Protostemonine effectively attenuates lipopolysaccharide-induced acute lung injury in mice

Abstract

Protostemonine (PSN) is the main anti-inflammatory alkaloid extracted from the roots of Stemona sessilifolia (known as "Baibu" in traditional Chinese medicine). Here, we reported the inhibitory effects of PSN on lipopolysaccharide (LPS)-induced macrophage activation in vitro and LPS-induced acute lung injury in mice. Macrophage cell line RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs) were treated with PSN (1, 3, 10, 30 and 100 μmol/L) for 0.5 h and then challenged with LPS (0.1 μg/mL) for 24 h. Pretreatment with PSN significantly inhibited LPS-induced phosphorylation of MAPKs and AKT, iNOS expression and NO production in the macrophages. C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) to induce acute lung injury (ALI). The mice were subsequently treated with PSN (10 mg/kg, ip) at 4 and 24 h after LPS challenge. PSN administration significantly attenuated LPS-induced inflammatory cell infiltration, reduced pro-inflammatory cytokine (TNF-α, IL-1β and IL-6) production and eliminated LPS-mediated lung edema. Furthermore, PSN administration significantly inhibited LPS-induced pulmonary MPO activity. Meanwhile, LPS-induced phosphorylation of p38 MAPK, iNOS expression and NO production in the lungs were also suppressed. The results demonstrate that PSN effectively attenuates LPS-induced inflammatory responses in vitro and in vivo; the beneficial effects are associated with the decreased phosphorylation of MAPK and AKT and the reduced expression of pro-inflammatory mediators, such as iNOS, NO and cytokines. These data suggest that PSN may be a potential therapeutic agent in the treatment of ALI.

Figure 1

PSN reduced phosphorylation of MAPKs and AKT in LPS-challenged RAW264.7 cells. (A) The chemical structure of PSN. (B and G) Western blot analysis of LPS-induced p-p38, p-ERK, p-JNK and p-AKT. RAW264.7 cells were pretreated with 30 μmol/L PSN or 0.1% DMSO for 30 min before stimulation with LPS (100 ng/mL) for indicated time. (H) The p-p38 and p-AKT were measured in RAW264.7 cells that were pretreated with PSN at 0, 1, 10 and 100 μmol/L for 30 min and then challenged with LPS (100 ng/mL) for 45 min. Whole cell extracts were prepared and the protein levels of p-p38, p-ERK, p-JNK and p-AKT were detected. β-Actin was used as loading control. Densitometric analysis of the ratios of p-p38/T-p38 (C, I), p-ERK/ERK (D), p-JNK/JNK (E), p-AKT/β-actin (G) and p-AKT/AKT (J) was performed using ImageJ software. Statistical analysis was performed using Student's t-test. n=3. ^*P<0.05, ^**P<0.01.

Figure 2

PSN reduced the phosphorylation of MAPKs and AKT in LPS-challenged BMDMs. (A) Western blot analysis of LPS-induced p-p38, p-ERK, p-JNK and p-AKT expression. BMDMs were pretreated with 30 μmol/L PSN or 0.1% DMSO for 30 min before stimulation of LPS (100 ng/mL). The expressions of p-p38, p-ERK, p-JNK and p-AKT were detected as above. Total p38, ERK, JNK and AKT antibodies were used as loading controls, respectively. (B–E) Densitometric analysis of the ratios of p-p38/T-p38, p-ERK/ERK, p-JNK/JNK, and p-AKT/AKT was performed using ImageJ software. Statistical analysis was performed using Student's t-test. n=3. ^*P<0.05, ^**P<0.01.

Figure 3

PSN inhibited iNOS expression, NO and pro-inflammatory cytokine production in LPS-stimulated macrophages. (A) PSN had no effect on toxicity of macrophages. RAW264.7 cells were treated with PSN at 0, 1, 3, 10, 30 and 100 μmol/L for 48 h. Cell viability was determined by MTT assay. (B and D) PSN decreased LPS-induced iNOS expression in RAW264.7 cells and BMDMs. Macrophages were pretreated with PSN at 0, 1, 3, 10, 30 and 100 μmol/L for 30 min before stimulation with LPS (100 ng/mL). After 24 h incubation, cell lysates were collected for Western blot analysis and supernatant was collected for NO detection. β-Actin was used as loading control. (C and E) Densitometric analysis of the ratios of iNOS and β-actin was performed using ImageJ software. (F and G) PSN reduced the NO production in LPS-induced RAW264.7 cells and BMDMs. (H–J) PSN inhibited LPS-induced mRNA expression of pro-inflammatory cytokines. RAW264.7 cells were treated with PSN at indicated concentrations before LPS challenge for 4 h. Total RNAs were then isolated, and the mRNA levels of IL-6 (H), TNF-α (I) and IL-1β (J) were quantified by quantitative real-time PCR. Statistical analysis was performed using Student's t-test. n=3. ^*P<0.05 ^**P<0.01, ^***P<0.001.

Figure 4

PSN attenuated inflammatory cell accumulation in lung tissues of ALI mice. (A) The schematic timeline of LPS-induced acute lung injury model. Mice were subjected to an intratracheal injection of LPS (5 mg/kg) and subsequent intraperitoneal injection of solvent or PSN (10 mg/kg). An additional injection of solvent or PSN was administered (12 h after LPS challenge) to mice that underwent a 24 h LPS treatment. (B, C) The total protein concentrations in BALF after LPS challenge for 4 h (B) and 24 h (C) were detected. (D, E) Myeloperoxidase (MPO) activity was determined according to the manufacturer's instructions 4 h (D) and 24 h (E) after LPS challenge. Values represent mean±SD. n=5 mice each group. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 5

PSN ameliorated pathological changes in ALI mice. The right lobes were fixed and processed for histological evaluation. (A–E) After H&E staining, the representative changes of the lungs from mice of different groups were shown with original magnification (100×) and partial enlarged detail (400×). (F) The degree of lung injury was quantitatively evaluated according to the histological analysis. Values are mean±SD. n=5. ^*P<0.05, ^**P<0.01.

Figure 6

PSN decreased expression of IL-6, TNF-α and IL-1β in mice treated with LPS for 4 h. After treatment with LPS and LPS/PSN for 4 h, mice were euthanized and the lung tissues were collected for homogenization. The total RNAs were isolated from lung homogenates, and the supernatant was stored. The mRNA levels of IL-6 (A), TNF-α (B), IL-1β (C) and protein production of IL-6 (D) and TNF-α (E) were then quantified by real-time PCR and ELISA assays, respectively. Values represent mean±SD. n=3. ^*P<0.05.

Figure 7

PSN decreased p38 phosphorylation, iNOS expression and NO production in the lungs of ALI mice. Sepsis-induced ALI were made by intratracheal injection of LPS (5 mg/kg) with or without PSN (10 mg/kg) administration for 4 h. Mice were then euthanized, and the lung tissues were collected. (A) Western blot analysis of p-p38 and iNOS protein expression in the lungs of control mice, ALI mice and PSN treated mice. (B and C) The ratio of p-p38/p38 and iNOS/β-actin were measured by densitometry. (D) The lung tissues were homogenized, and the supernatants were collected to detect the amounts of secreted NO. Data represent mean±SD. n=3. ^*P<0.05.