Intracellular ROS

The average fluorescence intensities: Control group (131.5450 ± 9.1995), LPS group (133.5750 ± 1.3647), LPS + Midazolam group (129.7600 ± 1.3152), LPS + Ro5-4864 group (24.5900 ± 2.2345), LPS + PK 11,195 group (34.1950 ± 0.4313), LPS + PK 11,195 + Midazolam group (71.4200 ± 8.0893), LPS + PK 11,195 + Ro5-4864 group (112.6250 ± 0.3748) and LPS + Cyclosporine-A group (87.4000 ± 1.7112). There was a significant difference in the average fluorescence intensities between the groups (p = 0.001). Compared with LPS group, LPS + Ro5-4864, LPS + PK 11,195, LPS + PK 11,195 + Midazolam, LPS + PK 11,195 + Ro5-4864 and LPS + Cyclosporine-A groups showed a decrease in average fluorescence intensities (p = 0.001, p = 0.001, p = 0.001, p = 0.002, p = 0.001). However, the average fluorescence intensity in LPS + Midazolam group was indistinguishable from LPS group (p = 0.421). (Fig. 1)

Fig. 1

ROS detected by fluorescent probe DCFH-DA. The average fluorescence intensities were presented as mean ± SD. nsp=0.421 vs. LPS group. *p = 0.001 < 0.05 vs. LPS group. **p = 0.002 < 0.05 vs. LPS group. Bar = 100 μm

The PBR agonist Ro5-4864, antagonist PK 11,195 and Cyclosporine-A counteracted the production of the intracellular ROS induced by LPS, resulting in a decrease in the intracellular ROS level. However, the central benzodiazepine receptor (CBR) agonist Midazolam was unable to blunt the production of the intracellular ROS induced by LPS. Interestingly, Ro5-4864 and PK 11,195 exhibited the comparable counteracting effects on the production of the intracellular ROS induced by LPS, and their combined use did not show a synergistic effect compared with their alone use. Moreover, the antagonist PK 11,195 did not reverse the effect of the agonist Ro5-4864.

Apoptosis rates of cells

The average total apoptosis rates: Control group (0.1000 ± 0.0397), LPS group (0.2370 ± 0.0796), LPS + Midazolam group (0.1980 ± 0.0397), LPS + Ro5-4864 group (0.1213 ± 0.0493), LPS + PK 11,195 group (0.1513 ± 0.0355), LPS + PK 11,195 + Midazolam group (0.1200 ± 0.0569), LPS + PK 11,195 + Ro5-4864 group (0.1120 ± 0.0435) and LPS + Cyclosporine-A group (0.1043 ± 0.0362). There was a significant difference in the average total apoptosis rates between the groups (p = 0.032). Compared with Control group, the average total apoptosis rate was increased in LPS group (p = 0.004). Compared with LPS group, LPS + Ro5-4864, LPS + PK 11,195, LPS + PK 11,195 + Midazolam, LPS + PK 11,195 + Ro5-4864 and LPS + Cyclosporine-A groups showed a decrease in the average total apoptosis rates (p = 0.011, p = 0.05, p = 0.011, p = 0.007, p = 0.005). There was no statistically significant difference in the average total apoptosis rates between LPS + Midazolam group and LPS group (p = 0.349). (Fig. 2)

Fig. 2

Cell apoptosis detected by flow cytometry. PerCP-Cy5−5: a kind of mixed dye. FITC: fluorescein isothiocyanate. The average total apoptosis rates were presented as mean ± SD. *p = 0.004 < 0.05 vs. Control group. nsp=0.349 vs. LPS group. **p = 0.011 < 0.05 vs. LPS group. ***p = 0.05 vs. LPS group. #p = 0.007 < 0.05 vs. LPS group. ##p = 0.005 < 0.05 vs. LPS group

The PBR agonist Ro5-4864, antagonist PK 11,195 and Cyclosporine-A exhibited an inhibitory effect on LPS-induced cell apoptosis, leading to a decrease in the average total apoptosis rates. However, the CBR agonist Midazolam did not show an anti-apoptotic effect. Similar to the results of the intracellular ROS detection, Ro5-4864 and PK 11,195 exhibited the comparable anti-apoptotic effects, and their combined use did not show a synergistic effect. Moreover, the antagonist PK 11,195 did not abrogate the anti-apoptotic effect of the agonist Ro5-4864 on cells treated with LPS.

Co-localization of the cell nuclear, TSPO and mitochondria

Under the fluorescence microscopy, the cell nuclear stained with DAPI emitted blue fluorescence. The TSPO labeled with FITC emitted green fluorescence, and the cell mitochondria stained with Mito-Tracker Red CMXRos emitted red fluorescence. The superposition of images of the green fluorescence (TSPO) and the red fluorescence (mitochondria) displayed yellow fluorescence, which indicated the co-localization of the TSPO and mitochondria. The TSPO was predominantly distributed on the cell mitochondria. (Fig. 3)

Fig. 3

Co-localization of the cell nuclear, TSPO and mitochondria detected by immunofluorescence. Blue: the cell nuclear stained with DAPI, Green: the TSPO labeled with FITC, Red: the cell mitochondria stained with Mito-Tracker Red CMXRos, Yellow: the superposition of the green fluorescence (TSPO) and the red fluorescence (mitochondria), Purple: the superposition of the blue fluorescence (nuclear) and the red fluorescence (mitochondria). Bar = 100 μm

MMP (ΔΨ) of cells

The average fluorescence intensity values: Control group (190.5200 ± 0.3818), LPS group (123.6800 ± 11.6531), LPS + Ro5-4864 group (176.5800 ± 1.1314) and LPS + PK 11,195 group (161.2800 ± 2.0223). The average fluorescence intensities exhibited statistically significant differences between the groups (p = 0.001). Compared with Control group, the average fluorescence intensity decreased in LPS group (p = 0.001). In contrast, LPS + Ro5-4864 and LPS + PK 11,195 groups showed an increase in average fluorescence intensities compared with LPS group (p = 0.001, p = 0.003). (Fig. 4)

Fig. 4

MMP (ΔΨ) detected by fluorescent probe Mito-Tracker Red CMXRos. The average fluorescence intensities were presented as mean ± SD. *p = 0.001 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. LPS group. ***p = 0.003 < 0.05 vs. LPS group. Bar = 100 μm

The PBR agonist Ro5-4864 and the antagonist PK 11,195 alleviated the decrease of ΔΨ induced by LPS. The effects of Ro5-4864 and PK 11,195 on counteracting the decrease of ΔΨ were comparable.

The absorbance of the cellular mitochondria

The OD values: Control group (0.1058 ± 0.0101), LPS group (0.0235 ± 0.0036), LPS + Ro5-4864 group (0.0528 ± 0.0048) and LPS + PK 11,195 group (0.0515 ± 0.0026). The differences of the OD values between the groups were statistically significant (p = 0.001). Compared with Control group, LPS group showed a decrease in the mitochondrial OD value (p = 0.001). In comparison with LPS group, LPS + Ro5-4864 group and LPS + PK 11,195 group exhibited an increase in the mitochondrial OD values (p = 0.001, p = 0.001). (Fig. 5)

Fig. 5

Mitochondrial swelling detected by absorbance. The OD values were presented as mean ± SD. *p = 0.001 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. LPS group

The PBR agonist Ro5-4864 and the antagonist PK 11,195 both demonstrated a prophylactic effect on LPS-induced mitochondrial swelling, attenuating the extent of the cellular mitochondrial swelling. The agonist Ro5-4864 and the antagonist PK 11,195 achieved the same effect on counteracting the mitochondrial swelling.

Subcellular structures of cells

Control group: The double-layer structures of the nuclear membranes were clearly visible, and the chromatin was distributed evenly. A large number of the structurally intact mitochondria were existed with the shape of the long shuttle, and the mitochondrial cristae were intact, as shown by the arrows (Fig. 6a). LPS group: The cell volumes were smaller. There were a few blebbings on the cell membranes, and the number of organelles in the cytoplasm was reduced. The nuclear membrane was ruptured, and the chromatin was aggregated in the periphery of the nuclear membrane. The number of mitochondria was reduced, and the structures of the double membranes and the cristae were disappeared. The matrix of the mitochondria was swollen and vacuolated. The mitochondria suffered from severe injury, as shown by the arrows (Fig. 6b). LPS + Ro5-4864 group: The structures of the nucleus membranes were clear. The number of mitochondria was reduced, and some of the structures of the mitochondrial double membranes and the cristae were broken. A small part of the mitochondrial matrix was swollen and vacuolated. The injury of the cell ultrastructure in this group lessened compared with LPS group, as shown by the arrows (Fig. 6c). LPS + PK 11,195 group: The structures of the nucleus membranes were clear. The number of mitochondria was reduced. A small number of mitochondria with inner and outer membranes ruptured, and the matrix spillage led to a lesser extent of the injury compared with LPS group, as shown by the arrows (Fig. 6d).

Fig. 6

Subcellular structures detected by TEM. a There existed a large number of mitochondria with the normal structures and the intact cristae in Control group. b Mitochondrial injury was severe in LPS group. c The PBR agonist Ro5-4864 alleviated LPS-induced mitochondrial injury. d The PBR antagonist PK 11,195 alleviated LPS-induced mitochondrial injury

Protein expression levelsTSPO, VDAC and ANT

The relative expression levels: TSPO: Control group (1.0567 ± 0.0091), LPS group (1.3824 ± 0.1406), LPS + Ro5-4864 group (0.4522 ± 0.0407) and LPS + PK 11,195 group (1.0828 ± 0.1886). VDAC: Control group (0.7762 ± 0.1928), LPS group (0.6682 ± 0.1966), LPS + Ro5-4864 group (0.7878 ± 0.2315) and LPS + PK 11,195 group (1.0212 ± 0.1706). ANT: Control group (0.7031 ± 0.0571), LPS group (0.6317 ± 0.2533), LPS + Ro5-4864 group (0.5495 ± 0.4010) and LPS + PK 11,195 group (0.5356 ± 0.0564). The differences of the TSPO expression between the groups were statistically significant (p = 0.001). The differences of the VDAC and ANT expression between the groups were not statistically significant (p = 0.249, p = 0.138). The expression level of TSPO increased in LPS group compared with Control group (p = 0.01) and decreased in LPS + Ro5-4864 group and LPS + PK 11,195 group compared with LPS group (p = 0.001, p = 0.015). However, the expression levels of VDAC and ANT did not differ between the groups. (Fig. 7)

Fig. 7

Western blot - TSPO (18KDa), VDAC (31KDa) and ANT (30KDa). The relative expression levels of proteins are presented as mean ± SD. TSPO: *p = 0.01 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. LPS group. ***p = 0.015 < 0.05 vs. LPS group. (The original uncropped blots were presented in the Additional file 1.)

LPS induced an increase in intracellular TSPO expression level. Pretreatments of cells with PBR agonist Ro5-4864 and antagonist PK 11,195 were all made a decrease in TSPO expression level, and the expression levels of VDAC and ANT were not affected.

Ratio of BCL-2 to BAX

The ratios of BCL-2 to BAX: Control group (1.8401 ± 0.2798), LPS group (0.7975 ± 0.4439), LPS + Ro5-4864 group (1.4461 ± 0.2938) and LPS + PK 11,195 group (1.8249 ± 0.2255). The differences of the ratios of BCL-2 to BAX between the groups were statistically significant (p = 0.013). The ratio of BCL-2 to BAX decreased in LPS group compared with Control group (p = 0.004) and increased in LPS + Ro5-4864 group and LPS + PK 11,195 group compared with LPS group (p = 0.038, p = 0.004). (Fig. 8)

Fig. 8

Western blot - Ratio of BCL-2 (52KDa) to BAX (21KDa). The relative expression levels of proteins are presented as mean ± SD. *p = 0.004 < 0.05 vs. Control group. **p = 0.038 < 0.05 vs. LPS group. ***p = 0.004 < 0.05 vs. LPS group. (The original uncropped blots were presented in the Additional file 1.)

LPS could lead to a decrease in the ratio of BCL-2 to BAX, which indicated that the pro-apoptotic protein was dominant. PBR agonist Ro5-4864 and antagonist PK 11,195 were found to reverse the ratio of BCL-2 to BAX, forming the predominance of expression of the anti-apoptotic protein.

Cytochrome C

The relative expression levels: In the mitochondria: Control group (0.9829 ± 0.1415), LPS group (0.2324 ± 0.0455), LPS + Ro5-4864 group (0.7493 ± 0.2537) and LPS + PK 11,195 group (0.6606 ± 0.2399). In the cytoplasm: Control group (0.7237 ± 0.1772), LPS group (1.1884 ± 0.0666), LPS + Ro5-4864 group (0.5644 ± 0.2465) and LPS + PK 11,195 group (0.4798 ± 0.1294). The differences of the Cytochrome C expression in the mitochondria and cytoplasm between the groups were statistically significant (p = 0.008, p = 0.004). The expression level of the mitochondrial Cytochrome C decreased in LPS group (p = 0.001), and the cytoplasmic Cytochrome C increased in LPS group (p = 0.01) compared with Control group. The mitochondrial Cytochrome C increased in LPS + Ro5-4864 group and LPS + PK 11,195 group (p = 0.01, p = 0.025), and the cytoplasmic Cytochrome C decreased in LPS + Ro5-4864 group and LPS + PK 11,195 group (p = 0.002, p = 0.001) compared with LPS group. (Fig. 9)

Fig. 9

Western blot - Cytochrome C (15KDa). The relative expression levels of proteins are presented as mean ± SD. In the mitochondria: *p = 0.001 < 0.05 vs. Control group. **p = 0.01 < 0.05 vs. LPS group. ***p = 0.025 < 0.05 vs. LPS group. In the cytoplasm: #p = 0.01 < 0.05 vs. Control group. ##p = 0.002 < 0.05 vs. LPS group. ###p = 0.001 < 0.05 vs. LPS group. (The original uncropped blots were presented in the Additional file 1.)

LPS induced a decrease expression level of Cytochrome C in the mitochondria and an increase of Cytochrome C transferred into the cytoplasm. PBR agonist Ro5-4864 and antagonist PK 11,195 could reverse this situation, which lead to an increase of Cytochrome C in the mitochondria but a decrease in the cytoplasm.

Cleaved Caspase-7

The relative expression levels: Control group (0.6638 ± 0.0894), LPS group (1.2731 ± 0.0815), LPS + Ro5-4864 group (0.6586 ± 0.0287) and LPS + PK 11,195 group (0.6729 ± 0.0683). The differences of the cleaved Caspase-7 expression in the cytoplasm between the groups were statistically significant (p = 0.001). The expression level of cytoplasmic cleaved Caspase-7 increased in LPS group compared with Control group (p = 0.001) and decreased in LPS + Ro5-4864 group and LPS + PK 11,195 group compared with LPS group (p = 0.001, p = 0.001). (Fig. 10)

Fig. 10

Western blot - Cleaved Caspase-7 (20KDa). The relative expression levels of proteins are presented as mean ± SD. *p = 0.001 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. LPS group. (The original uncropped blots were presented in the Additional file 1.)

LPS induced an increase expression level of cleaved Caspase-7 in the cytoplasm. PBR agonist Ro5-4864 and antagonist PK 11,195 resulted in a decrease of cleaved Caspase-7 in the cytoplasm.

Gene expression levels

The relative expression levels: BCL-2: Control group (0.9200 ± 0.0962), LPS group (0.3017 ± 0.0515), LPS + Ro5-4864 group (0.9969 ± 0.0222) and LPS + PK 11,195 group (0.3636 ± 0.1416). BAX: Control group (0.7097 ± 0.0328), LPS group (0.9866 ± 0.0803), LPS + Ro5-4864 group (0.9009 ± 0.1190) and LPS + PK 11,195 group (0.7570 ± 0.0268). The differences of BCL-2 and BAX expression between the groups were statistically significant (p = 0.001, p = 0.005). The expression level of BCL-2 decreased in LPS group (p = 0.001), and BAX increased in LPS group (p = 0.001) compared with Control group. The expression level of BCL-2 increased in LPS + Ro5-4864 group (p = 0.001) compared with LPS group. The expression level of BAX decreased in LPS + PK 11,195 group (p = 0.001) compared with LPS group. (Fig. 11)

Fig. 11

RT-qPCR - BCL-2 and BAX. The relative expression levels of genes are presented as mean ± SD. BCL-2: *p = 0.001 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. LPS group. BAX: #p = 0.001 < 0.05 vs. Control group. ##p = 0.001 < 0.05 vs. LPS group

LPS induced a decrease in BCL-2 gene expression level and an increase in Bax gene expression level. PBR agonist Ro5-4864 led to an increase in BCL-2 gene expression level, and PBR antagonist PK 11,195 had no effect on the expression of BCL-2 gene. PBR antagonist PK 11,195 led to a decrease in Bax gene expression level, and PBR agonist Ro5-4864 had no effect on the expression of Bax gene.

Serum IgE

The concentration of IgE (ng/ml): Control group (160.1333 ± 35.7417), OVA group (293.4667 ± 1.3663), OVA + Ro5-4864 group (159.3000 ± 13.0346), OVA + PK 11,195 group (170.6333 ± 20.1536) and OVA + Dexamethasone group (186.3000 ± 16.0468). The differences of the concentration of IgE were statistically significant (p = 0.001). The concentration of IgE increased in OVA group compared with Control group (p = 0.001) and decreased in OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group compared with OVA group (p = 0.001, p = 0.001, p = 0.001). ( Fig. 12)

Fig. 12

Serum ELISA. The concentration of IgE (ng/ml) and cortisol (µg/L) are presented as mean ± SD. IgE: *p = 0.001 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. OVA group. Cortisol: *p = 0.004 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. Control group. ***p = 0.021 < 0.05 vs. Control group. #p = 0.011 < 0.05 vs. OVA group

The concentration of IgE increased in OVA group and decreased after the treatment of the glucocorticoid. Both of Ro5-4864 and PK 11,195 made a decrease in the concentration of IgE in OVA-treated mice.

Serum cortisol

The concentration of cortisol (µg/L): Control group (148.1855 ± 13.2973), OVA group (198.7231 ± 12.8816), OVA + Ro5-4864 group (206.9220 ± 17.0949), OVA + PK 11,195 group (242.4059 ± 53.9319) and OVA + Dexamethasone group (187.6344 ± 16.8501). The differences of the concentration of cortisol were statistically significant (p = 0.001). The concentration of cortisol increased in OVA group, OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group compared with Control group (p = 0.004, p = 0.001, p = 0.001, p = 0.021). Compared with OVA group, the concentration of cortisol increased in OVA + PK 11,195 group (p = 0.011). However, the differences among OVA group, OVA + Ro5-4864 group and OVA + Dexamethasone group were not statistically significant (p = 0.612, p = 0.494). (Fig. 12)

The concentration of cortisol increased in mice subjected to OVA (including OVA group, OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group) compared with Control group. PK 11,195 demonstrated much greater potency for the secretion of cortisol, and Ro5-4864 showed weak facilitation.

IFN-γ, IL-5 and IL-17 in BALF

The concentrations of the cytokines: IFN-γ (ng/L): Control group (245.1563 ± 36.7071), OVA group (295.6771 ± 8.5819), OVA + Ro5-4864 group (244.6354 ± 58.1822), OVA + PK 11,195 group (278.7500 ± 109.6336) and OVA + Dexamethasone group (277.9688 ± 167.3692). IL-5 (pg/ml): Control group (14.9401 ± 5.3042), OVA group (27.5118 ± 6.9649), OVA + Ro5-4864 group (17.1415 ± 8.9518), OVA + PK 11,195 group (17.5886 ± 3.4816) and OVA + Dexamethasone group (16.3192 ± 5.6276). IL-17 (pg/ml): Control group (53.3413 ± 16.1508), OVA group (53.8088 ± 14.7874), OVA + Ro5-4864 group (38.5978 ± 14.9246), OVA + PK 11,195 group (31.2927 ± 16.0279) and OVA + Dexamethasone group (42.7644 ± 16.1169). The differences of the concentration of IFN-γ and IL-17 between the groups were not statistically significant (p = 0.063, p = 0.086). The differences of the concentration of IL-5 between the groups were statistically significant (p = 0.015). The concentration of IL-5 increased in OVA group compared with Control group (p = 0.002) and decreased in OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group compared with OVA group (p = 0.009, p = 0.012, p = 0.005). (Fig. 13)

Fig. 13

BALF ELISA. The concentration of IFN-γ (ng/L), IL-5 (pg/ml) and IL-17 (pg/ml) are presented as mean ± SD. IL-5: *p = 0.002 < 0.05 vs. Control group. **p = 0.009 < 0.05 vs. OVA group. ***p = 0.012 < 0.05 vs. OVA group. #p = 0.005 < 0.05 vs. OVA group

The concentration of IL-5 increased in OVA group and decreased after the treatment of the glucocorticoid. Both Ro5-4864 and PK 11,195 made a decrease in the concentration of IL-5 in OVA-treated mice, and the concentration of IFN-γ and IL-17 were not affected.

Inflammatory cells in BALF

Cell smears with Wright-Giemsa staining

The erythrocytes mixed in BALF were lysed with RBC lysis buffer before BALF cells centrifugation, and only inflammatory cells (bluish-purple particles) were visible on the BALF smear. A few inflammatory cells were on the smear of Control group. The inflammatory cells were increased and aggregated on the smear of OVA group compared with Control group. Compared with OVA group, the inflammatory cells were decreased and scattered on the smear of OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group. (Fig. 14)

Fig. 14

Inflammatory cells in BALF. Cell smears with Wright-Giemsa staining: The inflammatory cells were increased and aggregated on the BALF smear of OVA group, decreased and scattered after the treatment of the Dexamethasone. Both Ro5-4864 and PK 11,195 made a decrease and scatter of inflammatory cells on the BALF smear in OVA-treated mice. Cell counting: The total numbers of cells (106/ml) in the BALF are presented as mean ± SD. *p = 0.001 < 0.05 vs. Control group. **p = 0.001 < 0.05 vs. OVA group. Bar = 50 μm

Cell counting

The total numbers of cells (106/ml) in the BALF: Control group (1.6450 ± 0.4564), OVA group (7.8400 ± 1.4560), OVA + Ro5-4864 group (3.2600 ± 0.4445), OVA + PK 11,195 group (3.2517 ± 0.7447) and OVA + Dexamethasone group (2.4700 ± 1.1498). The differences of the total numbers of cells in the BALF were statistically significant (p = 0.001). The total numbers of cells increased in OVA group compared with Control group (p = 0.001) and decreased in OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group compared with OVA group (p = 0.001, p = 0.001, p = 0.001). (Fig. 14)

The total numbers of cells in the BALF increased in OVA group and decreased after the treatment of the glucocorticoid. Both Ro5-4864 and PK 11,195 made a decrease in the total numbers of cells in the BALF in OVA-treated mice.

Histopathological analysis

In Control group, the BECs were arranged orderly, and the bronchial walls were thin. The structures of alveolar walls were complete, and few inflammatory cells could be seen in the peribronchial and submucosal tissue spaces. Compared with Control group, the structures of the BECs were disordered with the exfoliation of parts of the BECs. The submucosal tissue spaces were edematous, and the bronchial walls were thickened. The pronounced infiltration with inflammatory cells had extended to the peribronchial and submucosal tissue spaces in OVA group. Compared with OVA group, the basal lamina was intact with a lesser exfoliation of BECs and a diminution of inflammatory cells infiltration in the peribronchial and submucosal tissue spaces in OVA + Ro5-4864 group, OVA + PK 11,195 group and OVA + Dexamethasone group. (Fig. 15)

Fig. 15

Histopathological examination. The lungs histopathological examination showed the injury of BECs and the infiltration of inflammatory cells in the peribronchial and submucosal tissue spaces in OVA group. The injury of BECs and the infiltration of inflammatory cells were abated after the treatments of Ro5-4864, PK 11,195 and Dexamethasone