Toona sinensis leaf aqueous extract displays activity against sepsis in both in vitro and in vivo models

Abstract

Toona sinensis (TS) leaves are used as a vegetable and in traditional Chinese medicine. However, in vivo experiments regarding the anti-inflammatory function of TS leaves have not previously been conducted. The aim of this study was to investigate the potential role of TS leaf extract (TSL) in the prevention of sepsis-induced lung injury in vivo and on macrophage activation in vitro. The results showed that oral gavage pretreatment with TSL in rats for 30 days improved the survival of cecal ligation and puncture-induced sepsis, potentially by attenuating sepsis-induced histological lung damage rather than inflammatory cell infiltration. Furthermore, we demonstrated that pretreatment with TSL attenuated the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase, thereby inhibiting nitric oxide production and release in murine macrophage-like RAW 264.7 cells. Interestingly, TSL did not affect the LPS-induced release of other cytokines (e.g., tumor necrosis factor α and interleukin 1β) but increased LPS-induced heme-oxygenase-1 expression. In conclusion, the study provides preliminary data for TSL on cecal ligation and puncture-induced sepsis. The beneficial impact of TSL needs extensive study to get solid evidence.

Introduction

Sepsis is the generalized inflammatory response elicited by an infectious process [1]. More than 75,000 patients die of septic shock every year in the USA, making this syndrome the leading cause of death in noncoronary intensive care units [2,3]. The respiratory system is the most frequently affected organ system, and lung dysfunction such as acute lung injury (ALI) or adult respiratory distress syndrome (ARDS) is the first step in the development of multiple organ failure [4–6]. Despite the many advances elucidating the pathogenesis and treatment of sepsis, no satisfactory therapy has been developed for ALI/ARDS to date [7].

In Gram-negative bacteria, LPS play a dominant role in the process of sepsis, and macrophages play an essential role in inflammation in response to LPS [8]. Macrophages mediate inflammatory responses by overproducing proinflammatory cytokines including interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNFα), and nitric oxide (NO) synthesized by inducible NO synthase (iNOS) [8]. It has been demonstrated that the induction of heme-oxygenase-1 (HO-1) expression in RAW264.7 macrophages inhibits LPS-activated sepsis [9]. Thus, HO-1, as a regulator of inflammation, is considered to be a therapeutic target.

Toona sinensis Roem (TS) or Cedrela sinensis, commonly known as Chinese mahogany or Chinese Toona, is a perennial deciduous arbor that belongs to the Meliaceae family [10,11]. TS is widely distributed over eastern Asia, and TS leaves have long been used as a vegetable in Taiwan, China, and Malaysia [12]. Previous pharmacological and bioactivity studies on the aqueous extract of TS leaves (TSL) have revealed anticancer [13], antioxidant [14–16], antidiabetes [17,18], and antivirus [19] activity, improvements in learning and memory in senescence-accelerated mice [20], and the reduction of steroidogenesis in mouse Leydig cells [21]. In addition, no studies have reported significant toxicity from TSL thus far [12,22]. TSL is also used as Chinese traditional medicine for the treatment of enteritis, dysentery, and eye infections [23]. Therefore, TSL might have beneficial effects on anti-inflammation. This study investigated the anti-inflammatory activity of TSL against lung injury in a cecal ligation and puncture (CLP)-induced septic model, and the macrophage release of NO/iNOS by the induction of LPS.

Materials and methods Preparation of TSL

The TSL, an advanced bioactive fraction, used in this study was purchased from Taiwan Toona Biotech Corporation (Kaohsiung, Taiwan), and the TS leaves were obtained from Tuku (Yunlin County, Taiwan) [24]. The preparation of TSL has been described in a previous study [24]. Briefly, the TS leaves were picked and washed with reverse osmosis water. Reverse osmosis water was then added to the TS leaves at a proportion of 4 L of water to 1 kg of leaves. The TS leaves were then boiled for 30 minutes, and the remaining liquid was concentrated over low heat and filtered with a sieve (70 mesh). The filtered concentrate was lyophilized with Virtis (Gardiner, NY, USA) apparatus to obtain a crude extract. The crude extract was then dissolved in reverse osmosis water before centrifugation at 1400 × g for 12 minutes, and the supernatant (TSL) was used in all of the experiments. The extract was further concentrated in a vacuum freeze dryer to form a powder, and stored at −20°C for subsequent analysis.

In vivo study Animals

Animal care and experimental procedures were conducted according to the guidelines for Care and Use of Laboratory Animals as established by the Kaohsiung Medical University, Kaohsiung, Taiwan. Prior to the induction of sepsis, adult male Sprague–Dawley rats (250–300 g) were housed in barrier facilities on a 12-hour light/dark cycle with food and water ad libitum. The rats were randomly assigned to four groups: sham-control (saline, sham-operated), TSL (TSL, sham-operated), CLP (saline, CLP procedure), and TSL+CLP (TSL and the CLP procedure), n = 15 in each group. In brief, oral gavage pretreatment with saline or TSL (1 g/kg/day) was given for 30 days prior to the CLP or sham procedure. The dosage of TSL used in this study was according to the safety evaluation of water extracts of Toona sinensis Roemor leaves in Dr. Liao's study [12].

Septic model

Sepsis was induced under anesthesia by CLP modified from the method of Wichterman et al. [25]. Laparotomy was performed through a midline abdominal incision. The cecum was then exposed, ligated with suture, and punctured twice with an 18-gauge needle. The cecum was re-positioned back into the abdominal cavity and the abdominal incision sutured. The control animals received a sham procedure that consisted of laparotomy, manipulation of the cecum, and closure. All of the animals received volume resuscitation with both intravenous and intraperitoneal saline (4 mL/100 g of body weight). They were returned to their cages and allowed free access to food and water.

Histopathological and histochemical assessments of the lungs

Based upon a previous study that identified 18 hours as the late-stage of sepsis following CLP [26], rats (3 replicates of 5 animals in each group) were sacrificed at 18 hours post-CLP or sham operation after being deeply anesthetized with 7% chloral hydrate (0.4 mL/100 g). Lung tissues were quickly removed and immersed in the periodate-lysine-2% paraformaldehyde fixative [27] at 4°C overnight. Then samples were rinsed with phosphate-buffered saline (PBS) and cryoprotected in 30% sucrose. Serial frozen sections (5 μm) were collected on gelatin-coated glass slides and stored at −20°C until use. For each animal, each slide contained six sections collected at 100 μm intervals throughout the entire tissue specimen. Two slides were selected for staining with hematoxylin and eosin for general cellularity and cell death. Two additional randomly selected slides were used for isolectin B4 staining to detect the presence of alveolar macrophages. This resulted in a total of 12 sections of lung tissue from each animal for each assessment.

Alveolar macrophages were identified by lectin binding using the method of Streit and Kreutzberg [28] for specific binding to the terminal α-galactose on the surface of the pulmonary macrophage [29,30]. Briefly, sections were washed in PBS buffer containing CaCl₂, MgCl₂, MnCl₂ (0.1 mM), and Triton X-100 for 20 minutes. The sections were then bathed in PBS buffer containing peroxidase-conjugated Griffonia simplicifolia isolectin B4 (0.025 mg/mL; Sigma-Aldrich, St Louis, MO, USA) at 4°C overnight, and then rinsed in PBS. Sites containing macrophage-bound peroxidase-lectin conjugates were visualized following 5 minutes of incubation with 3,3′-diaminobenzidine (DAB) substrate. The sections were then counterstained with thionine, dehydrated in ethanol, cleared in xylene, and mounted in Entellan Neu (Merck, Darmstadt, Germany).

In vitro study Cell culture

Murine RAW264.7 macrophages were cultured in growth media [Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Hyclone, ThermoFisher Scientific, South Logan, UT, USA), supplemented with 1.5 g/L NaHCO₃ and 1% penicillin and streptomycin (Gibco). Cells were seeded in 24-well plates or in 35-mm dishes and maintained at 37°C in a humidified environment containing 95% air and 5% CO₂. In all experiments, the cells were treated with various concentrations of TSL for 30 minutes prior to the addition of LPS (Escherichia coli, Serotype 055:B5; Sigma-Aldrich) for the corresponding time points in DMEM with 2% FBS.

Cell viability assay

Cells were cultured in 24-well plates at a density of 2 × 10⁵ cells/mL and treated with TSL (0–100 μg/mL) at 37°C for 6 hours and 24 hours. The stock solution of TSL was freshly prepared for all of the experiments (1 mg/mL in double distilled water). 3-4,5-dimethylthiazole-2-yl-2,5-diphenyl tetrazolium bromide was added to the culture at a final concentration of 0.5 mg/mL, and the cells were incubated at 37°C for 4 hours. Formazan crystals that formed in the cells were then solubilized with 500 μL of dimethylsulfoxide (Sigma-Aldrich) per well, and the absorbance at 570 nm was measured using a spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA).

NO analysis

NO was evaluated by measuring the amount of nitrite in the cell culture supernatant using Griess reagent (1% sulphanilamide and 0.1% naphthylethylene diamine dihydrochloride in 5% H₃PO₄). RAW264.7 cells were pretreated with TSL for 30 minutes, and then with or without LPS (1 μg/mL) for 6 hours and 24 hours. The culture supernatant was collected and stored at −20°C until use. A 100 μL aliquot of the cell culture supernatant was mixed with 100 μL of Griess reagent, followed by incubation for 10 minutes at room temperature. The absorbance at 540 nm was measured by a microplate reader (MRX ELISA reader; Dynex, Chantilly, VA, USA) and the inhibitory rates were calculated using a standard calibration curve prepared from different concentrations of sodium nitrite.

Measurement of IL-1β and TNF-α

Cells were pretreated with TSL for 30 minutes and then with or without LPS (1 μg/mL) for 6 hours and 24 hours. A 100 μL aliquot of the culture supernatant was collected to examine the levels of the cytokines using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (eBioscience Inc., San Diego, CA, USA).

Western blot analysis

After the various treatments, cells in 35-mm dishes were washed with PBS and collected in 50 μL of lysis buffer (0.15% TritonX-100, 2 mM MgCl₂, 25 mM HEPES, 60 mM PIPES, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 μg/mL aprotinin, 1 μg/mL pepstatin A, and 1 μg/mL leupeptin, pH 6.9) and centrifuged at 15,000g at 4°C for 30 minutes. Protein concentrations were measured using a protein assay kit (Bio-Rad Life Sciences, Hercules, CA, USA), and the samples were stored at −70°C until further analysis. Protein was applied at 10 μg/lane to 10% sodium dodecyl sulfate–polyacrylamide gel for electrophoresis, and then transferred to a polyvinylidene fluoride membrane. Membrane strips were blocked for 1 hour at room temperature with 5% non-fat milk in Tris-buffered saline (150 mM NaCl, 50 mM Tris base, pH 8.2) containing 0.1% Tween 20, then incubated overnight at 4°C with rabbit anti-heme oxygenase-1 (1:1000 dilution; Stressgen, Victoria, BC, Canada), mouse anti-iNOS (1:1000; BD Transduction Laboratories, Franklin Lakes, NJ, USA), and mouse anti-β-actin (1:20,000 dilution; Sigma) antibodies, in Tris-buffered saline-0.1% Tween20 with 5% bovine serum albumin. The strips were then treated with goat anti-rabbit or anti-mouse immunoglobulin G-horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were developed using an enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA) and exposed to Fuji X-ray films. The density of the bands on the polyvinylidene fluoride membrane was quantified by densitometry using Gel pro 3.1 (Media Cybernetics, Silver Spring, MD, USA). iNOS and HO-1 expressions were normalized on the basis of the β-actin levels. The band intensity of the LPS or control group was defined as 100%, and the densities of the bands in the test samples were expressed as a percentage of the value.

Statistical analysis

All qualitative data are representative of at least three independent experiments.

ELISA data and survival rates were analyzed by 2 × 2 analysis of variance (ANOVA), and post hoc analysis compared by the Bonferroni test. Western blot and nitrite levels were analyzed by Dunnett's test. The values were expressed as mean ± standard deviation. A p value <0.05 was considered to be statistically significant.

Results and discussion

In the present study, we used the CLP model as it closely resembles the pathophysiology of human sepsis and represents an indirect insult similar to the pathogenesis of ALI/ARDS [31–33]. We initially examined whether a survival advantage after CLP was conferred by TSL pretreatment. As shown in Fig. 1A, the survival rate of the CLP rats declined sharply at 18 hours post-CLP (67 ± 7%). More of the CLP rats pretreated with TSL for 30 days survived at 18 hours during this period (86 ± 8.6%; t = 4.63, p = 0.007). This indicates that the administration of TSL improved the survival of CLP rats. Following the histopathological and histochemical assessments in the lungs of the CLP rats with or without TSL treatment, we also found that TSL pretreatment reduced CLP-induced lung damage (Fig. 1B). The evidence from hematoxylin and eosin staining of the lung sections showed that CLP-induced septic rats had thickened alveolar septa with increased inflammatory and interstitial cell infiltration (Fig. 1B, upper panel) in line with our previous report [26]. In the CLP rats pretreated with TSL, these histopathological changes were minimized. Further evidence of inflammatory and/or immune responses in the lung tissue was provided by the histochemical images showing the presence of alveolar macrophages stained by isolectin-B4 (Fig. 1B, lower panel). Interestingly, we found that daily supplements of TSL in the rats prior to CLP surgery did not reduce the increased alveolar macrophage infiltration. Alveolar macrophages are regarded as being the most abundant antigen presenting cells in the airway and alveolar spaces, where they are critically involved in host defense, both as effector cells that bind and engulf pathogens and as sentinel cells that secrete proinflammatory cytokines and chemokines to recruit and activate inflammatory cells [34,35]. However, it has been reported that alveolar macrophages may serve to limit deleterious inflammatory responses within the lungs [36,37]. Taken together, these results suggest that the beneficial role of TSL in sepsis-induced ALI/ARDS might be related to anti-inflammatory/immune responses.

To elucidate the possible mechanism by which TSL mediates inflammatory signals in sepsis, we used LPS as the dominant inflammatory response to sepsis in the RAW264.7 macrophage cell line [8]. First, no cytotoxic effects were observed in 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays of the RAW264.7 cells treated with 10–100 μg/mL of TSL for 6 hours and 24 hours (Fig. 2A). In addition, TSL treatment increased HO-1 protein levels of the RAW264.7 cells in a concentration- and time-dependent manner (Fig. 2B) suggesting the antioxidase and anti-inflammation potential of TSL. Several lines of evidence have demonstrated that the HO-1 production by monocytes/leukocytes serves as an anti-inflammatory agent that controls cell or tissue injuries in the presence of oxidative stress and cytokinemia [9,38]. Therefore, we further detected the growth and decline of proinflammatory cytokines in the LPS-induced RAW264.7 cells pretreated with or without TSL. The concentration of nitrite was determined as NO production after 24 hours of LPS administration (Fig. 2C). The results showed that NO production was overproduced to a cytotoxic level (μM) when the RAW264.7 cells were administered with LPS alone. Pretreatment with TSL for 30 minutes dose-dependently downregulated NO production and the associated iNOS protein expression in the LPS-induced RAW 264.7 cells (Fig. 2D). Conversely, a consistent and increased HO-1 protein expression was observed in the TSL pretreatment groups compared with the LPS treatment alone group (Fig. 2D). This suggests that the negative correlation between iNOS and HO-1 protein expression might be due to the opposite regulation of iNOS and HO-1 in macrophage cells in response to cytokine exposure and oxidative stress [39].

In the present study, LPS-induced NO production/iNOS expression in the RAW264.7 cells was prevented by treatment with TSL. Therefore, we hypothesized that pretreatment with TSL may inhibit the production of proinflammatory cytokines, such as TNF-α or IL-1β, in LPS-induced RAW264.7 cells. Unexpectedly, TSL did not inhibit LPS-induced production of TNF-α or IL-1β production in the RAW264.7 cells (Fig. 2E and F). This indicates that the specific inhibition of iNOS protein expression and NO production and the induction of HO-1 protein might be responsible for the anti-inflammatory potential of TSL. Recently, the possibility that TNF-α might be able to downregulate iNOS expression and decrease infection-induced lung injury was reported [40]. Clinically, blocking TNF receptor fusion proteins in patients with septic shock can worsen patient survival [41]. Therefore, the findings suggest that the presence of TNF-α in RAW264.7 cells under LPS and TSL treatment might be beneficial for the anti-inflammatory effect of TSL. It would therefore be reasonable to suggest that TSL prevents LPS-induced NO release and iNOS protein expression in RAW264.7 macrophages through the negative regulation of TNF-α rather than through the suppression of TNF-α production [42,43].

In conclusion, the present study provides the first evidence that TSL has the capacity to improve lung injury in CLP-induced septic rats, and inactivate inflammatory responses in LPS-induced macrophages. The anti-inflammatory mechanism of TSL on the inhibition of NO production and iNOS expression might be through regulation of HO-1 or TNF-α activity. The study provides preliminary data for TSL on CLP-induced sepsis. The beneficial impact of TSL needs extensive study to get solid evidence.

Acknowledgments

The authors wish to thank Professor Hseng-Kuang Hsu, Professor G. Jean Harry, Ms Pei-Hui Wang, Belinda Wilson, and Mr Chao-Yuan Chang. This work was supported by the following grants: 1) National Science Council Taiwan (NSC 93-2320-B-037-031), 2) Kaohsiung Medical University, Taiwan (KMU-M-110014).