Tanshinone IIA ameliorates acute lung injury by inhibition of the NLRP 3 inflammasome

Tanshinone IIA is the phenanthrenequinone derivative extracted from the perennial plant Salvia miltiorrhiza Bunge (red sage). We investigated whether inhibition of the nucleotide-binding oligomerization domain (NOD)-like receptor family protein 3 (NLRP3) inflammasome mediates the protective effect of tanshinone IIA in acute lung injury (ALI) induced in rats by oleic acid (OA) injection. Compared with the control treatment, OA injection induced pulmonary histological impairment, increased the lung wet/dry weight ratio (7.0±1.1 vs 4.3±0.6 ) and CO2 partial pressure (PaCO2) (52±6.4 vs 40±3.6 mmHg), decreased arterial O2 partial pressure (PaO2) (63±8.4 vs 100±3.0 mmHg), and increased tumor necrosis factor α (TNFα) (8.8±2.3 vs 5.2±1.5 pg/mL), monocyte chemoattractant protein-1 (MCP-1) (36.1±4.9 vs 25.2±6.6 pg/mL) and interleukin-1β (IL-1β) (15.9±3.2 vs 4.6±1.3 pg/mL) in the bronchoalveolar lavage (BAL) fluid. Tanshinone IIA provided protection against ALI, observed as a reduction in the lung wet/dry weight ratio and CO2 partial pressure, and increased O2 partial pressure. The cytokine increase was also prevented. Tanshinone IIA attenuated increased protein levels of NLRP3, caspase-1 and IL-1β in pulmonary tissues, suggesting that it ameliorates ALI by preventing NLRP3 inflammasome activation.


INTRODUCTION
Acute lung injury (ALI) is a clinical disorder responsible for one of the major causes of in-hospital hypoxemic respiratory insufficiency, resulting in morbidity and mortality [1].ALI features impairs the permeability of the alveolar-capillary barrier, followed by pulmonary edema with fluid containing a large amount of proteins, resulting in hypoxemia [2].Despite marked improvement in the treatment of ALI, ALI-caused mortality is high [3].Therefore, investigating the mechanism of pulmonary damage in ALI should help improve the clinical treatment.
The main pathology in ALI is observed as a disturbed balance between pro-and anti-inflammatory processes, accompanied by stimulation of apoptosis [4,5].IL-1β is one of the early inflammatory cytokines induced in ALI that causes the secretion of other cytokines [6].IL-1β is activated by the NLRP3 inflammasome [7].Inflammasome, comprised of NLRP3, apoptosis-associated speck-like protein and procaspase-1, is an important intracellular multiprotein complex of the innate immune system, which is increased in pulmonary tissue.Upon stimulation, the NLRP3 inflammasome cleaves procaspase-1 to produce caspase-1, which mediates the generation of the biologically active form of IL-1β from pro-IL-1β [8][9][10].Numerous studies have demonstrated the essential role of inflammasome in ALI [11][12][13][14].Prevention of the activation of the NLRP3 inflammasome is a potential target for treating ALI [15,16].
Tanshinone IIA is the phenanthrenequinone derivative extracted from Salvia miltiorrhiza Bunge that is extensively used in Chinese traditional medicine for treating several diseases.Tanshinone IIA has various biological effects, such as anti-inflammatory and antioxidant effects.The protective effect of tanshinone IIA against ALI has been verified in animal models of ALI induced by lipopolysaccharide (LPS) [17][18][19][20], seawater exposure [21,22] and paraquat [23].However, the mechanism of the ameliorative effect of tanshinone IIA on ALI has not been fully investigated.
Tanshinone IIA was found to inhibit activation of inflammasome and to ameliorate cardiac damage in canines with myocardial ischemia-reperfusion injury [24].These results suggested an attenuating effect of tanshinone IIA on inflammasome.In view of the major role of NLRP3 in ALI progression, we hypothesized that tanshinone IIA might ameliorate ALI by inhibiting inflammasome.We used the ALI model induced by oleic acid (OA) in rats to examine the effect of tanshinone IIA on inflammasome.

Animals and experimental procedure
Male Sprague-Dawley (SD) rats (250±10 g) were obtained from Vital River (Beijing, China).The rats were housed under standard conditions with 12-h light/ dark cycles (lights on at 6:00), controlled temperature (28~30°C) and free access to food and tap water.SD rats were randomly divided into four groups as follows: control, tanshinone IIA, OA, OA+tanshinone IIA.Control rats were intravenously injected with physiological saline (1 mL/kg body weight); OA rats were intravenously injected with OA to induce lung injury (125 μL/kg body weight in 1 mL saline; Sigma-Aldrich, St Louis, MO, USA).Tanshinone IIA (10 mg/ kg body weight; Xian Guanyu Bio-tech, Xian, China) was intravenously injected or injected 30 min before the injection of OA.The rats were killed 6 h after OA treatment.

Blood and lung tissue sampling
Arterial blood samples (300 μL) were collected from the abdominal aorta of rats after sacrifice into a heparinized syringe for examining arterial PaO 2 and PaCO 2 in the lung by blood gas analysis.The lungs were excised, the right lung was weighed to measure the lung wet/dry (W/D) weight ratio to assess edema, and the left lung was cut into two parts.The first part was fixed in 10% formalin to observe lung injury by hematoxylin-eosin (H&E) staining by light microscopy.The second part was stored at -80°C for protein extraction.

Bronchoalveolar lavage (BAL) preparation
After blood collection, a cannula was inserted into the trachea.The lungs were lavaged three times with 5 mL phosphate buffered saline (PBS; pH 7.4) to obtain BAL fluid.The protein content of BAL fluid was estimated [25], and the presence of cytokines (TNFα, MCP-1 and IL-1β) using a Bio-Plex Kit (Bio-Rad, Hercules, CA) was determined.

Lung histology
Specimens fixed in 10% formalin were dehydrated, cut into 5-μm-thick sections and stained with H&E for routine histology.

Lung W/D weight ratio
The wet weight of right lungs was recorded immediately after removal.The lungs were then placed in an incubator at 60°C for 72 h and the dry weight was measured.The W/D lung ratio was presented as W/D lung mass, which shows the fraction of wet lung weight caused by water.

Western blot analysis
Pulmonary tissue homogenate was centrifuged at 12000 ×g for 15 min and the supernatant was mixed with 5 × loading buffer (Applygen Technologies Inc. Beijing, CN).The mixture was boiled for 10 min and cooled to room temperature.Protein concentration was estimated with a Bradford protein assay kit.Equal amounts of protein (20~100 μg) were loaded on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAG), transferred to nitrocellulose membranes and incubated with NLRP3, caspase-1 and IL-1β (all 1:1000, Abcam, Cambridge, UK).Secondary antibodies (Kirkegaard & Perry Laboratories Inc., Gaithersburg, US) were used at 1:5000 dilution.Chemiluminescence was exposed to X-ray film (Kodak Scientific) and the blots were scanned and quantified using NIH ImageJ.

Statistical analysis
Statistical analysis involved the use of GraphPad Prism ver.5.00 for Windows (GraphPad Software, San Diego, CA, USA).Data are expressed as the mean±SD.Comparisons among more than 2 groups were analyzed by one-way ANOVA followed by the Newman-Keuls test.P<0.05 was considered statistically significant.

Effect of tanshinone IIA on the histological indicators of injury in OA-treated rats
Controls and tanshinone IIA-treated rats showed normal lung architecture, open alveoli and thin alveolar walls (Fig. 1A and B).Rats with OA-induced ALI showed widespread inflammatory infiltration, thickened alveolar walls and septa, with hemorrhaging and a proteinaceous exudate filling some alveoli (Fig. 1C).The pathogenesis of lung injury was ameliorated with tanshinone IIA treatment, with reduced infiltration (Fig. 1D).

Effect of tanshinone IIA on pulmonary impairment in OA-induced ALI
As compared to the controls, ALI rats showed a significantly increased ratio in W/D lung weight (Fig. 2A), decreased PaO 2 (Fig. 2B) and increased PaCO 2 (Fig. 2C) (all p<0.05).Compared to the group administered OA alone, in the OA+tanshinone IIA group the ratio of W/D lung weight was significantly decreased (Fig. 2A), the PaO 2 was increased (Fig. 2B) and the PaCO 2 was decreased (Fig. 2C) (all p<0.05).The ratio of W/D lung weight (Fig. 2A), PaO 2 (Fig. 2B) and PaCO 2 (Fig. 2C) were similar in the tanshinone IIA-treated and control groups (all p>0.05).

Effect of tanshinone IIA on inflammatory cytokines in BAL fluid
As compared with the controls, the OA treatment significantly increased the levels of TNFα, MCP-1 and IL-1β in the BAL fluid (Fig. 3, p<0.05).The tanshinone IIA treatment reversed the OA-induced protein levels (Fig. 3, p<0.05).The levels of TNFα, MCP-1 and IL-1β were similar in tanshinone IIA-treated and control groups (all p>0.05).

DISCUSSION
We report on the ameliorative effect of tanshinone IIA in OA-induced ALI in rats, which may be mediated by the inhibition of inflammasome activation.Tanshinone IIA significantly ameliorated OA-induced pulmonary histological pathology, edema, impaired gas exchange, and it increased the levels of cytokines in BAL fluid.The protein levels of NLRP3, caspase-1 and IL-1β in pulmonary tissues were increased in OA-treated rats and attenuated by tanshinone IIA treatment.The protective effect of tanshinone IIA on lung injury has been confirmed in different animal models, including lung injury induced by LPS [17][18][19][20], seawater exposure [21,22] and paraquat [23].In the OA-induced ALI model in rats, we demonstrated the ameliorative effect of tanshinone IIA on ALI, confirmed by an alleviation of pathological histological indicators, pulmonary edema and gas exchange, and decreased levels of inflammatory cytokines in BAL fluid.From our and previous results, tanshinone IIA could be an effective agent against ALI.
To further investigate the mechanism of tanshinone IIA in ameliorating ALI, we examined the protein levels of NLRP3, caspase-1 and IL-1β in pulmonary tissues, which showed activation of inflammasome.The essential role of the NLRP3 inflammasome in ALI has been investigated [11][12][13][14], and it has been considered a novel target for ALI treatment [15,16].We found that   tanshinone IIA significantly reduced the OA-increased protein levels of NLRP3, caspase-1 and IL-1β in pulmonary tissues, suggesting that tanshinone IIA might ameliorate ALI by inhibiting inflammasome activation.
One limitation of our work was the inability to confirm the causal role of inflammasome involved in the ameliorative effect of tanshinone IIA.Hu et al. [24] also reported that tanshinone IIA inhibited the increase in inflammasome in the myocardium induced by ischemia-reperfusion, which might mediate the cardioprotective effect of tanshinone IIA.Therefore, further research should investigate the direct inhibitory effect of tanshinone IIA on the NLRP3 inflammasome and whether the protective effect of tanshinone IIA against ALI can be blunted when controlling inflammasome activation by genetic methods or agonist treatment.

CONCLUSION
Our results demonstrate that tanshinone IIA significantly ameliorated OA-induced ALI.OA-activated inflammasome in pulmonary tissues was attenuated by the tanshinone IIA treatment.Considering the essential role of the NLRP3 inflammasome in the progression of ALI, tanshinone IIA might ameliorate ALI by inhibiting inflammasome.These results point to a novel strategy for ALI therapy.
All animal procedures were in compliance with the Animal Management Rule of the Ministry of Health, People's Republic of China (documentation No. 55, 2001) and the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Animal Care Committee of Taizhou People's Hospital.