Paclitaxel alleviated sepsis-induced acute lung injury by activating MUC1 and suppressing TLR-4/NF-κB pathway

Abstract

Purpose: It has been reported that approximately 40% of ALI (acute lung injury) incidence resulted from sepsis. Paclitaxel, as a classic anti-cancer drug, plays an important role in the regulation of inflammation. However, we do not know whether it has a protective effect against CLP (cecal ligation and puncture)-induced septic ALI. Our study aims to illuminate the mitigative effects of paclitaxel on sepsis-induced ALI and its relevant mechanisms.

Materials and methods: The survival rates and organ injuries were used to evaluate the effects of paclitaxel on CLP mice. The levels of inflammatory cytokines were tested by ELISA. MUC1 siRNA pre-treatment was used to knockdown MUC1 expression in vitro. GO203 was used to inhibit the homodimerization of MUC1-C in vivo. The expression levels of MUC1, TLR 4 and p-NF-κB/p65 were detected by Western blot.

Results: Our results showed that paclitaxel improved the survival rates and ameliorated organ injuries especially lung injury in CLP-induced septic mice. These were accompanied by reduced inflammatory cytokines in sera and BALF (bronchoalveolar lavage fluid). We also found paclitaxel could attenuate TLR 4-NF-κB/p65 activation both in lung tissues of septic mice and LPS-stimulated lung type II epithelial cell line A549. At the upstream level, paclitaxel-upregulated expression levels of MUC1 in both in vivo and in vitro experiments. The inhibitory effects of paclitaxel on TLR 4-NF-κB/p65 activation were reversed in lung tissues of septic mice pre-treated with MUC1 inhibitor and in MUC1-knockdown A549 cells. Protection of paclitaxel on sepsis-induced ALI and decrease of inflammatory cytokines were also abolished by inhibition of MUC1.

Conclusion: Collectively, these results indicated paclitaxel could significantly alleviate acute lung injury in CLP-induced septic mice and LPS-stimulated lung type II epithelial cell line A549 by activating MUC1 and suppressing TLR-4/NF-κB pathway.

Keywords: MUC1; NF-κB; TLR 4; acute lung injury; sepsis.

Figure 1

Effects of paclitaxel on survival rates of septic mice. Survival curve of septic mice during a 7-day period. CLP mice were administered with 75, 150, 225, 300 μg/kg paclitaxel or vehicle (sterile 0.9% saline) 2 hrs after CLP or 150 μg/kg paclitaxel immediately after CLP. Sham, n=10; CLP and CLP + paclitaxel, n=20. Survival rates among groups were analyzed using Kaplan–Meier analysis and log-rank tests. We compared CLP group with CLP + paclitaxel group and calculated P-value marked in the figures. P-value between CLP group and CLP + paclitaxel (75 µg/mL) group was 0.276. P-value between CLP group and CLP + paclitaxel (150 µg/mL) group was 0.024. P-value between CLP group and CLP + paclitaxel (150 µg/mL, immediate postoperative administration) group was 0.073. P-value between CLP group and CLP + paclitaxel (225 µg/mL) group was 0.527. P-value between CLP group and CLP + paclitaxel (300 µg/mL) group was 0.940.

Figure 2

Protection of paclitaxel on organ injuries in septic mice. (A–D) Representative morphological images at 6 and 24 hrs by HE staining were presented to assess the injury severity of lungs, heart, kidney and liver. (E, G, I, K) Histological scores of organ injury were calculated. Lung wet/dry ratio (F), sera creatine kinase (H), sera creatinine (J) and sera alanine aminotransferase (L) were also tested. Samples were harvested at 6 and 24 hrs after CLP. Data represent means ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Effects of paclitaxel on inflammatory cytokine concentrations in sera and BALF of septic mice. The levels of IL-1β, IL-6, TNF-α, IL-10 in sera (A) and bronchoalveolar lavage fluid (BALF) (B) were analyzed by ELISA. Data represent means ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.

Figure 4

Paclitaxel inhibited TLR 4-NF-κB/p65 activation and upregulated MUC1 in sepsis-induced ALI. (A–D) TLR 4, p-NF-κB/p65 expression levels at 6 and 24 hrs after CLP in lung tissues were detected by Western blot.  *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Paclitaxel inhibited TLR 4-NF-κB/p65 activation and upregulated MUC1 in LPS-stimulated lung type II epithelial cell line. (A–C) A549 cells were stimulated with different doses of LPS (1, 2.5, 5, 10 μg/mL) for 24 hrs. (D–F) A549 cells were incubated with 2.5 μg/mg LPS for 6, 12, 24, and 48 hrs. (G–J) A549 cells were treated with different doses of paclitaxel (1, 2.5, 5, 10, 15, 20 nM) after stimulated by 2.5 μg/mL LPS for 24 hrs. TLR 4, p-NF-κB/p65 and MUC1 expression levels were detected by Western blot. Data represent means ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Mitigative effects of paclitaxel on lung injury induced by CLP were reversed by MUC1 inhibitor. Different doses of GO203 (specific inhibitor MUC1) (10, 20, 30 mg/kg) and its negative control CP2 (10, 20, 30 mg/kg) were injected intraperitoneally in mice prior to CLP and paclitaxel treatment. (A–C) TLR 4 and p-NF-κB/p65 were detected by Western blot. (D–G) Representative morphological images at 24 hrs by HE staining were presented to assess the injury severity of lungs, heart, kidney and liver. (H–K) Histological scores of organ injury were calculated. Samples were harvested at 24 hrs after CLP. Data represent means ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.

Figure 7

Downregulation of inflammatory cytokines by paclitaxel in sera and BALF of CLP-induced septic mice was reversed by MUC1 inhibitor. The levels of IL-1β, IL-6, TNF-α, IL-10 in sera (A) and bronchoalveolar lavage fluid (BALF) (B) were analyzed by ELISA. Data represent mean ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.

Figure 8

Mitigative effects of paclitaxel in LPS-stimulated A549 cells were reversed by MUC1 knock-down. (A–D) A549 cells were transfected with NC-siRNA or MUC1-siRNAs for 24 hrs. Expression levels of TLR 4, p-NF-κB/p65 and MUC1 were detected by Western blot. (E–H) A549 cells transfected with NC-siRNA or MUC1-siRNAs for 24 hrs were treated with LPS (2.5 μg/mL) and paclitaxel (5 nM) for another 24 hrs. Expression levels of TLR 4, p-NF-κB/p65 and MUC1 were detected by Western blot. (I) The levels of IL-1β, IL-6, TNF-α, IL-10 in the culture supernatants were analyzed by ELISA. Data represent mean ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.