The therapeutic effect of Bletilla striata extracts on LPS-induced acute lung injury by regulation of inflammation and oxidation

Abstract

Bletilla striata, a widely used traditional herbal medicine, has been shown to exhibit various biological activities but its antioxidative and anti-inflammatory activities have not been well studied. In this study, we isolated the active ingredients from B. striata, and identified the ingredient structures from the highest antioxidative fraction BM60 using HPLC-ESI-HRMS and then investigated the antioxidative and anti-inflammatory responses and the underlying mechanisms of fraction BM60 both in vitro and in vivo. The BM60 treatment reduced the production of NO in NR8383 macrophages. In addition, acute lung inflammation was induced in mice by intratracheal instillation of lipopolysaccharide (3.0 mg kg⁻¹), and treatments with BM60 at the doses of 35, 70, 140 mg kg⁻¹ significantly reduced macrophages and neutrophils in the bronchoalveolar lavage fluid (BALF) and markedly ameliorated lung wet-to-dry weight (W/D) ratios, myeloperoxidase (MPO) activity and pulmonary histopathological conditions. Moreover, the treatment might be attributed to the down-regulations of neutrophil infiltration, malondialdehyde (MDA) and up-regulations of superoxide dismutase (SOD) in lung tissues. In addition, the BM60 treatment inhibited the infiltration of inflammatory cells and tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) production. The results from histopathological and immunochemical analyses confirmed the above biochemical assay data. The preliminary mechanistic study indicated that BM60 suppressed LPS-induced nuclear factor-κB (NF-κB) activation in a dose-dependent manner. Therefore, this work prompted further evaluation of B. striata to discover individual active ingredients for preventing acute lung injury in the future.

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Introduction

Lung inflammatory responses, characterized by the accumulation of immune cells, are associated with many respiratory diseases, including chronic obstructive pulmonary disease and emphysema.¹ The inflammatory process plays a key role in the development of ALI, and the main pathological change of ALI is acute leakage inflammatory response with leakage of protein into the alveolar space, inflammatory cell accumulation, interstitial edema, and disruption of epithelial integrity.² Now, pulmonary disease is a common clinical problem associated with significant morbidity and mortality in some emergency infectious diseases.^3,4

Lipopolysaccharide (LPS), a well-known endotoxin, elicits immune responses by promoting the secretion of pro-inflammatory cytokines,³ such as tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), chemokines, and inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), which are synthesised by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively.⁵ Accordingly, intratracheal instillation of LPS is considered as an extremely common modeling method of ALI in mice and the symptoms of LPS-stimulated ALI in mice is nearly consistent with those observed in humans. Nuclear factor-kappa B (NF-κB), one of the multipotent transcriptional factors, is required for maximal transcription of TNF-α, IL-1β, and IL-6, and plays an important role in the pathogenesis of lung diseases.⁶ Therefore, it has been suggested that inhibitors of NF-κB function may be useful as anti-inflammatory agents. The intratracheal instillation of LPS has been widely used to study the pathogenesis and prevention of ALI in mice. Oxidative stress is defined as a status of an imbalance between cellular anti-oxidative capacity and reactive oxygen species (ROS) formation caused by the dysregulation of antioxidant system.⁷ Thereby, the amelioration of the imbalance condition by enhancing cellular antioxidant capacity or scavenging ROS may make some difference for a variety of pathology and disease models.^8,9

Bletilla striata (Thund.) Reichb. f. (Orchidaceae), a perennial herb, mainly distributes in China, North Korea, Japan and Burma.¹⁰ The tubers of Bletilla striata are a traditional Chinese herbal medicine in the treatment of ulcers, bleeding, burns and other skin wounds, and is also known to be involved in antioxidant¹¹ and antitumor activities.¹² Chinese Pharmacopoeia states that Bletilla striata possesses the functions of hemostasia, detumescence, healing and enhancement of bodily function.¹⁰ Pharmacological researches have shown that Bletilla striata polysaccharides are an important functional factor in traditional Chinese medicine. Phytochemical analyses of B. striata identified nearly 80 secondary metabolites. The dominant chemical components of this genus include phenanthrenes,^13,14 bibenzyls,¹⁵ flavonoids, and biphenanthrenes,^16,17 which are extremely common in the family Orchidaceae. Moreover, a few triterpenoids, steroidal saponins, lignans, and glucosyloxybenzyl 2-isobutylmalates have been reported for the species.^18–20

It was reported that polysaccharide could induce the proliferation of vascular endothelial cells and growth factor,²¹ immuno-enhancement²² and act on macrophage and simulate cells to expression some pro-inflammatory cytokines.^23–25 Additionally, the EtOAc-soluble fraction of the 80% ethanol extract of Bletilla has been reported to exert anti-oxidative and anti-inflammatory activity in vitro.¹⁰ However, there are little reports on the fraction of the ethanol extract from Bletilla striata via oral administration for its in vivo anti-inflammatory activity and related mechanisms. Therefore, we investigated the anti-inflammatory activities of Bletilla striata extract in a LPS-induced pulmonary inflammation animal model. The major compounds in the tested extracts were tentatively identified by high performance liquid chromatography-mass spectrum (HPLC-HRMS) analysis for the tubers of this species. To better understand the therapeutic use of B. striata, we investigated the anti-inflammatory responses and the underlying mechanisms of the different partition fractions in vivo.

Results and discussion

Characterization of Bletilla striata extracts

Total phenolic contents of B. striata extracts were determined using Folin–Ciocalteu reagent. It is found that BM60 has the highest phenolic content, followed by BM70, BM80, BM90, while BM30 has the lowest phenolic content (Table 1). The phenolic in BM60 were found to be 27.51 ± 3.12 mg gallic acid equivalent per gram of dry weight.

Table 1 Total phenolic contents expressed as mg GAE per g

Fractions	BM30	BM60	BM70	BM80	BM90
mg GAE per g	0.27 ± 0.24	27.51 ± 3.12	24.68 ± 3.34	10.76 ± 2.11	6.35 ± 1.59

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The DPPH free radical scavenging activity of BM60, BM70, and BM80 were tested at five different concentrations (0.05, 0.10, 0.20, 0.30, 0.40 mg mL⁻¹) and the results were summarized in Table 2. Compared with BM70 and BM80, BM60 exhibited more significant free radical scavenging activity and results showed that the scavenging activity was concentration dependent and statistically significant (p < 0.05).

Table 2 DPPH free radical scavenging activity (%)

C (μg mL^−1)	BM60	BM70	BM80
50	26.29 ± 2.63	12.21 ± 0.30	9.308 ± 0.47
100	38.52 ± 2.31	18.52 ± 2.17	12.98 ± 0.38
200	67.75 ± 2.42	30.33 ± 4.66	17.60 ± 1.13
300	78.66 ± 1.85	41.54 ± 1.02	25.06 ± 1.45
400	84.10 ± 2.40	50.23 ± 3.17	32.18 ± 1.59

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Based on the results above, BM60 was selected for the further analysis. From an analytical point of view, one of the most powerful tools is mass spectrometry since it provides high sensitivity, opportunities of coupling with liquid chromatography. Single stage MS joined with UV detector is seldom employed for full structural characterization, but if standards or reference data are available it is efficient for confirming or identifying target ingredients.^26,27 The major compounds in BM60 were detected and identified using HPLC-ESI-HRMS, by comparing their chemical formulas and indices of hydrogen deficiency with those of commercial standards and literature data. Fig. 1 shows the HPLC-ESI-HRMS total ion current (TIC) chromatograms and the corresponding HPLC chromatograms (254 nm). Table 3 shows the molecular formula deduced from a molecular ion peak in the HPLC-ESI-HRMS spectrum considering the elution order.

Fig. 1 TIC chromatograms and corresponding HPLC chromatograms (254 nm) of BM60.

Table 3 Characterization of compounds in the BM60

Peak no.	Rt	m/z	Theo. mass	ppm	RDB equiv.	Composition
1	6.07	744.3077	744.3073	0.50	10.5	C34H46O17
2	7.60	490.2278	490.2283	−1.00	5.5	C22H32O11
3	9.03	504.2438	504.2439	−0.27	5.5	C23H34O11
4	9.59	301.0707	301.0707	0.12	10.5	C16H12O6
5	10.40	257.0809	257.0808	0.25	9.5	C15H12O4
6	11.20	255.0653	255.0652	0.45	10.5	C15H10O4
7	11.81	259.1330	259.1329	0.50	7.5	C16H18O3
8	12.89	363.1228	363.1227	0.27	13.5	C22H18O5
9	13.38	243.1016	243.1016	0.12	8.5	C15H14O3
10	14.17	241.0861	241.0859	0.74	9.5	C15H12O3
11	14.36	257.0809	257.0808	0.25	9.5	C15H12O4
12	15.17	421.1644	421.1646	−0.39	13.5	C25H24O6
13	15.34	271.0966	271.0965	0.42	9.5	C16H14O4
14	16.07	271.0966	271.0965	0.42	9.5	C16H14O4
15	17.02	349.1434	349.1434	−0.10	12.5	C22H20O4
16	17.49	485.1955	485.1959	−0.75	16.5	C30H28O6
17	17.84	241.0861	241.0859	0.74	9.5	C15H12O3
18	17.88	347.1279	347.1278	0.33	13.5	C22H18O4
19	18.45	245.1173	245.1172	0.32	7.5	C15H16O3
20	19.48	483.1795	483.1802	−1.48	17.5	C30H26O6
21	20.63	351.1592	351.1591	0.33	11.5	C32H22O4
22	21.79	351.1592	351.1591	0.33	11.5	C32H22O4
23	23.25	457.2007	457.2010	−0.55	15.5	C29H28O5

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Previous chemical investigations of B. striata led to the isolation of nearly 80 secondary metabolites.²⁸ In this study, our analysis revealed a high content in polyphenols and 23 compounds were detected from BM60 and their proposed structures are listed in Fig. 2, which belong to bibenzyls (7, 16, 19, 21–23),^29–33 phenanthrenes (5, 6, 10, 11, 13, 14, 17, 18),^30,34 dihydrophenanthrenes (8, 9, 12, 15),^30,35–37 biphenanthrenes (20),^16,17 flavonoids (4),³⁷ and glucosyloxybenzyl 2-isobutylmalates (1–3).^29,32

Fig. 2 Proposed structures of compounds identified in BM60 (1–23).

Phenolic compounds are known to be the most potent antioxidants from herbs, the activity of which is related to the presence of a number of phenolic hydroxyl groups which are attached to the ring structures. Previous phytochemical studies on Bletilla species have led to the isolation of phenanthrene derivatives, bibenzyls, flavonoids, phenolic compounds, cyanidin glycosides, triterpenoids, and anthocyanins. It is noteworthy that the EtOAc-soluble fraction from the 80% EtOH extract of B. striata has been reported to exert anti-oxidative and anti-inflammatory activity in vitro.¹⁰ However, there is no report to explain the therapeutic effect of extract on acute lung injury in vivo. As to our knowledge, the present work was the first study focused on the protective effect on LPS-induced acute lung injury and the potential mechanism through the regulations of inflammation and oxidative state.

Effect of BM60 on cell viability and nitric oxide production

The NR8383 cells were treated with various concentrations (1–100 μg mL⁻¹) of BM60 for 24 h and 48 h. The cell viability was determined by the CCK8 assay. As shown in Fig. 3A and B, in order to exclude BM60 mediated cytotoxicity, non-lethal concentrations (1, 10, 25 μg mL⁻¹) were used in the subsequent experiments. In the progress of inflammation, the cells were pretreated with BM60 (1, 10 and 25 μg mL⁻¹) before LPS stimulation and measured NO production. In Fig. 3C, the model group, stimulated by LPS, had significantly increased level of NO production as opposed to the control group. Compared with the model group, BM60 slightly repressed NO production at low concentration, but the inhibitory effect of BM60 on NO production was strongly elevated at concentrations of 10 and 25 μg mL⁻¹, dose-dependently. BM60 at the concentration of 10 and 25 μg mL⁻¹ showed 46% and 68%, respectively, reductions in NO production.

Fig. 3 (A) The effects of BM60 on the cell viability of NR8383 cells at 24 and 48 h, as revealed by the CCK8 assays. (B) The NR8383 cells were pretreated with BM60 for 2 h before 1 μg mL⁻¹ LPS challenge. (C) Effect of BM60 on LPS-induced NO production in NR8383 cells. CS: control group, the cells without any treatments; LPS: model group, the cells challenged with LPS. ^##p < 0.01 vs. CS; *p < 0.05, **p < 0.01 vs. LPS. All the experiments were repeated three times at least.

Anti-inflammatory and antioxidative activities in vivo

In the design of experimental studies, the experimental animals were concurrently treated to assess the effects of extracts from BS on lung injuries induced by LPS. The inflammatory mediators such as iNOS and MPO as well as oxidative defensive enzymes such as SOD and MDA were determined in vivo to evaluate anti-inflammatory and anti-oxidative effects of extracts. Additionally, inflammatory mediators such as TNF-α, IL-1β, and IL-6 were also examined in lung to further substantiate the study results. Finally, the histopathological analysis and NF-κB p65 immunohistochemistry of mouse lung tissues was performed to verify the treatment effects of extracts on the attenuation of lung injury.

Neutrophils, one of the most important components of the initial innate immune response in the lung against bacterial infections are the earliest immune cells to be recruited to the site of injury and express multiple cytotoxic products.³⁸ In LPS-induced ALI, the neutrophils accumulated in the lungs, changed the expressions of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 and finally led to the pulmonary injury.⁶ In this study, we found that the neutrophils clearly increased in lung tissues after LPS exposure and the effect of BM60 on the number of inflammatory cells in BALF was shown in Fig. 4A. The number of total cells and neutrophils increased significantly after LPS treatment. BM60 dose dependently inhibited the number of total cells and neutrophils in BALF induced by LPS. As expected, BM60 pretreatment significantly decreased the neutrophils in lung tissues.

Fig. 4 (A) Total cell and neutrophil accounts in BALF. (B) Effects of BM60 on LPS-induced lung wet-to-dry ratios. CS: control group, mice without any treatments; MS (LPS): model group, mice challenged with LPS; PS: positive control group, mice treated with Dex. All values expressed as the mean ± SD. ^##p < 0.01 vs. CS; *p < 0.05, **p < 0.01 vs. MS.

Lung wet-to-dry ratio was measured to independently evaluated the severity of pulmonary edema, which is a typical symptom of local and systemic inflammation.³⁹ As shown in Fig. 4B, LPS treatment significant increased the lung wet/dry ratio, compared with the control group (p < 0.01). However, the lung wet/dry ratios of the mouse treated by BM60 and Dex exhibited variability when compared with those in the LPS group, and those differences achieved statistical significance. The data showed that the extracts of BM60 could decrease the LPS-induced lung wet-to-dry ratio, which suggested that BM60 have a protective effect on LPS-induced ALI.

As a trusty index of oxidative stress, such as MDA, SOD, and GSH-Px, which regulate ROS, may be of great importance in reducing the harmful effects of ROS.^40–42 In this study, the mice treated by intratracheal LPS presented a significant rise in MDA in lung (Fig. 5A). On the contrary, the level of SOD was dramatically decreased (Fig. 5B). The treatment with BM60 (35, 70, and 100 mg kg⁻¹) seems to decrease the levels of MDA, and increase the level of SOD in lung in dose-dependent fashion with the same tendency to Dex (1 mg kg⁻¹, p.o.) which used as a positive control. It was previously reported that phenolic compounds had the best antioxidant potential among the phytochemicals, which is consistent with the results obtained in the therapeutic treatment evaluations in this study.

Fig. 5 Effects of BM60 on the levels of MDA and SOD in the lungs. CS: control group, mice without any treatments; MS (LPS): model group, mice challenged with LPS; PS: positive control group, mice treated with Dex. All values expressed as the mean ± SD. ^##p < 0.01 vs. CS; *p < 0.05, **p < 0.01 vs. MS.

MPO, as a major constituent of neutrophil cytoplasmic granules, plays an important role in the initiation and progression of acute and chronic inflammatory diseases.⁴³ The total activity of MPO in a tissue is therefore a direct measure of neutrophil sequestration in that tissue. In the present study, the activity of MPO was significantly increased in lung tissues after LPS challenge. As expected, the extracts was capable of attenuating the activity of MPO and the neutrophil infiltration in lung tissues, indicating the protective effects of BM60 against LPS-induced inflammatory damage to the lungs in treatment modes (Fig. 6A).

Fig. 6 Effects of BM60 on MPO activity in lung homogenate, and cytokines in BALF. CS: control group, mice without any treatments; MS (LPS): model group, mice challenged with LPS; PS: positive control group, mice treated with Dex. Values are expressed as means ± SD. ^##p < 0.01 vs. CS; *p < 0.05, **p < 0.01 vs. MS.

Additionally, the levels of inflammatory cytokines in BALF were measured as key markers to evaluate the degree of inflammatory responses.^44,45 Inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 are well-characterized cytokines involved in the inflammatory process of acute lung injury. TNF-α is the earliest and primary endogenous mediator of the process of an inflammatory reaction produced by the immune system. TNF-α, mainly produced by monocytes/macrophages, can elicit the inflammatory cascade, cause damage to the vascular endothelial cells, and induce alveolar epithelial cells to produce other cellular factors, such as IL-6. Interleukin-1 (IL-1) is a prototypical pro-inflammatory cytokine that stimulates both local and systemic responses, also known as endogenous pyrogen, leukocyte endogenous mediator, mononuclear cell factor and lymphocyte activating factor.³ The data presented in Fig. 6B–D demonstrate that BM60 may significantly inhibit the production of TNF-α, IL-1β, and IL-6. The levels of TNF-α, IL-1β, and IL-6 evidently increased in BALF after LPS exposure, while BM60 treatment significantly decreased these cytokines in BALF compared to the model group.

In addition, histopathological study also confirmed the protective effect of extracts on acute lung injury (Fig. 7). The results of the present study indicate that the histopathological damage and neutrophil infiltration induced by LPS was attenuated by the extracts treatments, indicating that extracts could be used in therapeutic treatment of the lung injury.

Fig. 7 The architecture pictures of the lung tissues with different treatments. (A) CS: blank control group, without any treatments; (B) MS: model group, challenged with LPS; (C) PS: positive control group, treated with Dex; (D) BM60 (L); (E) BM60 (M); (F) BM60 (H).

NF-κB comprises a family of transcription factors that act as regulators of pro-inflammatory mediators.^46,47 It is well known that NF-κB P65 is a key signaling pathway accounting for the expression of proinflammatory cytokines induced by LPS. Therefore, the possibility of BM60 inhibiting the production of TNF-α, IL-1β, and IL-6 by interfering with the activation of NF-κB was investigated. As can be seen from the result of NF-κB p65 immunohistochemistry (Fig. 8) and western blot (Fig. 9), BM60 was found to down regulate NF-κB P65 expression and suppress the inflammatory and apoptosis activities in LPS-induced ALI by blocking the NF-κB pathway in lung.⁴⁸

Fig. 8 The results of NF-κB p65 immunohistochemistry assays. (A) CS: blank control group, without any treatments; (B) MS: model group, challenged with LPS; (C) PS: positive control group, treated with Dex; (D) BM60 (L); (E) BM60 (M); (F) BM60 (H).

Fig. 9 Effects of BM60 on the protein expression of NF-κB activities in LPS-induced mice. CS: control group, mice without any treatments; MS (LPS): model group, mice challenged with LPS; PS: positive control group, mice treated with Dex. **p < 0.01 vs. MS. All the experiments were repeated three times at least.

The analysis results of these tests above demonstrated that BM60 treatment group was found to significantly ameliorate the levels of lipid peroxidation and inflammatory cytokines compared with those in LPS-induced group, and the experimental data suggested that the conservatory effect on acute lung injury after LPS stimulation ascribed to the change in the mediation of concerning biological indicators.

Experimental section

Plant materials and reagents

Bletilla striata (BS) was supplied by Changsha central hospital (Hunan, China). Dexamethasone (Dex) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Lipopolysaccharide (LPS, from Escherichia coli 055:B5, L2880, lyophilized powder) was purchased from Sigma (St. Louis, MO, USA). Mouse tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) Enzyme-Linked Immunosorbent Assay (ELISA) kit was purchased from Shanghai Xi tang Biotechnology Co. (Shanghai, China). The levels of myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using commercial kits was provided by Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Anti-NF-κB p65 and anti-β-actin monoclonal antibodies were acquired from Proteintech Biotechnology (Rocky Hill, USA). All other chemicals used in the experiments were of reagent grade.

Experimental cells and animals

The murine macrophage cell line NR8383 was obtained from ATCC (Manassas, VA USA) and maintained in F-12K medium supplemented with 15% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units per mL penicillin and 100 μg mL⁻¹ streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂.

Male SD mice (180–200 g) were obtained from the Center of Experimental Animals of Shandong University (Jinan, China). The mice were acclimatized for 5 days under standard laboratory conditions with free access to food and water before the experiments. All procedures in this study complied with the institutional guidelines of the Animal Care and Use Committee of Shandong University and the Committee have approved the experiments.

Extraction and partition of B. striata

B. striata (50 g) was refluxed with 95% ethanol (200 mL) three times for 1 h. The ethanol extract was filtered and concentrated in vacuo to give the ethanol extract (3.15 g). The concentrated ethanol extract was suspended in water and then extracted with petroleum ether (0.35 g), ethyl acetate (1.02 g), and n-butyl alcohol (1.10 g), respectively. The ethyl acetate extract was partitioned successively with different proportions of methanol (30%, 60%, 70%, 80%, 90%) through MCI (BM). The 60% fraction (BM60, 0.077 g) was concentrated in a rotary evaporator and the resulting brown resin was obtained. 30% (BM30, 0.358 g), 70% (BM70, 0.013 g), 80% (BM80, 0.052 g), 90% (BM90, 0.032 g).

Determination of total phenolic content

Total phenolic content was determined by Folin–Ciocalteu assay.⁴⁹ In brief, 8 μL of reagent and 20 μL of the proper dilution of test sample were added to each well of a 96-well plate. The contents of the wells were pipetted up and down to mix and then allowed to stand for 10 min. After this, 200 μL of a 2% aqueous sodium carbonate solution was added to each well. The contents of the wells were mixed and incubated at room temperature for 10 min. Absorbance was read at 620 nm on a the microplate reader (Infinite M200, Tecan, Switzerland). The results are expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE per g).

DPPH radical scavenging assay

The free radical scavenging activity of BM from B. striata was measured using the previous methods^50,51 with slight modification. Initially, 1 mL of methanol solution containing each of the samples at different concentrations (0.05, 0.10, 0.20, 0.30, 0.40 mg mL⁻¹) was mixed with 2 mL solution of DPPH radical in ethanol (0.10 mM), respectively. After mixed by vortex, the mixture was then incubated for 30 min at room temperature in the dark, and the absorbance at 519 nm of the resulting solution was measured in triplicate. Lower absorbance of the mixture indicated higher free radical scavenging activity of the sample. DPPH radical scavenging activity was calculated according to the following formula:
where A₀ is the absorbance of DPPH solution without the tested samples.

HPLC-ESI-HRMS analysis

In order to ensure the major components of BM60 used in this study, high performance liquid chromatography (HPLC) analysis was performed. Briefly, the sample solutions were put into the HPLC system (Agilent 1260 HPLC system, Santa Clara, CA, USA), which is equipped with a DAD system and a Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 μm) was used for HPLC analysis. HPLC was performed at a constant flow rate of 0.6 mL min⁻¹. The sample injection volume was 10 μL, and the mobile phase was composed of deionized water with 0.1% formic acid (A) and acetonitrile (B). The gradient elution program was as follows: 0–18 min, 45% (B); 18–25 min, 45–80% (B). The column temperature was set at 30 °C, and the detection wave length was set at 254 nm.

LC-HRMS experiments were carried out on a Thermo Electron LTQ/Orbitrap XL hybrid mass spectrometer (Thermo-Finnigan, Bremen, Germany) equipped with an electrospray ionization interface. An Accela HPLC system (ThermoElectron) was equipped with an autosampler, a vacuum degasser unit and a quaternary pump. The mass spectrometer employing positive ionization was calibrated across m/z 100–2000 using the manufacturer's calibration standards mixture (caffeine, MRFA and Ultramark 1621 in an acetonitrile–methanol–water solution containing 1% acetic acid) allowing for mass accuracies < 5 ppm in the external calibration mode. The ionization voltage was 3.5 kV, and the capillary temperature was set at 300 °C. Nitrogen was used as both the sheath gas (40 units) and auxiliary gas (15 units). The resolving power was 15 000 for full-scan and MS acquisition was set with ascan range of m/z 100–900.

CCK8 assay for cell viability and proliferation

Cells were cultured in F-12K supplemented with 15% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 units per mL penicillin and 100 μg mL⁻¹ streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂. After incubation with BM60 with or without LPS for 24 h, the cell viability was determined with Cell Counting Kit 8 (CCK8). Briefly, cells were seeded in a 96-well plate and processed according to the experimental design. On the day of detection, 10 μL assay reagent was added into the cell supernatant, and the OD 450 nm value was detected 1.5 h later. The absorbance in cultures treated with LPS alone was regarded as 100% cell viability. This experiment was repeated in quadruplicate wells, and the result was expressed as the mean value ± standard deviations (SD).

Quantification of nitric oxide production

Nitrite accumulation in culture media was used as an indicator of NO production.⁵² NR8383 cells were seeded onto a 24-well plate (5 × 10⁵ cells per mL) and incubated at 37 °C for 24 h. The cells were pretreated with extracts (1, 10 and 100 μg mL⁻¹) for 4 h and then stimulated with 1 μg mL⁻¹ of LPS for 24 h. NO concentrations in the supernatant were measured using the Griess reagent system (Promega, Madison, WI, USA). Absorbance was measured by the microplate reader at 540 nm. The NO concentrations were determined using a NaNO₂ standard curve.

Experimental groups and administration

Acute lung injury was induced in mice by non-exposure intratracheal instillation of LPS (3.0 mg kg⁻¹). Thirty-six mice were randomly divided into six groups and each group contains six mice: one group of healthy mice injected once daily with a physiological saline was used as a blank control (CS); and one group of the LPS-treated mice orally administrated once daily with a physiological saline was used as a model control (MS); 1 mg kg⁻¹ bodyweight dexamethasone (Dex, i.p.) was used as a positive control (PS) after the intratracheal instillation of LPS. Administration of the BM60 extract of 35, 70, and 140 mg kg⁻¹ (L, M, H) was performed by oral gavages. All animals were orally administrated daily for one week. 24 h after the last oral gavage dosing, the mice were sacrificed for further experiments.

Collection of bronchoalveolar lavage fluid and cell counting

After being anesthetized, the rats were exsanguinated and the right lung was lavaged with 8 mL cold phosphate-buffered saline (PBS) for four times. The recovery rate was 90% and retrieved PBS was kept on ice. After lavages, the BALF samples were centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The number of total cells was counted using a hemocytometer and differential cell counts were measured on slides prepared by cytocentrifugation and Diff-Quick staining using light microscopy. The cell-free supernatants were removed and stored at −80 °C for further use.

Measurement of wet-to-dry ratio of the lungs

The middle lobe of the left lung was excised and weighed to measure the wet weight, and then dried at 60 °C for 48 h to obtain the stable dry weight. The index of pulmonary edema was evaluated by the wet weight-dry weight ratio (W/D) of the lung tissues.

Measurement of MPO activity in lung and inflammatory cytokine in BALF

MPO activity is a significant marker for neutrophil accumulation in inflammatory tissues. Briefly, lung tissues were homogenized and fluidized with extraction buffer to obtain 10% homogenate. The homogenate was then centrifuged at 4500 rpm at 4 °C for 20 min, and the supernatant assayed with an MPO commercial kits according to the instructions recommended by the manufacturers. The enzymatic activity was examined at 460 nm using a microplate reader. All the samples were assayed in triplicate.

The levels of TNF-a, IL-1β, and IL-6 in BALF were determined with the relevant ELISA kits according to the manufacturer's instructions. The optical density of each well was assayed at 450 nm with a microplate reader. Finally, the contents were calculated according to the standard curves.

Determination of antioxidant system and lipid peroxidation products

The homogenate was collected as above to assay MDA and SOD levels. In brief, centrifuged at the speed of 3000 rpm for 10 min at 4 °C, the supernatant was transferred and stored at −80 °C. Subsequently, the following operations were conducted according to the instructions of commercial kits.

Histopathological and immunohistochemistry evaluation

Lung specimens were excised, fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin and sectioned at 4 mm thickness by rotary microtome. The sections were stained with hematoxylin and eosin (H&E) stain for histopathological analysis. Subsequently, 5 μm paraffin-embedded sections were cut for NF-κB p65 immunohistochemistry. The sections were treated with 3% H₂O₂ for 10 min and incubated with the IgG against mouse NF-κB p65 (1 : 200) at 4 °C overnight. After washing three times, the sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. All the sections were analyzed after diaminobenzidine staining. The pulmonary histopathological and immunohistochemistry were examined using light microscopy at a magnification of 200×.

Western blot analysis

The lung tissues from different rats were homogenized in liquid nitrogen and incubated in lysis buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland) to obtain protein. Protein concentrations were determined using a BCA protein kit (Bio-Rad, USA). Subsequently, 20 μg of proteins were loaded, separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transblotted onto polyvinylidene fluoride (PVDF) membrane. Then the membrane was blocked in 5% skimmed milk for 1 h, and incubated with incubated with primary antibodies (1 : 1000) at 4 °C overnight. After being washed three times using PBST solution, the membranes were incubated with secondary antibodies (1 : 10 000) at room temperature for 1 h. Finally, blots were developed by enhanced chemiluminescence (GEhealthcare). Bio-Rad Quantity One Software was used for the densitometric analysis.

Statistical analysis

All data are presented as a mean ± SD and statistical analysis was evaluated using the statistical software GraphPad Prism. Group differences were performed using an unpaired t-test. A p value of p < 0.05 or p < 0.01 was considered statistically significant.

Conclusions

In this work, the common Chinese herb B. striata was extracted by using ethanol and resulting fractions were tested for antioxidative activities. The fraction BM60 with the highest free radical scavenging activity was then selected for further evaluation. The active ingredients in the BM60 were identified by using HPLC-ESI-HRMS and their structures were proposed based on various analyses and literature data. The major active ingredients in the BM60 was tentatively identified as phenolic compounds with the major backbones of bibenzyls, phenanthrenes, and dihydrophenanthrenes, the structures of which are consistent with reported literature data. The antioxidative and anti-inflammatory effects of the B. striata extract BM60 and underlying mechanisms both in vitro and in vivo were then evaluated. Our study results show that the B. striata fraction BM60 had strong antioxidative and anti-inflammatory activities against LPS-induced lung oxidative stress and inflammation both in NR8383 macrophage cells and mouse models. The results from histopathological and immunochemical analyses confirmed those enzymatic assay data. In addition, the preliminary mechanistic study indicated that the therapeutic effects of the fraction BM60 on ameliorating mouse acute lung injury may be partially attributed to blocking the NF-κB pathway. Since the antioxidative and anti-inflammatory pharmacological activities of BM60 came from holistic summation of all the components in the extract, it would be very difficult to define the pharmacological efficacy at the individual molecular level. Therefore, further studies should be conducted in order to distinguish the therapeutic roles of individual active compounds in BM60, which could then lead us to discover the new therapeutic agents for inflammation related lung diseases such as lung injury, lung cancer and COPD.

Acknowledgements

This work was supported by the funds from National “Major Science and Technology Project–Prevention and Treatment of AIDS, Viral Hepatitis, and Other Major Infectious Diseases” (Grant #2013ZX10005004), Major Project of Science and Technology of Shandong Province (Grant #2015ZDJS04001), Science & Technology Enterprise Technology Innovation Fund of Jiangsu Province (Grant #BC2014172), Small & Medium Enterprise Technology Innovation Project of Lianyungang City (Grant #CK1333).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra17059e

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