Journal of Ethnopharmacology 155 (2014) 1483–1491

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Research Paper

The total alkaloids of Aconitum tanguticum protect against lipopolysaccharide-induced acute lung injury in rats Guotai Wu a,1, Lidong Du b,1, Lei Zhao b, Ruofeng Shang a, Dongling Liu b, Qi Jing b, Jianping Liang a,n, Yuan Ren b,nn a Key Laboratory of New Animal Drug Project of Gansu Province, Key Laboratory of Veterinary Pharmaceutics Discovery, Ministry of Agriculture, Lanzhou Institute of Animal Science and Veterinary Pharmaceutics Science, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730050, PR China b Key Laboratory of Pharmacology and Toxicology of Traditional Chinese Medicine of Gansu Province, Gansu University of Traditional Chinese Medicine, 35 Dingxi Road, Chengguan District, Lanzhou, Gansu 730000, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 4 June 2014 Received in revised form 5 July 2014 Accepted 18 July 2014 Available online 4 August 2014

Ethnopharmacological relevance: Aconitum tanguticum has been widely used as a remedy for infectious diseases in traditional Tibetan medicine in China. The total alkaloids of Aconitum tanguticum (TAA) are the main active components of Aconitum tanguticum and have been demonstrated to be effective in suppressing inflammation. Our aim was to investigate the protective effects of TAA on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats. Materials and methods: TAA was extracted in 95% ethanol and purified in chloroform. After vacuum drying, the TAA powder was dissolved in dimethyl sulfoxide. Adult male Sprague-Dawley rats were randomly divided into six groups. Rats were given dexamethasone (DXM, 4 mg/kg) or TAA (60 mg/kg, 30 mg/kg) before LPS injection. The PaO2and PaO2/FiO2 values, lung wet/dry (W/D) weight ratio and histological changes in lung tissue were measured. The cell counts, protein concentration, tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and interleukin-1b (IL-1b) in bronchoalveolar lavage fluid (BALF), and myeloperoxidase (MPO) activity in lung tissue were determined at 6, 12 or 24 h after LPS treatment. In addition, the NF-κ B activation in lung tissue was analyzed by western blot. Results: In ALI rats, TAA significantly reduced the lung W/D ratio and increased the value of PaO2 or PaO2/ FiO2 at 6, 12 or 24 h after LPS challenge. TAA also reduced the total protein concentration and the number of total cells, neutrophils or lymphocytes in BALF. In addition, TAA decreased MPO activity in the lung and attenuated histological changes in the lung. Furthermore, TAA inhibited the concentration of TNF-a, IL-6 and IL-1b in BALF at 6, 12 or 24 h after LPS treatment. Further study demonstrated that TAA significantly inhibited NF-κ B activation in lung tissue. Conclusions: The current study proved that TAA exhibited a potent protective effect on LPS-induced ALI in rats through its anti-inflammatory activity. & 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article: 6-benzoylheteratisine (PubChem CID: 5487064) Heteratisine (PubChem CID: 441735) Atisine (PubChem CID: 6426913) Aconitine (PubChem CID: 245005) Hypaconitine (PubChem CID: 23337) Benzoylaconitine (PubChem CID: 49868330) Benzoylhypacoitine (PubChem CID: 3047329) Benzoylmesaconine (PubChem CID: 3036967) Hordenine (PubChem CID: 68313) Heterophylline (PubChem CID: 251575) Keywords: Total alkaloids of Aconitum tanguticum Acute lung injury TNF-α IL-6 IL-1β NF-κ B

Abbreviations: TAA, total alkaloids of Aconitum tanguticum; AC, aconitine; HDTAA, high dose of TAA; LDTAA, low dose of TAA; ALI, acute lung injury; LPS, lipopolysaccharide; H–E, hematoxylin–eosin; BALF, bronchoalveolar lavage fluid; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-1β, interleukin-1β; W/D, lung wet/dry ratio; MPO, myeloperoxidase; NF-κ B, nuclear factor-kappa B; DMSO, dimethyl sulfoxide; PBS, phosphate buffer solution; DXM, dexamethasone; ELISA, enzyme-linked immunosorbent assay; TCM, traditional Chinese medicine n Correspondence to: Lanzhou Institute of Husbandry and Pharmaceutical Sciences, CAAS, 335 Jiangouyan Road, Qilihe District, Lanzhou 730050, PR China. Tel./fax: þ 86 931 2115287. nn Corresponding author. Tel./fax: þ86 931 8762653. E-mail addresses: [email protected] (J. Liang), [email protected] (Y. Ren). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2014.07.041 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Aconitum tanguticum (Maxim.) Stapf has long been used for the treatment of infectious fever, pneumonia, enteritis, hepatitis, prevalent influenza and common inflammation. It is a traditional Tibetan medicine in China and is widely distributed in the southeastern Qinghai-Tibet Plateau, southern Gansu, western Sichuan and northern Yunnan provinces (Zhang et al., 2012a). Phytochemical studies found that Aconitum tanguticum contains 22 types of alkaloids, three types of flavonoids, 35 types of essential oils and some polysaccharides (Luo et al., 2012a). The main ingredients of

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Aconitum tanguticum were mono- or dimeric diterpenoid alkaloids (Pelletier and Joshi, 1991; Wang et al., 2002, 2005; Li et al., 2004). Pharmacological studies showed that Aconitum tanguticum exhibited various biological effects such as antibiosis, antivirus and antiinflammation (Zhang et al., 2009, 2010). Diterpenoid alkaloids are considered to be the main composition of anti-inflammatory and analgesic (Ha and Li, 2010; Luo et al., 2012b). Acute lung injury (ALI) is a severe inflammatory disease characterized by neutrophilic infiltration, pulmonary edema, and hypoxemia (Dushianthan et al., 2011; Vadasz and Sznajder, 2011). ALI is associated with the development of multiple organ dysfunction syndromes, which plays a pivotal role in the death of patients with shock, sepsis, and multiple transfusions (Lee and Downey, 2001; Gu and Song, 2012). ALI is characterized by the activation of multiple inflammatory cells and production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) (Shinbori et al., 2004; Wright et al., 2004), and proteases (Matsuda et al., 2011). The major cytokine is TNF-α, which induces pulmonary endothelial cell activation, migration of leukocytes, neutrophil degranulation and capillary leakage, alveolar cell perfusion and oxygen exchange (Ma et al., 2009). Thus, inhibition of these factors may potentially be a therapeutic target in lung injury. Recently, natural products of plant origin with immense ethnopharmacological importance have been given top priority as treatments for inflammatory diseases (Talhouk et al., 2007; Li et al., 2010). In particular, the understanding of the antiinflammatory effects of alkaloids has made great progress (Meng et al., 2003). Cyclooxygenase-2, TNF-α, IL-1 and prostaglandin E2 (PGE2) can be inhibited by TAA (purity 4 75%) in arthritic rats (Zeng et al., 2009). In addition, auricular edema, permeability of celiac blood capillaries in mice and paw swelling in rats were inhibited by the total alkaloids of Aconitum naviculare, which is often used instead of Aconitum tanguticum in Tibetan pharmaceutical applications due to the similar main chemical compositions (Qu, 2009). Although Aconitum tanguticum is used to treat diseases related to inflammation in the Tibetan folk of China and TAA has exhibited a therapeutic effect on inflammation, research has mainly led to partial findings. Whether TAA has protective effect on ALI and what the underlying mechanisms of TAA action are have never been investigated. In the present study, we studied the effects of TAA on an experimental model of ALI induced by LPS and tried to clarify the mechanism involved. Our results might provide a pharmacological basis for its folkloric use in the treatment of ALI.

alkaline solution was extracted by chloroform, and 40.8 g of gray powder was obtained. 2.1.2. Quality control of TAA TAA (50 mg) was precisely weighed and dissolved in 5% potassium hydroxide in methanol (100 ml), then boiled under reflux for 1.0 h until the methanol had evaporated. The residue was adjusted to pH 3–4 with 0.5 mol/L sulfuric acid, then transferred to a separator funnel. The residue was extracted twice with 10 ml ether, then the ether liquid was combined, and the ether was removed. The residual powder was dissolved in 10 ml methanol and allowed to sit for 15 min. The test sample was finally filtered through a 0.45 μm micro-porous membrane before analysis. An Aichrom C18 (4.6  150 mm2, 5 μm) chromatographic column was employed. The column temperature was kept at 35 1C, the mobile phase was methanol–acetic acid–isopropanol– 0.05 mol/L aqueous potassium dihydrogen phosphate (67:4:4:173), the flow rate was l.0 ml/min, the detection of wavelength was 230 nm, and the injection volume was 10 μl. Three replicates of each sample were assessed. Approximately 5 mg of AC was precisely weighed and the general procedure was carried out. Benzoic acid was used as the internal standard (Chen et al., 2009). 2.1.3. Components and toxicity of TAA A study from another group showed the content for each main alkaloid in TAA was specific, such as AC, hypAC, mesacomhne, atisine, 6-acetylheteratisine, and 6-benzoylheteratisine and so on. The results of these studies will be reported in another paper. The acute toxicity of TAA was known from our previous studies and preliminary experiments when administered to mice via intraperitoneal injection, LD50 ¼338.80 mg/kg, 95% confidence limit 315.87–363.39 mg/kg. 2.2. Rats

2. Materials and methods

Male Sprague Dawley (SD) rats (6 weeks old, 200–220 g) were purchased from the Center of Experimental Animal of Gansu University of TCM (Lanzhou, Gansu, China). Rats were housed in groups of 5 under standard conditions (12 h light/dark cycle, temperature 25 70.5 1C, and relative humidity 55 75%) with standard food and water ad libitum and allowed to adapt to experiment environment for 7 days. All of the procedures for the animal study were approved by the Institutional Animal Care and Use Committee of Gansu University of TCM (Lanzhou, Gansu, China). All experiments were performed in accordance with the guidelines for the care and use of laboratory animals of the National Institute of Health.

2.1. Material and chemicals

2.3. Experimental design

2.1.1. Preparation of TAA Aconitum tanguticum was obtained from Qinghai Jinhe Tibetan Medicine Pharmaceutical Co., Ltd. (Qinghai, China), and identified by Dr. Feng lin Liu, Gansu University of Traditional Chinese Medicine(TCM). A voucher specimen (reference number 13081201) has been deposited in the herbarium stock room of the College of Pharmacy, Gansu University of TCM, Lanzhou, China. The extraction and purification of TAA were carried out according to previous reports (Wang et al., 2002). Briefly, the whole Aconitum tanguticum plant (20 kg) was powdered and immersed into 200 L 95% ethanol for 24 h, then boiled under reflux for 2.0 h twice. The solution was filtered and concentrated. The concentrate was extracted with 2% hydrochloric acid solution several times, and Na2CO3 was added to the filter liquor to adjust the pH to 9–10. The solution was allowed to sit for 24 h. After precipitation, the

Male SD rats were assigned to six groups and treated as follows: (1) normal control group ( accession to a small amount of dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO) in saline, the final concentration of DMSO was 0.05%, v/v), (2) LPS alone group (LPS 5 mg/kg), (3) TAA alone group (TAA 60 mg/kg, the final concentration of DMSO was 0.05%, v/v), (4) LPSþdexamethasone (LPSþDXM, 4 mg/kg) treatment group, (5) LPSþ HDTAA (60 mg/kg, a high dose of TAA) treatment group, and (6) LPSþLDTAA (30 mg/kg, a low dose of TAA) treatment group. The doses of these drugs and LPS were based on our previous studies and preliminary experiments. The dose of 1/20 LD50 (LD50 ¼338.80 mg/kg) could not obviously protect against LPSinduced ALI in rats, so close to 1/10 LD50 and 1/5 LD50 as low dose and high dose, respectively, were selected in the present study. The TAA powder was dissolved in a small amount of DMSO, and was diluted to the required concentration in normal saline (the final

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concentration of DMSO was 0.05%, v/v). The corresponding drugs or saline were intraperitoneally injected 1 h before intravenous LPS (5 mg/kg, from Escherichia coli in 50 ml saline, serotype O55:B5, Sigma, St. Louis, MO) or sterile saline (control, 10 ml/kg) was administered as previously described (Li et al., 2009; Huang et al., 2010; Wang et al., 2011a, 2011b). All rats were anaesthetized by the intraperitoneal administration of 1.0 ml/100 g 10% pentobarbital sodium solution, after which they were injected intravenously with 5.0 mg/kg LPS at 6, 12 or 24 h. The arterial blood was collected and analyzed for blood gas variables (PaO2, and PaO2/FiO2). Then, the rats were sacrificed, and the trachea was isolated. Bronchoalveolar lavage fluid (BALF) was obtained from the left bronchial tube with phosphate buffer solution (PBS), and the total cell counts, neutrophil and lymphocyte counts, total protein concentration and proinflammatory cytokines in BALF were measured. The right middle lung lobes were stored in liquid nitrogen at 80 1C until subsequent analysis. The right upper lung lobes were used to quantify the magnitude of the pulmonary edema. The right lower lung lobes were used for histological evaluation. 2.4. Analysis of PaO2 and PaO2/FiO2 in arterial blood The arterial blood was collected and analyzed for blood gas variables (PaO2, and PaO2/FiO2) with a fully automatic blood gas analyzer (Instrumentation Laboratory Co., USA) 2.5. Measurement of lung wet/dry (W/D) weight ratio Rats were sacrificed at 6, 12 or 24 h, after LPS treatment. Right upper lung lobe tissue samples were excised and weighed immediately after removal (wet weight), then placed in an oven at 80 1C for 48 h to determine the stable dry weight. The wet-to-dry weight ratios were calculated to quantify the magnitude of the pulmonary edema (Staub, 1978). 2.6. Measurement of cell counts and protein concentrations in BALF BALF was obtained by placing a 20-gauge catheter into the trachea. The left lung was lavaged three times with cold PBS in a total volume of 3.0 ml (1.0 ml  3). The recovery rate of BALF was above 90%. A 0.2-ml aliquot from each sample was used to determine the total cell count with a hemacytometer, and cytospins were prepared for neutrophil and lymphocyte counts by staining using a modified Giemsa method. At least 200 cells were counted per slide. The remainder was immediately centrifuged at 3000 rpm for 15 min at 4 1C. The total protein concentration in the BALF supernatant was quantified by the Bradford method with bicinchoninic acid (BCA) as a standard. The rest of supernatant was aliquoted and frozen at  80 1C for subsequent analysis of cytokine levels (Casal et al., 2000, Hashimoto et al., 2004).

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to the manufacturer's instructions (Dakewe Biotech, Beijing, China).

2.9. Lung pathological analysis Rats were sacrificed 6, 12 or 24 h after LPS challenge. The in vitro tissues of the right lower lung lobes were fixed with 4% paraformaldehyde for 24 h. The samples were dehydrated and embedded in paraffin. Sections (4-mm thickness) were cut and stained with hematoxylin and eosin (H&E) and examined with light microscopyat 200  . The total surface of the slide was scored by two blinded pathologists with expertise in lung pathology in the following four categories: alveolar congestion, hemorrhage, neutrophil infiltration into the airspace or vessel wall, and thickness of alveolar wall/hyaline membrane formation. Each category was graded on a 0–4 scale, where 0¼ no injury; 1 ¼injury up to 25% of the field; 2 ¼injury up to 50% of the field; 3 ¼injury up to 75% of the field; and 4 ¼diffuse injury (Gupta et al., 2007).

2.10. Western blot analysis Rats were sacrificed 6, 12 or 24 h after LPS challenge. Parts of the right lung tissues were flash frozen in liquid nitrogen immediately after removal and stored at  80 1C until use. The extraction of nuclear proteins from lung tissues was performed using a Nuclear and Cytoplasmic Protein Extraction Kit according to the manufacturer's protocol (Beyotime Institute of Biotechnology, Shanghai, China). The protein content in the supernatant of the lysed lungs was determined by the BCA method. Samples were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat milk in TBST (0.05%) for 1 h at room temperature, the membranes were incubated with primary antibody (anti-p65 and anti-β-actin, Cell Signaling Technology, AMRESCO, CA, USA) and then reincubated with conjugated secondary antibodies. Protein bands were detected with an ECL detection kit (AMRESCO, USA). The protein signals were quantified by scanning densitometry using a FluorChem Q System (Sangon Biotech, Shanghai, China).

2.11. Statistical analysis All data are expressed in term of means 7S.E.M and were analyzed by using one-way analysis of variance followed by the Student–Newman–Keuls test. A two-tailed P value o0.05 was considered statistically significant. SPSS 18.0 for Windows was used for the statistical analyses (SPSS Inc., Chicago, IL, USA).

3. Results 2.7. Measurement of MPO in lung homogenates 3.1. Evaluation of TAA quality The right middle lung lobes were homogenized in cool normal saline (1:10 lung tissue:normal saline). The homogenate was then centrifuged at 3000 rpm for 10 min at 4 1C. The MPO activities in the lung homogenates were examined using a MPO determination kit (Nanjing JianCheng Bioengineering Institute, China) according to the manufacturer's instructions (Shen et al., 2009). 2.8. Determination of pro-inflammatory cytokines in BALF The levels of TNF-a, IL-6 and IL-1β in the BALF were determined using commercially available rat TNF-α, IL-6 and IL-1β enzyme-linked immunosorbent assay (ELISA) kits according

The chromatogram of benzoic acid is shown in Fig. 1A. Benzoic acid was found in TAA and AC after boiling under reflux for 1 h. The reflux turned AC or TAA into benzoic acid in alkaline conditions (Fig. 1B and C). These results are in agreement with previous reports (Chen et al., 2009). Using AC as the standard, the content of total ester alkaloids in TAA was determined quantitatively as 80.6 mg/100 mg (80.6%) by HPLC. The aconite calibration curves were linear between 0.06718 and 0.09349 μg (r ¼0.9996). The average recovery of aconite was 97.10% with RSD ¼1.86 (n ¼ 6). Furthermore, the content for each main alkaloid in TAA has been determined in another study group (data not available).

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Fig. 1. HPLC chromatogram of TAA. The retention time of benzoic acid is specified on the chromatogram for the comparison (A). AC (B) or TAA (C) is transformed into benzoic acid in the alkaline conditions after boiling under reflux.

3.2. TAA increased the PaO2 and PaO2/FiO2 in arterial blood To evaluate lung function, we measured the PaO2 (Fig. 2A) and PaO2/FiO2 (Fig. 2B) in arterial blood. The PaO2 and PaO2/FiO2 were

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Fig. 2. TAA increased the PaO2 and PaO2/FiO2 in arterial blood and decreased the lung W/D ratio in rats with LPS-induced ALI. Rats were given TAA (60 mg/kg and 30 mg/kg) or DXM (4 mg/kg) as described in Section 2. The arterial blood of each rat was obtained, and rats were sacrificed at 6, 12 or 24 h after LPS challenge. The PaO2 (A) and PaO2/FiO2 (B) in arterial blood and the lung W/D weight ratios (C) were measured. Each bar represents the mean 7 S.E.M. (n¼ 10 per group). Statistical analysis was performed by one-way ANOVA. nPo 0.05, nnPo 0.01 vs. the CON group at the same time point; #P o0.05, ##Po 0.01 vs. the LPS group at the same time point.

significantly decreased in groups that received LPS compared with those of the control. However, the PaO2 and PaO2/FiO2 at 6, 12 and 24 h increased significantly after administration of TAA. TAA improved lung function in ALI rats. There was no significant

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Fig. 4. TAA decreased MPO activity in lung homogenates. Rats were given TAA (60 mg/kg and 30 mg/kg) or DXM (4 mg/kg) as described in Section 2 and sacrificed at 6, 12 or 24 h after LPS challenge. The right middle lung lobes were homogenized in cool normal saline (1:10 lung tissue: normal saline). The homogenate was then centrifuged at 3000 rpm for 10 min at 4 1C. The MPO activities in the lung homogenates were examined using an MPO determination kit. Each bar represents the mean 7 S.E.M. (n¼ 10 per group). Statistical analysis was performed with a one-way ANOVA. nPo 0.05, nnPo 0.01 vs. the CON group at the same time point; # Po 0.05, ##Po 0.01 vs. the LPS group at the same time point.

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difference in PaO2 and PaO2/FiO2 between the control and TAA alone groups. 3.3. TAA decreased the lung W/D ratio

CON TAA LPS LPS+DXM LPS+HDTAA LPS+LDTAA

To observe lung edema, the lung W/D ratios were measured (Fig. 2C). The W/D ratios significantly increased in LPS groups compared with those of controls. However, the lung W/D ratio was decreased significantly after administration of TAA at 6, 12 and 24 h. There was no significant difference in the lung W/D ratio of the control and TAA alone groups. TAA reduced the lung edema in ALI rats.

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3.4. TAA prevented LPS-induced vascular leakage in the lung

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The effect of TAA on LPS-induced lung vascular leakage was tested. TAA exhibited protective effects against LPS-induced increases in BALF total cell counts (Fig. 3A), neutrophils (Fig. 3B), lymphocytes (Fig. 3C) and protein content (Fig. 3D) at different time points. There was no significant difference in protein or cell accumulation in BALF between the control and TAA alone groups. 3.5. TAA decreased MPO activity in lung homogenates To assess neutrophil accumulation in lung tissue, MPO activity was measured. As shown in Fig. 4, there is no significant change in homogenate MPO in the control group and TAA alone group. LPS

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Fig. 3. TAA decreased the number of total cells, neutrophils and lymphocytes and the total protein concentration in the BALF of rats with LPS-induced ALI. Rats were given TAA (60 mg/kg and 30 mg/kg) or DXM (4 mg/kg) as described in Section 2 and sacrificed at 6, 12 or 24 h after LPS challenge. The trachea was isolated, the left bronchial tube was ligated, and the BALF was obtained. The total cell counts (A), neutrophils (B), lymphocytes (C) and total protein concentration (D) in the BALF were examined. Each bar represents the mean 7S.E.M. (n ¼10 per group). Statistical analysis was performed by one-way ANOVA. nPo 0.05, nn Po 0.01 vs. the CON group at the same time point; #P o0.05, ##Po 0.01 vs. the LPS group at the same time point.

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The concentration of TNF-α, IL-6 and IL-1β in BALF increased significantly at 6, 12 and 24 h after LPS injection. Pretreatment with TAA significantly attenuated the increase of TNF-α, IL-6 and IL-1β in BALF at the same time points. There was no significant difference in TNF-α, IL-6 and IL-1β in BALF in the control and TAA alone groups (Fig. 5).

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Finally, we determined whether the suppression of neutrophilic lung inflammation by TAA was associated with NF-κ B-p65 activation in the lung (Fig. 7A). The LPS group showed a significant increase in NF-κ B-p65 expression in the cytoplasm compared with that of the normal control group. Pretreatment with TAA markedly inhibited the expression of NF-κ B-p65 in the LPStreated group (Fig. 7B).

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Histological examination confirmed lung injuries in LPSinduced ALI. These injuries included pulmonary edema, infiltration of the tissue and alveoli with inflammatory cells and signs of tissue injury, as well as thickening of the alveolar wall in the lung. The histological appearance of the lung in the control and TAA alone groups was normal. TAA attenuated lung damage significantly in rats with LPS-induced ALI (Fig. 6). The results showed that LPS administration induced pulmonary edema, infiltration of inflammatory cells in the lung tissue and alveoli, and alveolar damage at 6, 12 and 24 h (Fig. 6C). However, TAA pretreatment significantly protect against lung injury (Fig. 6E and F). There was no obvious change in lung structure in the control or TAA alone groups (Fig. 6A and B).

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4. Discussion

LPS+DXM LPS+HDTAA LPS+LDTAA

We confirm that TAA protects against LPS-induced ALI rats. Moreover, results inferred that TAA suppressed neutrophil infiltration in the lung, and suppressed the key pro-inflammatory transcription factors NF-κ B, TNF-α, IL-6 and IL-1β. However, there are three aspects of this problem that have to be addressed. The first question involves methods of the ALI rat model induced by PLS, the second problem relates to the process of ALI and changes of related therapeutic effects, and the third aspect deals with the chemicals and toxicity of TAA. ALI rats can be successfully induced by intravenous injection of LPS. LPS is a principle component of the outer membrane of gramnegative bacteria and plays a key role in eliciting inflammatory responses (Saluk-Juszczak and Wachowicz, 2005). Moreover, LPS is an important inducer of lung injury that can be employed in the investigation of ALI (Kitamura et al., 2001; Wu et al., 2002). In the research of modeling method of ALI animals, LPS stimulation methods included intravenous injection (Li et al., 2009; Huang et al., 2010; Xin et al., 2011; Wang et al., 2011a, 2011b), intraperitoneal injection (Cunha et al., 2011), intratracheal instillation (Fu et al., 2012; Xu et al., 2014), intranasal instillation (Liu et al., 2010; Wan et al., 2013) and aerosol inhalation (Liu et al., 2013). Although the method previously described has achieved success, we believe that ALI has been caused by sepsis due to the serious systemic infection (Rojas et al., 2005). Thus the basic procedure is that LPS enters the blood circulation, then activates neutrophils, afterwards results in acute lung injury. Further, intravenous injection of LPS was completed to invade the bloodstream in contrast LPS intratracheal or intranasal instillation, especially easy operation was

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Fig. 5. TAA decreased the proinflammatory cytokines in the BALF of rats with LPSinduced ALI. Rats were given TAA (60 mg/kg and 30 mg/kg) or DXM (4 mg/kg) as described in Section 2 and sacrificed at 6, 12 or 24 h after LPS challenge. The trachea was isolated, the left bronchial tube was ligated and the BALF was obtained. The levels of the inflammatory cytokines TNF-α (A), IL-6 (B) and IL-1β(C) in the BALF were analyzed by TNF-α, IL-6 and IL-1βELISA kits. Each bar represents the mean 7 S.E.M. (n¼ 10 per group). Statistical analysis was performed by one-way ANOVA. n Po 0.05, nnPo 0.01 vs. the CON group at the same time point; #Po 0.05, ##Po 0.01 vs. the LPS group at the same time point.

caused a significant increase in MPO activity levels in lung homogenates compared with those of the control. Pretreatment with TAA significantly decreased MPO activity.

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the best advantage. As for the dosage of LPS, it is not the same in different treatments. The intranasal instillation or aerosol inhalation was 10–50 μg/kg (Fu et al., 2012; Wan et al., 2013; Xu et al., 2014), the intravenous injection was 4–6 mg/kg (Huang et al., 2010; Wang et al., 2011a, 2011b), even 40 mg/kg (Xin et al., 2011). In this study, the ALI rats were induced by intravenous injection of LPS (5 mg/kg), which was similar to the pathogenesis of sepsis and there was no death in ALI rats. This approach causes pulmonary inflammation as an acute injury that begins after 4 h and is maximal at 24 h. Some limitations of this study are whether there is other important organs acute injury in ALI rats, and whether it is optimistic for the survival of ALI rats after 24 h. TAA protection against LPS-induced ALI in rats is the dynamic, comprehensive process in the present study. ALI is not a specific disease in lung but a dynamic complex clinical syndrome. Firstly, we monitored pulmonary function and morphology to better reflect the changes of ALI process at 6 h, 12 h, and 24 h. The findings showed that pretreatment with TAA either increased the PaO2 (Fig. 2A) or PaO2/FiO2 (Fig. 2B), indicating that TAA protected the alveolar oxygen exchange function in ALI. We found that TAA decreased the lung W/D ratio (Fig. 2C), which indicates that TAA could inhibit the leakage of serous fluid into lung tissue and attenuate the development of pulmonary edema. The parallel results that rats exposed to LPS exhibited a massive recruitment of inflammatory cells including neutrophils and lymphocytes at 6 h, 12 h, and 24 h. In contrast, treatment with TAA inhibited the LPS-induced increase in the number of total cells and neutrophils or lymphocytes in the BALF (Fig. 3A–C). Results imply that the total cell counts, neutrophils and lymphocytes in the BALF play an important role in the development of most cases of ALI and are considered to be central to the pathogenesis of ALI/ARDS (Abraham, 2003; Cepkova and Matthay, 2006). In addition, rats exposed to LPS presented high protein content in the BALF. LPS-induced increases in total protein in the BALF were inhibited by TAA (Fig. 3D). These findings indicate that TAA prevents vascular leakage in LPS-treated lungs. Consistent with histological analysis of the lung, there was substantial infiltration of neutrophils in rats with LPS-induced ALI; TAA successfully abated lung inflammation and reduced tissue neutrophilis, which corroborated our findings in the BALF (Fig. 6B). These findings confirm that the protective effect of TAA on rats with ALI induced by LPS is related to the attenuation of inflammatory cell sequestration and migration into the lung tissue. Secondly, TAA significantly attenuated the increased IL-6 and IL-1β production and also regulated the secretion of TNF-α in the BALF of ALI rats at 6 h, 12 h, and 24 h (Fig. 5). The results that infer pro-inflammatory cytokine production in BALF were consistent with the severity of the acute lung inflammation and ALI (Krafft et al., 1996; Goodman et al., 2003;

Fig. 6. TAA improved lung histology in rats with LPS-induced ALI (magnification 200x, H-E staining). Rats were given TAA (60 mg/kg and 30 mg/kg) or DXM (4 mg/ kg) as described in Section 2 and sacrificed at 6, 12 or 24 h after LPS challenge. Tissue from the right lower lung lobe was fixed with 4% paraformaldehyde for 24 h. The samples were dehydrated and embedded in paraffin. Sections (4-mm thickness) were cut and stained with hematoxylin and eosin (H&E) and examined by light microscopy to determine the histological scores (A). Each bar represents the mean7 S.E.M. (n¼ 6 per group). Statistical analysis was performed by one-way ANOVA. nP o0.05, nnPo 0.01 vs. the CON group at the same time point; #P o0.05, ## P o 0.01 vs. the LPS group at the same time point. Histologically, the lung sections demonstrated characteristics of acute alveolar damage and acute inflammation after LPS administration. These features included the following: hemorrhage; edema; appearance and accumulation of neutrophils; and markedly thickened alveolar wall in the LPS group (D1–D3). DXM (E1–E3), HDTAA (F1–F3) or LDTAA (G1– G3) treatment prevented the above tissue changes in LPS-induced ALI, and some parts of the tissues exhibited normal lung histological features. Lungs from the normal control group (B) and TAA group (C1–C3) displayed normal histological features. The figure is representative of three replications.

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time

CON

TAA

LPS

LPS+DXM

LPS+HDTAA

LPS+LDTAA

6h NFκB-p65 12 h

24 h β-actin

Relative protein expression

1.6 1.4 1.2 1.0 0.8 0.6 6h

12h

24h

Fig. 7. TAA inhibited NF κ B-p65 activation in the lung of rats with LPS-induced ALI. Rats were given TAA (60 mg/kg and 30 mg/kg) or DXM (4 mg/kg) as described in Section 2 and sacrificed at 6, 12 or 24 h after LPS challenge. The extraction of nuclear proteins from the remainder of the right lung tissue was performed with a Nuclear and Cytoplasmic Protein Extraction Kit and subjected to Western blotting. The protein content in the supernatant of the lysed lungs was determined by the BCA method. Blots are representative of three replications in each group. Each bar represents the mean 7 S.E.M. (n¼10 per group). Statistical analysis was performed by one-way ANOVA. nP o0.05, nn P o0.01 vs. the CON group at the same time point; #Po 0.05, ##Po 0.01 vs. the LPS group at the same time point.

Shinbori et al., 2004; Wright et al., 2004). In addition, TNF-α signaling affects various cytokines that also play a role in ALI (Wang et al., 2007; Ma et al., 2009). Moreover, the activation of NFκ B, which is an important transcription factor in regulation of inflammation and oxidative stress, is an indispensable event in ALI/ARDS (Schwartz et al., 1996; Matsuda et al., 2011; Wang et al., 2011a, 2011b; Peng et al., 2012). In the present study, we also report that LPS caused a significant increase in the nuclear translocation of NF-κ B-p65 in the lung tissue 6, 12 and 24 h after LPS challenge, whereas TAA treatment significantly reduced NF-κ B activation (Fig. 7). In addition, we also found that pretreatment with TAA significantly elevated the MPO activity in the lung (Fig. 4). The results support decreased MPO activity which is associated with favorable prognosis of lung injury (Abraham, 2003; Pérez et al., 2012). Notwithstanding its limitation, this study does suggest that anti-inflammatory cytokines (IL-4, IL-10, and IL11) production and oxidant activity have been considered the important component of the pathogenesis of ALI. However, these problems can be solved if we consider that ALI is the result of the imbalance between the systemic inflammatory response and compensatory anti-inflammatory response. Despite its preliminary character, this study can clearly indicate that the protective effects of TAA on ALI are induced by LPS through the improvement of the pathological changes in the lung, inhibition of NF-κ B activation, and decreasing inflammatory cell infiltration, vascular leakage and pro-inflammatory cytokine release. Another problem we have to face is the chemicals and toxicity of TAA, Aconitum tanguticum has slight toxicity in clinical use, for the content of AC is relatively low (Luo et al., 2012a). In toxicity studies, our results show that TAA can protect lung function by its anti-inflammatory activity without a significant cellular toxicity in lung tissue (Figs. 1–6). In addition, a study from another group showed that the acute toxicity of TAA was known when administered to mice via intraperitoneal injection, LD50 ¼338.80 mg/kg,

95% confidence limit 315.87–363.39 mg/kg. However, there was no particular toxicity to the liver or kidney. Unfortunately, the acute toxicity of AC is very serious, TD50 ¼ 10.45 μ g/kg, LD50 ¼97.98 μ g/ kg (Zhang et al., 2012b). And preliminary studies indicate that double ester alkaloid toxicity is very serious, especially AC, while the monoester benzoylaconine toxicity is 1/200–1/500 of AC, and have the same anti-inflammatory, antihypertensive activity (Li et al., 2014). So we speculate that the content of AC in TAA is less, but alkaloids containing benzoyl are the main components. Based on the above results, we found that TAA might be a useful therapy for lung injury and could be developed as a potent yet safe suppressor of lung inflammation in ALI/ARDS.

5. Conclusion In summary, the present data proved the protective effects of TAA on acute lung injury induced by LPS through the improvement of the pathological changes in the lung, inhibition of NF-κ B activation, and decreasing inflammatory cell infiltration, vascular leakage and pro-inflammatory cytokine release. This study suggests that TAA may be a useful therapy for lung injury.

Acknowledgments This study was supported by the “Five-Year” plan for national science and technology projects in rural areas of China (No. 2011AA10A214) and the Program of Gansu Province Key Laboratory of Pharmacology and Toxicology of TCM (No. ZDSYS-KJ-2012004). The authors are grateful to Dr. Yali She for providing technical assistance in pathological observations and to Dr. Xiaoming Sun for English language revisions.

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The total alkaloids of Aconitum tanguticum protect against lipopolysaccharide-induced acute lung injury in rats.

Aconitum tanguticum has been widely used as a remedy for infectious diseases in traditional Tibetan medicine in China. The total alkaloids of Aconitum...
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