Natural bear bile powder attenuates lipopolysaccharide-induced acute lung injury in mice by regulating the NF-κB and Nrf-2/HO-1 signaling pathways

doi:10.12122/j.issn.1673-4254.2026.07.16

Abstract

Abstract:

Objective To investigate the mechanism that mediates the protective effect of bear bile powder (BBP) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Methods Cultured RAW264.7 cells were pretreated with, different concentrations of BBP, or dexamethasone (Dex) for 1 h before LPS challenge. Sixty male C57BL/6 mice were randomized into 5 groups (n=12), including a control group, a LPS-induced ALI model group, a Dex treatment group, and two BBP treatment groups treated with low- (30 mg/kg) or high-dose (120 mg/kg) BBP. The levels of inflammatory factors and oxidative stress-related indicators in the cells and mouse lung tissues were determined, and mouse lung histopathology, wet/dry weight ratio, and BALF cell count were examined; the expressions of NF-κB and Nrf-2/HO-1 pathways were detected using qPCR, Western blotting, and immunohistochemistry. Results In LPS-stimulated RAW264.7 cells, BBP (12.5 μg/mL) significantly reduced the levels of TNF-α, COX-2, IL-1β, IL-6, MDA, NO and ROS, enhanced SOD activity, increased mRNA expressions of IκB-α, Nrf-2, and HO-1, and lowered mRNA expressions of TNF-α, IL-1β, Keap-1, and NF-κB (p65). The ALI mouse models showed severe lung pathologies and edema, increased BALF cell counts, and increased levels of TNF‑α, COX-2, IL-1β, IL-6, MDA, and NO, lowered SOD activity with reduced protein expressions of IκB-α, Nrf-2, and HO-1 and increased expressions of Keap-1 and p65. Treatment with high-dose BBP significantly ameliorated lung pathologies in the mouse models and improved the aberrant alterations in pulmonary expressions of inflammatory factors, oxidative stress-related indicators and the NF-κB and Nrf-2/HO-1 pathways. Conclusion BBP protects against LPS-induced ALI in mice possibly by targeting the Nrf-2/HO-1 and NF-κB pathways, suggesting its potential as a therapeutic agent for ALI.

Key words: natural bear bile powder, acute lung injury, nuclear factor-κB signaling pathway, Nrf-2/HO-1 signaling pathway

Yuhang SUN, Xingzhen LU, Xinyue ZHANG, Jianpeng GUO, Xin JIN. Natural bear bile powder attenuates lipopolysaccharide-induced acute lung injury in mice by regulating the NF-κB and Nrf-2/HO-1 signaling pathways[J]. Journal of Southern Medical University, 2026, 46(7): 1622-1633.

Figures/Tables 11

Tab.1 Primers for RT-PCR

Primer	Sequence
p65	F: 5′ - TGCGATTCCGCTATAAATGCG-3′ R: 5′ - ACAAGTTCATGTGGATGAGGC-3′
TNF-α	F: 5′- CAGGCGGTGCCTATGTCTC -3′ R: 5′- CGATCACCCCGAAGTTCAGTAG-3′
IL-1β	F: 5′ - GAAATGCCACCTTTTGACAGTG-3′ R: 5′ - TGGATGCTCTCATCAGGACAG-3′
IκBα	F: 5′ - CGGCACCGAGTACCTTGAC -3′ R: 5′ - CACCGGCTGCATACAACTTCT-3′
Nrf-2	F: 5′-CTTTAGTCAGCGACAGAAGGAC-3′ R: 5′ - AGGCATCTTGTTTGGGAATGTG-3′
HO-1	F: 5′ - GATAGAGCGCAACAAGCAGAA -3′ R: 5′ - CAGTGAGGCCCATACCAGAAG -3′
Keap-1	F: 5′ -CGGGGACGCAGTGATGTATG -3′ R: 5′ - TGTGTAGCTGAAGGTTCGGTTA-3′
GAPDH	F: 5′ - AGGTCGGTGTGAACGGATTTG-3′ R: 5′ - GGGGTCGTTGATGGCAACA-3′

Fig.1 Results of CCK-8 assay. A: Cell viability assessed by CCK-8 assay in RAW 264.7 cells treated with different concentrations of BBP. B: Cell viability detected by CCK-8 assay of LPS-induced RAW 264.7 cells treated with different concentrations of BBP. Data are presented as Mean±SD (n=3). #P<0.05, ##P<0.01, ###P<0.001 vs Control group; **P<0.01 vs Model group.

Fig.2 Effect of BBP on inflammatory cytokine levels in LPSinducedRAW264.7 cells Mean±SD, n=3). ##P<0.01, ###P<0.001 vs Control group; **P<0.010.01, ***P<0.010.001 vs Model group.

Fig.3 Effect of BBP on MDA, SOD and NO in LPS-induced RAW264.7 cells (Mean±SD, n=3). ##P<0.01, ###P<0.001 vs Control group; *P<0.05, **P<0.01, ***P<0.001 vs Model group.

Fig.4 Effect of BBP pretreatment on ROS in RAW264.7 cells assessed using DCFH-DA fluorescent probe (Scale bar=100 μm).

Fig.5 qPCR for detecting mRNA expressions of the Keap-1, HO-1, Nrf-2,NF-κB, TNF-α, IκB-α and IL-1β in LPS-induced RAW264.7 cells (Mean±SD, n=3). ##P<0.01, ###P<0.001 vs Control group; **P<0.010.01, ***P<0.010.001 vsModel group.

Fig.6 Effects of BBP on LPS-induced histopathological changes in lung tissues of ALI mice. A: HE staining for examining histological alterations in mouse lung tissues (Scale bar=200 μm). B: Lung injury score. C: Lung wet-to-dry ratio. Data are presented as Mean±SD (n=6). ###P<0.001 vs Control group; **P<0.01, ***P<0.001 vs Model group.

Fig.7 　Effects of BBP on changes cell counts in BALF from LPSinducedALI mice. A: Number of total cells in the BALF. B: Number ofneutrophils in the BALF. C: Levels of BALF protein. D: MPO activitydetected using corresponding assay kits in the lung tissue. Data arepresented as Mean±SD, n=6). ##P<0.01, ###P<0.001 vs Control group; *P<0.05, **P<0.01, ***P<0.001 vs Model group.

Fig.8 Effects of BBP on levels of inflammatory factors (IL-1β, COX-2,TNF‑α and IL-6) and oxidative stress indicators (NO, SOD andMDA) in lung tissues of LPS-induced ALI mice. Data are presentedas Mean±SD, n=6). ##P<0.01 vs control group; *P<0.05, **P<0.01 vsmodel group.

Fig.9 　Effects of BBP on expression levels of Nrf-2, HO-1, IL-1β, and NF-κB (p65) in lung tissues of mice with ALI. (a) IHC results of Nrf-2, HO-1,IL-1β and NF-κB (p65) in lung tissues of mice in each group (Scale bar=50 μm). (b) Proportion of positive cells for (A) HO-1, (B) NF-κB (p65),(C) IL-1β, and (D) Nrf-2 in lung tissues of mice in each group (%). Dataare presented as Mean±SD (n=6). ##P<0.01, ###P<0.001 vs Control group; *P<0.05, **P<0.01 vs Model group.

Fig.10 Effect of BBP on expressions of Keap-1, Nrf2, HO-1, IκB-α and NF‑κB (p65) in mouse lung tissues. Data are presented as Mean±SD (n=6). ##P<0.01, ###P<0.001 vs Control group; *P<0.05, **P<0.01 vs Model group.

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