Cells and strains

MSSA (ATCC 25923; American Type Culture Collection, Manassas, VA, USA) strains producing staphylococcal pneumonia were isolated from clinical samples, grown in Luria Bertani (LB) medium and conserved at ‑80˚C in 50% LB broth and 50% glycerol. The BEAS-2B cell line was purchased from the Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China) and cultivated in RPMI‑1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The media were supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and maintained in a humidified incubator at 37˚C with 5% CO₂. NIH3T3 cells were purchased from Wuhan University Preservation Center (Wuhan, China). The Murine CMV (MCMV) Smith strain (ATCC-VR-1399) was ordered from the American Type culture Collection. The MCMV strain was propagated on NIH3T3 cells and titrated on the mouse embryo fibroblasts as described previously (16).

MTT assay

The BEAS-2B cells were cultured in RPMI-1640 supplemented with 10% FBS. The cells were stimulated for 24 h at 37˚C with different concentrations of Evo (20‑100 µM; 20, 40, 60, 80 and 100 µM). Subsequently, 20 µl of 5 mg/ml MTT solution was added and the plate was incubated at 37˚C for 4 h. Following aspiration and washing with PBS, 150 µl of DMSO was added to each well. The absorbance was determined spectrophotometrically at 490 nm on an ELX800 UV universal microplate reader.

Infection of cells with MSSA

The BEAS-2B cells incubated with 1 mL of RPMI-1640 containing the MSSA with the indicated multiplicity of infection (1×10⁵ CFU) at 37°C in 5% CO₂. After 3 h, the suspension was aspirated, and the cells were washed with PBS and incubated with Evo (10, 20 and 40 µm) for 24 h.

Animal model

Pathogen-free female BALB/c mice (n=90; 6 weeks old; 22-25 g) were supplied by the Animal Center of Soochow University (Changzhou, China; certificate no. SCXK 2015-0004). The mice were housed individually in steel microisolator cages at 22°C with a 12/12‑h light/dark cycle, and fed a standard laboratory diet and water Animal welfare and experimental procedures and was approved by the Animal Experimental Ethical Committee of Soochow University.

The procedures of animal model establishment were performed as previously described (16). Following anesthetization, the BALB/c mice were infected by intra-peritoneal injection with 3×10⁴ PFU MCMV Smith strain or saline. At 4 months post-MCMV primary infection, all mice underwent cecal ligation and puncture (CLP) to trigger viral reactivation in the MCMV groups. Briefly, the mice were anesthetized under sodium pentobarbital following cutaneous asepsis. A 5-mm incision in the lower-right quadrant of the abdomen was introduced, and the cecum was localized and ligatured 3 mm above its extremity using an 18G needle. The abdomen was then sutured, and cutaneous asepsis was performed with 5% alcoholic povidone. After 24 h, the animals were administered with an intraperitoneal injection of 0.01 mg/g of tramadol. At 14 days post-CLP, the surviving mice in all groups received an intranasal inoculation with 50 µL 5×10⁸ CFU MSSA to induce severe pneumonia.

Experimental designs

The mice were randomly divided into five groups: Control group (saline + CLP + MSSA); model group (MCMV + CLP + MSSA); and Evo groups (MCMV + CLP + MSSA + 5, 10, or 20 mg/kg Evo). Each group was divided into two sub-groups: 5 and 15 days. Following intra-nasal inoculation with MSSA, the mice were intragastrically administrated with different concentrations of Evo (5, 10, and 20 mg/kg), which were selected according to the preliminary experiments, whereas mice in the control and MCMV groups received an equal volume of distilled water of 0.5% Tween-80, once per day for a period of 15 days. In the 15-day cohort, the daily weight was closely monitored every 5 days and spontaneous mortality of the animals was noted during this period. On days 5 and 15, all surviving mice were sacrificed with a lethal dose of thiopental, and samples of blood and the lungs were subjected to further analysis.

Histological analysis

The animals were sacrificed at 5 and 15 days post-treatment. The lungs were removed and embedded in paraffin, cut into 3 mm sections, and stained with hematoxylin and eosin. Subsequently, pathological changes were evaluated under a light microscope.

Bacterial counts in lung tissues

The lung tissues were crushed and homogenized in 1 ml of PBS to determine the lung bacterial count. Serial 10-fold dilutions of the lung homogenates were cultured using a 5% horse blood (BioMerieux, SA., Marcy-l'Etoile, France) agar plate at 37°C under anaerobic conditions. Following bacterial culture, identification of the colonies was performed by culture on Chapman medium (Bertin Pharma (Montigny le Bretonneux, France) and confirmed using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry.

ELISA analysis

The concentrations of cytokines in the cell culture medium and mouse serum collected by cardiac puncture at 5 and 15 days post-infection with MSSA were determined by ELISA for mouse tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, interferon (IFN)-α and IFN-γ following the manufacturer's protocol. Serum was separated by centrifugation at 400 x g for 10 min at 4°C and stored at −80°C. The 96-well microplates were read using an ELX800 UV universal microplate reader at 450 nm.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA of the lung tissue was obtained using TRIzol reagent. The reverse transcription reactions for mRNA were performed using PrimeScript™ RT Master mix (Promega Corporation, Madison, WI, USA). The RT-qPCR analysis was performed using Taq polymerase (Takara Biotechnology Co., Ltd., Dalian, China) consisting of a final volume of 20 µl, containing 2 µl cDNA, 10 µl SYBR-Green Mix, 4 µl primer mix and 4 µl ddH2O (Takara Biotechnology Co., Ltd.). Primers specific for the TNF-α, IL-6, IL-1β, glyco-protein-B (gB) gene and β-actin are listed in Table I. β-actin was selected as an internal control to normalize target gene expression. Thermocycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec, then a melting curve analysis from 60°C to 95°C every 0.2°C for 1.5 min was obtained. The transcript amount was normalized to U6 and β-actin and quantified using the 2^−ΔΔCq method (17).

Western blot analysis

The western blot analysis was performed as described previously (15). Protein samples were extracted from the cells or tissues with Protein Extraction Reagent (Pierce; Thermo Fisher Scientific, Inc.). Subsequently, the protein concentration was measured using a bicinchoninic acid protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China). Total protein (20 µg) in each sample was separated using SDS-PAGE (10% gel) and transferred onto a polyvinyli-dene difluoride filter membrane. The membrane was blocked using 5% non-fat milk at 25°C for 1 h, and then incubated with primary antibodies overnight at 4°C. Antibodies against IκBα (cat. no. 9242S; 1:1,000; Cell Signaling Technology, Inc.), p-IκBα (cat. no. 8219; 1:1,000; Cell Signaling Technology, Inc.), NF-κB p65 (cat. no. ab16502; 1:500; Abcam), JNK (cat. no. ab176662; 1:1,000; Abcam), p-JNK (cat. no. ab207477; 1:1,000; Abcam), Erk (cat. no. ab176641; 1:1,000; Abcam), p-Erk (cat. no. ab192591; 1:1,000; Abcam), p38 (cat. no. ab32142; 1:1,000; Abcam), p-p38 (cat. no. ab178867; 1:1,000; Abcam), β-actin (cat. no. ab8226; 1:3,000; Abcam) and Lamin A (cat. no. ab26300; 1:3,000; Abcam) were used. β-actin and Lamin A were used as internal controls. Then, the membrane was incubated with anti-goat HRP-conjugated antibody (cat. no. AR1017; 1:5,000; Boster Systems, Inc., Pleasanton, CA, USA) at 25°C for 2 h. Detection was executed with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The immunoreactive bands were detected by enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Evo inhibits NF-κB and MAPK pathways in vitro

As shown in Fig. 2A, MSAA infection markedly induced the nuclear translocation of NF-κB/p65 and p-IκBα, whereas Evo led to marked repression of these trends in a dose-dependent manner. In addition, Evo reduced the phosphorylation of JNK, ERK and p38 MAPKs (Fig. 2B).

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Figure 2

Evo inhibits NF-κB and MAPK pathways in vitro. Effects of Evo on the expression of proteins of the (A) NF-κB and (B) MAPK pathways in BEAS-2B cells infected with MSSA. Data are shown as the mean ± standard deviation. ^##P<0.01, vs. control group; ^*P<0.05 and ^**P<0.01, vs. MSSA group. Evo, evodiamine; MSSA, methicillin-susceptible Staphylococcus aureus; NF-κB, nuclear factor-κB; MAPK, mitogen-activated protein kinase; IκB, inhibitor of NF-κB; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; p-, phosphorylated.

Evo reduces mortality rates

As shown in Fig. 3A, the accumulative mortality rates during 15 days with Evo doses of 5, 10 and 20 mg/kg were 65, 44 and 29%, respectively, whereas the mortality rate in the MCMV group was 73%. Evo (10 and 20 mg/kg) dose-dependently improved the survival rate, compared with that in the MCMV groups.

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Figure 3

Effects of Evo in mice. Effects of Evo on (A) mortality rate, (B) body weight loss, (C) the morphology (black arrows indicate abscesses) and (D) histological changes of lung tissues (x200) of mice inoculated with MSSA post-cytomegalovirus reactivation. The lungs of mice were collected 5 and 15 days following staphylococcal pneumonia. Data are shown as the mean ± standard deviation. ^##P<0.01, vs. control group; ^*P<0.05 and ^**P<0.01, vs. Model group. Evo, evodiamine; MSSA, methicillin-susceptible Staphylococcus aureus.

Evo decreases body weight loss

Compared with the control group, the MCMV mice showed a trend towards increased weight loss. The weight loss in the Evo (10 and 20 mg/kg) groups were lower than that in the MCMV group, whereas the weight loss in the Evo (5 mg/kg) group showed no statistical significance (Fig. 3B).

Histological assessment

Morphological examination of the lungs from the MCMV mice sacrificed on days 5 and 15 post-MSSA pneumonia revealed voluminous right or left lung abscesses (Fig. 3C). Overall, 30.5% mice had developed abscessing pneumonia in the MCMV group whereas none was noted in the control group. Following the administration of Evo, with the exception of the Evo (5 mg/kg) group, the lung abscesses were largely improved.

As shown in Fig. 3D, no evident histological abnormalities were observed in the control group. By contrast, the lung tissues of the MCMV group exhibited severe pathologic changes, including infiltration of inflammatory cells, alveolar wall thickening and alveolar hemorrhage. However, these histopathological changes were significantly reversed following treatment with Evo (10 and 20 mg/kg).

Evo inhibits the secretion of inflammatory cytokines

As shown in Fig. 4, the levels of TNF-α, IL-1β and IL-6 were significantly increased in the MCMV mice compared with those in the control group. By contrast, with the exception of Evo (5 mg/kg), pretreatment with Evo (10 and 20 mg/kg) efficiently reduced the production of inflammatory cytokines (Fig. 4A-C). In addition, the levels of IFN-α and IFN-γ in the Evo (10 and 20 mg/kg)-treated mice were reduced in comparison with those in the MCMV group (Fig. 4D and E).

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Figure 4

Evo inhibits the secretion of inflammatory cytokines in mice. Evo decreased the secretion of inflammatory cytokines in serum of mice with staphylococcal pneumonia following cytomegalovirus reactivation. The levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) IFN-α and (E) IFN-γ were detected by enzyme-linked immunosorbent assay. Data are shown as the mean ± standard deviation. ^#P<0.05 and ^##P<0.01, vs. control group; ^*P<0.05 and ^**P<0.01, vs. Model group. Evo, evodiamine; MSSA, methicillin-susceptible Staphylococcus aureus; TNF-α, tumor necrosis factor-α; IL, interleukin; IFN, interferon.

Evo decreases lung bacteria count

As shown in Fig. 5, the lung bacterial count of mice at 5 days post-MSSA pneumonia was higher in the MCMV group. Treatment with Evo (10 and 20 mg/kg) facilitated bacterial clearance (Fig. 5A). On day 15, the lung bacterial counts of the Evo groups were also significantly decreased (Fig. 5B).

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Figure 5

Effects of Evo on lung bacteria count. Evo reduced the lung bacteria count at (A) 5 days and (B) 15 days in mice with staphylococcal pneumonia following cytomegalovirus reactivation. Data are shown as the mean ± standard deviation. ^##P<0.01, vs. control group; ^**P<0.01, vs. MCMV group. Evo, evodiamine; MSSA, methicillin-susceptible Staphylococcus aureus.

Evo inhibits the mRNA expression of gB

The mRNA levels of gB in the lung tissues were determined using RT-qPCR analysis to confirm whether Evo inhibited staphylococcal pneumonia following CMV reactivation. As shown in Fig. 6A and B, the mRNA content of the gB gene in the MCMV group was significantly increased, whereas no virus was detected in the control group. By contrast, Evo (10 and 20 mg/kg) at 15 days post-infection had effectively inhibited the amplification of the gB gene.

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Figure 6

Effect of Evo on mRNA levels of gB. mRNA levels of gB at (A) 5 days and (B) 15 days in mice with staphylococcal pneumonia following cyto-megalovirus reactivation were determined using reverse transcription-quantitative polymerase chain reaction analysis. The graph shows the quantification of results normalized to β-actin levels. Data are shown as the mean ± standard deviation. ^##P<0.01, vs. control group; ^*P<0.05 and ^**P<0.01, vs. Model group. Evo, evodiamine; gB, glycoprotein-B.

Not applicable.