RES group received HS/RES plus vehicle. The HS/RES + CEP group received HS/RES plus cepharanthine (5 mg/kg, iv). The HS/RES + CEP + SnPP group received HS/RES plus cepharanthine (5 mg/kg, iv) plus the potent HO-1 enzymatic activity inhibitor tin protoporphyrin (SnPP, 30 mg/kg, iv; Enzo Life Sciences, Farmingdale, NY, USA). All rats received intravenous injection of vehicle, cepharanthine, or cepharanthine plus SnPP immediately before resuscitation or at corresponding time points in the Sham groups. At 300 min following resuscitation, arterial blood (0.5 mL) was drawn from 12 randomly selected rats in each group. Arterial blood
gas (ABG) was immediately analyzed with a blood gas analyzer (Gem Premier 3000; Instrumentation Laboratory, Bedford, MA, USA). The lung samples were then harvested immediately after euthanization with pentobarbital (100 mg/kg, iv). The remaining 6 rats of each group were used for lung permeability measurements.
2.4. Lung tissues collection and bronchoalveolar lavage (BAL) For the above-mentioned 12 rats from each group, the left main bronchus was tied and the left lung was excised. The superior and inferior lobes of the left lung were separated, and the inferior lobe was snap frozen in liquid nitrogen and stored at 80 °C. The left superior lobe was used for wet/dry weight ratio measurement. Then, 6 of these 12 rats from each group received right lung perfusion with 4% formaldehyde and then excised. For the remaining 6 rats, the right lung was lavaged 5 times with 3 mL sterile saline and the BAL fluid (BALF) was collected [24]. An aliquot of BALF was diluted 1:1 with trypan blue dye for counting total cell number. The remaining BALF was collected and centrifuged. The protein concentration of the supernatant was measured using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, IL, USA).
2.5. Histologic analysis The formalin-fixed and paraffin-embedded lung tissues were serial sectioned and stained with hematoxylin and eosin. Histologic features including alveolar wall edema, vascular congestion, hemorrhage, and polymorphonuclear (PMN) leukocyte infiltration were examined under a light microscope using our previously published protocol [24]. Each histologic feature was scored on a 5-grade scale: 0 (normal) to 5 (severe). The overall lung injury in each rat was classified as normal to minimal when the sum of the scores was 0–5, mild when 6–10, moderate when 11–15 and severe when 16–20.
2.6. Wet/dry weight ratio and myeloperoxidase (MPO) activity assay Wet/dry weight ratio (i.e., lung water content) and MPO activity (i.e., quantification of tissue PMN accumulation) were analyzed by protocols we have previously described [25]. The freshly harvested left superior lobe was weighed and then dried in the oven at 80 °C for 24 h. The lobe was then weighed again in dry condition. The wet/dry weight ratio was then calculated. For MPO activity assay, the snap-frozen lung tissues were homogenized and centrifuged. The suspension was then sonicated and the supernatant was obtained and incubated in a water bath at 60 °C for 2 h. MPO activity was measured using a MPO fluorometric detection kit (Enzo Life Science) according to the manufacturer’s instructions.
2.7. Lung permeability Lung permeability was determined by the Evans blue dye (EBD) extravasation [26]. In brief, rats received 30 mg/kg (iv) of EBD (Sigma) at 300 min after resuscitation. A blood sample (1 mL) was drawn at 5 min after EBD injection to determine the plasma EBD concentration. The rats were then euthanized at 20 min after EBD injection and BAL was performed, as above-mentioned. The collected BALF was then centrifuged at 1500 rpm at 4 °C for 10 min. The EBD concentrations of the BALF and the plasma were then analyzed by spectrophotometry at 620 nm. The EBD concentration of the BALF was then compared to that of the plasma to determine lung permeability.
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2.8. Inflammatory molecules For inflammatory molecule assay, the snap-frozen lung tissues were processed by a protocol we have previously described [24]. The concentrations of tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1b), interleukin-6 (IL-6) and prostaglandin E2 (PGE2) were determined using enzyme-linked immunosorbent assay kits (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. 2.9. Reverse transcription-polymerase chain reaction (RT-PCR) Transcriptional expression of cyclooxygenase-2 (COX-2), i.e., the enzyme regulating PGE2 expression, and HO-1 of the lung tissues were determined using RT-PCR. The protocols of primer sequences and amplification for COX-2, HO-1, and b-Actin were performed according to our previous report [27]. After separation, band intensities of PCR-amplified cDNA were quantified by densitometric techniques (Scion Image for Windows, Scion Corp, Frederic, MD, USA). 2.10. HO activity assay HO activity was analyzed from the microsomal fraction by measuring the formation of bilirubin, using a protocol we have previously described [5]. The lung tissues were homogenized and the homogenates were centrifuged. After disposing of the supernatant, the microsomal pellet was re-suspended in a solution of 100 mM potassium phosphate buffer and 2 mM MgCl2 (pH 7.4). The bilirubin concentration was measured as difference in absorbance between 450 and 600 nm and was calculated with an excitation coefficient of 27.3 mM1cm1. HO activity was expressed as pmol of bilirubin formed per mg of tissue protein. Protein concentration was measured using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, IL, USA). 2.11. Statistical analysis Data were expressed as means ± standard deviations. Differences among groups were tested using one-way analysis of variance (ANOVA). If one-way ANOVA revealed significant differences among groups, then the post-hoc tests with the Bonferroni multiple comparison tests were performed. Differences were considered significant at P < 0.05. 3. Results 3.1. Hemodynamic data One-way ANOVA revealed that the baseline MAP and heart rate (HR) were not significantly different among these five groups (data not shown). The MAP and HR of the Sham and the Sham + CEP groups were stable during the whole experiment. One-way ANOVA revealed significant group differences in the end MAP (P < 0.001) and HR (P < 0.001). Multiple comparison tests revealed that the end MAP and HR of the HS/RES group were significantly lower than those of the Sham group (both P < 0.001, Table 1). Moreover, the end MAP and HR of the HS/RES + CEP group were significantly higher than those of both the HS/RES group (both P < 0.001, Table 1) and the HS/RES + CEP + SnPP group (both P < 0.001, Table 1). 3.2. ABG data The ABG data including the values of pH, PaO2, PaCO2 and base excess (BE) were measured. One-way ANOVA revealed significant
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group differences in the pH, PaO2, BE and PaCO2 (all P < 0.001). Multiple comparison tests revealed that the pH, PaO2, and base excess (BE) of the Sham and the Sham + CEP groups were not significantly different (Table 1). The pH, PaO2 and BE of the HS/RES group were significantly lower than those of the Sham group (all P < 0.001, Table 1). The pH, PaO2 and BE values of the HS/RES + CEP group were significantly higher than those of the HS/RES group (all P < 0.001), whereas the PaCO2 values between the two groups were not significantly different (Table 1). In addition, the pH, PaO2, and BE values of the HS/RES + CEP + SnPP group were significantly lower than those of the HS/RES + CEP group (all P < 0.001), whereas the PaCO2 value of the HS/RES + CEP + SnPP group was significantly higher than that of the HS/RES + CEP group (P < 0.001, Table 1). 3.3. Histologic lung injury score Histologic analysis disclosed normal to minimal lung injury in rats of the Sham and the Sham + CEP groups (Fig. 1A and B). Histologic analysis also disclosed moderate to severe lung injury in rats of the HS/RES and the HS/RES + CEP + SnPP groups (Fig. 1C and E) and mild injury in rats of the HS/RES + CEP group (Fig. 1D). Lung injury scores (Fig. 1F) basically paralleled the histologic analysis. 3.4. BALF analysis, wet/dry weight ratio, MPO activity and lung permeability One-way ANOVA revealed a significant group difference in the BALF total cell numbers (P < 0.001). The BALF total cell numbers of the Sham and the Sham + CEP groups were low (Fig. 2A). Multiple comparison tests revealed that the BALF total cell numbers of the HS/RES group was significantly higher than that of the Sham group (P < 0.001, Fig. 2A). In contrast, the BALF total cell numbers of the HS/RES + CEP group was significantly lower than of the HS/RES group (P < 0.001, Fig. 2A). However, the BALF total cell numbers of the HS/RES + CEP + SnPP group was significantly higher than that of the HS/RES + CEP group (P = 0.005, Fig. 2A). Data of the BALF protein concentration (Fig. 2B), wet/dry weight ratio (Fig. 2C), MPO activity (Fig. 2D) and lung permeability (Fig. 2E) essentially matched the data of the BALF total cell numbers (Fig. 2A), except that the wet/dry weight ratio of the HS/RES and the HS/RES + CEP groups were not significantly different (Fig. 2C). 3.5. Inflammatory molecules data One-way ANOVA revealed a significant group difference in the pulmonary TNF-a concentration (P < 0.001).The pulmonary TNF-a concentration of the Sham and the Sham + CEP groups were low (Fig. 3A). As expected, multiple comparison tests revealed that the pulmonary TNF-a concentration of the HS/RES group was significantly higher than that of the Sham group (P < 0.001, Fig. 3A). Moreover, the pulmonary TNF-a concentration of the HS/RES + CEP group was significantly lower than that of the HS/RES group (P < 0.001, Fig. 3A). In contrast, the pulmonary TNF-a concentration of the HS/RES + CEP + SnPP group was significantly higher than that of the HS/RES + CEP group (P < 0.001, Fig. 3A). Data of the pulmonary concentrations of IL-1b (Fig. 3B), IL-6 (Fig. 3C), COX-2 mRNA (Fig. 3D) and PGE2 (Fig. 3E) essentially matched the data of TNF-a (Fig. 3A). 3.6. HO-1 expression One-way ANOVA revealed significant group differences in the pulmonary HO-1 mRNA and HO activity (P < 0.001). The pulmonary concentrations of HO-1 mRNA and HO activity of the Sham group were low. Multiple comparison tests revealed that the
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Table 1 Hemodynamic and arterial blood gas data at the end of experiment. Group (n = 12)
MAP (mmHg)
HR (beats/min)
pH
PaO2 (mm Hg)
PaCO2 (mm Hg)
Base excess (mM)
Sham Sham + CEP HS/RES HS/RES + CEP HS/RES + CEP + SnPP
113 ± 13 112 ± 15 83 ± 3* 103 ± 10# 81 ± 3*,y
332 ± 14 348 ± 16 266 ± 11* 294 ± 21*,# 250 ± 7*,y
7.26 ± 0.04 7.26 ± 0.03 7.15 ± 0.03* 7.23 ± 0.04# 7.14 ± 0.05*,y
130 ± 11 126 ± 10 99 ± 8* 123 ± 7# 88 ± 7*,#,y
47 ± 3 47 ± 3 45 ± 2 44 ± 2 49 ± 3#,y
8.1 ± 1.4 7.9 ± 0.9 12.6 ± 1.4* 8.8 ± 1.2# 14.5 ± 1.8*,#,y
Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. HR: heart rate. MAP: mean arterial pressure. Data were means ± standard deviations. * P < 0.05 vs. the Sham group. # P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. y P < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
Fig. 1. Representative microscopic findings of the lung tissues stained with hematoxylin-eosin (10) and the lung injury scores (n = 6 in each group). (A) The Sham group. (B) The Sham plus cepharanthine (Sham + CEP) group. (C) The hemorrhagic shock/resuscitation (HS/RES) group. (D) The HS/RES + CEP group. (E) The HS/RES + CEP plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (HS/RES + CEP + SnPP) group. (F) The lung injury scores. DMSO: dimethylsulfoxide. Data were means ± standard deviations. ⁄P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/ RES + CEP group.
pulmonary concentrations of HO-1 mRNA and HO activity of the Sham + CEP group were significantly higher than those of the Sham group (P < 0.001 and =0.013, Fig. 4A and B). The pulmonary concentrations of HO-1 mRNA and HO activity of the HS/RES group were also significantly higher than those of the Sham group (both P < 0.001, Fig. 4A and B). Moreover, the pulmonary concentrations of HO-1 mRNA and HO activity of the HS/RES + CEP group were significantly higher than those of the HS/RES group (P = 0.028 and 0.002, Fig. 4A and B). The pulmonary HO activity of the HS/RES + CEP group was significantly higher than that of the HS/RES + CEP + SnPP group (P = 0.002, Fig. 4B), whereas the pulmonary HO-1 mRNA concentrations of the two groups were not significantly different (P = 0.995, Fig. 4A). 4. Discussion This study replicates the findings of earlier studies [8,9,24], and demonstrates conclusively that this model of HS/RES causes increases in pulmonary pro-inflammatory cytokines and acute lung injury in rats. The situation closely mimics the clinical
situation of the early phase of hemorrhagic shock. The novel finding of our study is that iv cepharanthine administered immediately before resuscitation significantly ameliorated all experimental indices of pulmonary inflammation and lung injury caused by HS/RES. Moreover, our data demonstrates the active involvement of the HO-1 dependent pathway in mediating the protective effects of cepharanthine against HS/RES. The shock event and subsequent post-resuscitation event are two dependent yet distinct patho-physiologies of the early phase of hemorrhagic shock [1]. Tissue ischemia induced by the hemorrhagic event and subsequent reperfusion event, performed as a component procedure of resuscitation, can lead to the release of toxic mediators from ischemic tissues and subsequently trigger the local and distal inflammation cascades [2,28]. The resultant systemic pro-inflammatory response from both cascades can cause persistent hypotension, despite resuscitation [29,30]. Hemodynamics and anatomy combine to determine that the lung tissues are extremely vulnerable to the insults of HS/RES. Synergistic interaction between local pulmonary inflammatory molecules and the infiltrated neutrophils can damage pulmonary microvascular
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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Fig. 2. (A) The total cell number in bronchoalveolar lavage fluid (BALF; n = 6 in each group), (B) the total protein concentration in BALF (n = 6), (C) the wet/dry weight ratio of the lungs (n = 12), (D) the myeloperoxidase (MPO) activity of the lungs (n = 12) and (E) the lung permeability (n = 6). Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. DMSO: dimethylsulfoxide. Data were means ± standard deviations. ⁄ P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
Fig. 3. The concentrations of (A) tumor necrosis factor-a (TNF-a), (B) interleukin-1b (IL-1b), (C) IL-6, (D) cyclooxygenase-2 (COX-2) mRNA and (E) prostaglandin E2 (PGE2) in rat lungs (n = 12 in each group). Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. DMSO: dimethylsulfoxide. Data were means ± standard deviations. ⁄P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/ RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
endothelium and exacerbate acute lung injury that affecting gas exchange [8]. The impaired gas exchange in addition to the postresuscitation hypotension may elicit a vicious cycle of systemic and pulmonary pro-inflammatory response. In agreement with previous reports [2,8,24], our data demonstrated that HS/RES upregulates expression of a range of inflammatory molecules in lung tissues and causes lung injury. Moreover,
the rats in HS/RES group had significant hemodynamic compromise and lower PaO2 after resuscitation. The significantly lower MAP and HR as well as lower PaO2 in rats of HS/RES group could be a consequence of the vicious cycle from greater systemic and pulmonary inflammation. As lung injury impairs gas exchange [31], the lower PaO2 corresponded to the reduced delivery of O2 from the lungs to the bloodstream after HS/RES. Importantly, our
Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008
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molecules upregulation induced by HS/RES. These data provide clear evidence to highlight the involvement of the HO-1 dependent pathway in mediating the protective effects of cepharanthine against pulmonary inflammation and acute lung injury induced by HS/RES. The limitations of the present study should be acknowledged. Firstly, our investigation was limited to the early phase of HS/RES. The question of whether cepharanthine exerts therapeutic effects in the delayed phase of HS/RES remains un-answered. Secondly, this study highlights the effect of cepharanthine on modulating HO-1 expression. However, the underlying mechanisms remain to be elucidated. Since HO-1 induction is tightly controlled by nuclear factor E2-related factor 2 (Nrf2) [7,35] and our results demonstrate that cepharanthine can induce HO-1 expression in sham-operated rat lungs, it is probable that cepharanthine can modulate Nrf2 expression. 5. Conclusions Cepharanthine significantly mitigates pro-inflammatory cytokine response in acute lung injury induced by HS/RES in rats. The mechanism may involve the HO-1 dependent pathway. Conflict of interests The authors declare that there is no conflict of interest regarding the publication of this paper. Fig. 4. (A) Representative gel photography and densitometric analysis data illustrate the transcriptional expression of heme oxygenase-1 (HO-1) and (B) the microsomal bilirubin concentrations in rat lungs (n = 12 in each group). Sham: the sham group. Sham + CEP: the Sham plus cepharanthine group. HS/RES: the hemorrhagic shock/resuscitation group. HS/RES + CEP: the HS/RES plus cepharanthine group. HS/RES + CEP + SnPP: the HS/RES plus cepharanthine plus the potent heme oxygenase-1 activity inhibitor tin protoporphyrin (SnPP) group. DMSO: dimethylsulfoxide. b-Actin was used as the internal standard. Data were means ± standard deviations. ⁄P < 0.05 vs. the Sham group. #P < 0.05 the HS/RES + CEP or HS/RES + CEP + SnPP group vs. the HS/RES group. yP < 0.05 the HS/RES + CEP + SnPP group vs. the HS/RES + CEP group.
study revealed that both the biochemical indices of inflammation and the physical lung injury induced by HS/RES were significantly attenuated by iv cepharanthine. These results provide basis for further research to incorporate cepharanthine into the treatment of HS/RES-induced acute lung injury. The inducible HO-1 enzyme has anti-oxidative and antiinflammatory properties, as it tightly regulates the catabolism of heme to produce carbon monoxide, iron, and biliverdin [6,7]. However, endogenous HO-1 induction takes part in a regulatory rather than therapeutic role against hemorrhagic shock [4,23]. Only further enhancement of HO-1 expression can be beneficial in reducing systemic inflammatory response after HS/RES [23,32]. Data from this study, in accordance with the previous report [23], confirmed that HS/RES induces HO-1 expression in rat lungs. Our data also disclosed that, through as yet unknown mechanisms, cepharanthine can induce HO-1 expression in rat lungs (as seen in the Sham + CEP group). Our data further disclosed that cepharanthine can further enhance HO-1 induction in rat lungs after HS/RES. In addition, SnPP is commonly used in experimental models to antagonize the function of HO-1 [33,34]. Our preliminary examination of SnPP at commonly used dose (30 mg/kg) showed that SnPP has no significant effects and side effects in rat lungs of the Sham and HS/RES groups (please see supplemental material). However, the expected findings from the present study disclosed that SnPP can significantly counteract the effects of cepharanthine on mitigating hemodynamic compromise, O2 exchange, lung injury indices and pulmonary inflammatory
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Please cite this article in press as: M.-C. Kao et al., Cepharanthine mitigates pro-inflammatory cytokine response in lung injury induced by hemorrhagic shock/resuscitation in rats, Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.09.008