PEBL, a component-based Chinese medicine, reduces virus-induced acute lung injury by targeting FXR to decrease ACE2 levels

Abstract

Introduction: Despite the growing clinical need, the therapeutic efficacy of drugs for acute lung injury (ALI) remains inadequate. Traditional Chinese Medicine (TCM) holds potential in managing ALI due to its unique therapeutic properties. However, the intricate nature of TCM formulations hinders global adoption. Component-based Chinese medicine (CCM) offers a promising pathway for TCM's internationalization. Phillyrin-Emodin-Baicalin-Liquiritin (PEBL), a CCM with significant anti-inflammatory activity, is derived from the well-established TCM formula Liang-Ge-San. Whether PEBL effectively addresses viral ALI, however, remains unclear.

Objectives: This study aims to investigate the therapeutic effects and underlying mechanisms of PEBL on viral ALI.

Methods: The efficacy of PEBL against Poly(I:C)-induced ALI was assessed by analyzing cytokine production, macrophage infiltration, pulmonary damage, and mortality. Bioinformatics and network pharmacology were employed to identify key targets and signaling pathways. The molecular mechanisms were further validated using Poly(I:C)-treated RAW264.7 cells, Tg(coro1α: GFP) zebrafish, BALB/c mice, and models of Influenza A/Puerto Rico/8/1934 (H1N1) virus strain (PR8)-induced ALI in BALB/c mice and SARS-CoV-2 Omicron XBB.1.16 subvariant (XBB)-induced ALI in hACE2-transgenic C57BL/6 mice.

Results: PEBL mitigated Poly(I:C)-induced ALI, as evidenced by reduced cytokine levels, diminished macrophage infiltration, alleviated lung damage, and decreased mortality. Virtual screening identified the farnesyl X receptor (FXR) and angiotensin-converting enzyme 2 (ACE2) as key therapeutic targets for viral pneumonia. Mechanistically, PEBL downregulated FXR expression, inhibiting FXR binding to ACE2 promoters, which subsequently suppressed NF-κB-p65 nuclear translocation and cytokine production. In vivo, PEBL attenuated cytokine production by inhibiting ACE2 transcription through FXR downregulation, leading to alleviation of Poly(I:C)-induced ALI in both zebrafish and mice. Additionally, PEBL significantly improved symptoms of ALI caused by PR8 and XBB infections, by disrupting the FXR/ACE2 signaling axis, resulting in reduced weight loss, lower lung indices, diminished viral load and titer, fewer pulmonary lesions, and suppressed NF-κB-p65 nuclear translocation, along with decreased cytokine storm.

Conclusions: This study provides the first evidence that PEBL offers protective effects against ALI induced by acute respiratory viruses. PEBL prevents FXR from binding to ACE2 by inhibiting FXR transcription, which reduces macrophage infiltration, cytokine storm formation, and inflammatory injury, thereby ameliorating viral ALI. These findings underscore the potential of PEBL as a candidate for further exploration in the treatment of viral ALI.

Keywords: ACE2; Acute lung injury; Cytokine storms; FXR; PEBL; Virus.

Graphical abstract

Fig. 1

Synergistic effects of PEBL components in Poly(I:C)-induced ALI in zebrafish. (A) Chemical structures of the TCM monomers in PEBL. Both PEBL and its individual components exert protective effects against Poly(I:C)-induced acute lung injury (ALI) in zebrafish, with PEBL showing the most significant protection. (B) Quantitative analysis of macrophage infiltration in the swim bladder section of 5 dpf Tg(coro1α: GFP) larvae at 4 hpi, evaluating the intervention effects of PEBL and its individual components (n = 10). (C) Survival analysis of 5 dpf Tg(coro1α: GFP) larvae over 72 hpi, assessing the protective effects of PEBL and its individual components (n = 30). (D) Fluorescence imaging of macrophages in the swim bladder section from various treatment groups at 4 hpi, indicated by the red circle. Macrophages are located within the swim bladder region. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Poly(I:C); ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 vs. PEBL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

PEBL alleviates Poly(I:C)-induced ALI in a dose-dependent manner and modulates cytokine levels in macrophage inflammation. (A) Experimental design for PEBL treatment in ALI zebrafish. (B) Dose-dependent reduction in mortality by PEBL. Survival plot of 5 dpf Tg(coro1α: GFP) larvae at 72 hpi (n = 30). (C) Dose-dependent reduction in macrophage recruitment by PEBL. Quantitative analysis of macrophage infiltration in the swim bladder section at 4 hpi (n = 10). (D) Fluorescence images of macrophages in the swim bladder section at 4 hpi following different concentrations of PEBL, marked by the red circle. (E-J) PEBL reduces Poly(I:C)-induced cytokine elevation in RAW264.7 cells (n = 3). mRNA levels of IL-1β, IL-6, and TNF-α in cells were measured by qPCR (E-G), while protein concentrations of these cytokines in culture media were quantified using ELISA (H-J). ^##P < 0.01, ^###P < 0.001 vs. Poly(I:C); ^**P < 0.01, ^***P < 0.001 for group comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Identification of FGs in viral pneumonia. (A) Volcano plots showing DEGs. (B) Heatmap illustrating module-trait relationships. Red indicates positive correlations; blue represents negative correlations. (C) Histogram displaying gene significance across various modules. (D) Intersection of the brown module with DEGs to identify viral pneumonia FGs. (E) Network connectivity count within the PPI network. FXR, NR1H4. (F) GO analysis of FGs. (G) KEGG pathway analysis of FGs. (H) Metascape analysis of FGs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

FXR and ACE2 as predicted key targets for PEBL in antiviral pneumonia. (A-C) Identification of HGs in viral pneumonia. 113 machine learning algorithm combinations were assessed through 10-fold cross-validation (A). ROC curves for FGs identified by the optimal diagnostic models (B). Correlation analysis visualizing relationships among the FGs (C). (D) Common therapeutic targets identified for the four monomers in PEBL. (E) NR1H4 (FXR) and ACE2 as primary therapeutic targets of PEBL for viral pneumonia. (F-I) Optimal docking conformations of PEBL monomer molecules with FXR protein. The docking diagrams for FXR protein with phillyrin (F), emodin (G), baicalin (H), and liquiritin (I).

Fig. 5

PEBL suppresses Poly(I:C)-induced FXR and ACE2 expression and NF-κB-p65 nuclear translocation in RAW264.7 cells. (A-E) PEBL reduces the mRNA (A-B) and protein (D-E) levels of FXR and ACE2 and diminishes NF-κB-p65 nuclear translocation (C, E). (F-H) PEBL suppresses the protein distribution of FXR and ACE2, inhibits the nuclear translocation of NF-κB-p65. Representative images show the localization of FXR (F, green), ACE2 (G, green), NF-κB-p65 (H, green), and DAPI (blue), captured by immunofluorescence at 40 × magnification using confocal microscopy. Scale bar = 10 μm. UDCA was used as a positive control. Nuc, nucleus; Cyt, cytoplasm; Mem, membrane. n = 3; *P < 0.05, ^**P < 0.01, ^***P < 0.001 for group comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

PEBL suppresses Poly(I:C)-induced FXR binding to ACE2 by inhibiting FXR transcription in RAW264.7 cells. (A-B) FXR overexpression reverses the effect of PEBL on the protein levels of ACE2 and NF-κB-p65. n = 3. (C-D) FXR overexpression reverses the inhibitory effect of PEBL on ACE2 distribution and NF-κB-p65 nuclear translocation. Representative images show the localization of ACE2 (C, green), NF-κB-p65 (D, green), and DAPI (blue), captured by immunofluorescence at 40 × magnification using confocal microscopy. Scale bar = 10 μm. (E-H) PEBL requires FXR to decrease ACE2 expression and mitigate Poly(I:C) infection. In FXR-KD cells (F, H), no significant change in ACE2 expression was observed following treatments with CDCA, Poly(I:C), UDCA, or PEBL, compared to WT cells (E, G). WT, wild-type RAW264.7 cells; n = 3. (I) Co-IP analysis reveals no binding between FXR and ACE2 proteins. (J-K) PEBL reduces Poly(I:C)-induced FXR binding to the ACE2 promoter, confirmed by ChIP-qPCR and agarose gel electrophoresis.Nuc, nucleus; Cyt, cytoplasm; Mem, membrane; OSTα, positive control; ACE2-NC, negative control; C, control; P, Poly(I:C). n = 6; *P < 0.05, ^**P < 0.01, ^***P < 0.001 for group comparisons; ns, non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

PEBL alleviates Poly(I:C)-induced ALI by blocking FXR/ACE2 signaling in zebrafish. (A) Experimental strategy diagram illustrating the potential mechanism of PEBL in ALI zebrafish. (B-I) PEBL reduces the mRNA levels of IL-1β, IL-6, TNF-α, IFN-γ, IFN-φ1 (mammalian-derived IFN-α/IFN-β), and CXC-64 (other fish-derived IP10) by inhibiting FXR expression, which in turn downregulates ACE2 levels, thereby alleviating ALI. UDCA was used as a positive control; n = 3; *P < 0.05, ^**P < 0.01, ^***P < 0.001 for group comparisons.

Fig. 8

PEBL mitigates Poly(I:C)-induced ALI by inhibiting FXR/ACE2 signaling in mice. (A) Experimental strategy diagram for assessing the effects of PEBL treatment in ALI mice. (B) CDCA antagonizes the mortality-reducing effect of PEBL. Survival plot of Poly(I:C)-induced ALI mice at 10 dpi (n = 10). (C) CDCA counteracts the PEBL-mediated improvement in pulmonary structural morphology. Lung tissue was stained with H&E; scale bar = 50 μm. (D) CDCA reverses the PEBL-mediated reduction in macrophage infiltration. Macrophage presence in lung tissue was assessed via IHC, with CD68 as a macrophage marker; scale bar = 50 μm. (E-P) CDCA inhibits the PEBL-mediated decrease in IL-1β, IL-6, TNF-α, IFN-γ, IFN-α, and IP10 levels. Cytokine protein levels in serum were measured by ELISA, and cytokine mRNA levels in lung tissue were quantified by qPCR. (Q-V) CDCA reverses the PEBL-mediated reductions in FXR, ACE2, and SHP expression, as well as NF-κB-p65 nuclear translocation, as confirmed by qPCR and Western blot in lung tissue. (W-X) CDCA inhibits the PEBL-mediated suppression of FXR distribution and NF-κB-p65 nuclear translocation, as observed by IF in lung tissue. Representative images show the localization of FXR (W, green), NF-κB-p65 (X, green), and DAPI (blue), captured by immunofluorescence at 40 × magnification using confocal microscopy. Scale bar = 20 μm. UDCA, positive control; dpi, days post-injection; PEBL-25, 25 mg/kg PEBL; PEBL-50, 50 mg/kg PEBL; Nuc, nucleus; Cyt, cytoplasm; Mem, membrane. n = 8; ^##P < 0.01, ^###P < 0.001 vs. Poly(I:C); *P < 0.05, ^**P < 0.01, ^***P < 0.001 for group comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

PEBL mitigates PR8 infection-induced ALI by inhibiting FXR/ACE2 signaling. (A) Experimental protocol diagram to assess the effects of PEBL in PR8-infected ALI mice. (B) CDCA antagonizes the protective effects of PEBL, including inhibition of weight loss (B, n = 7), reduction in mortality (C, n = 7), decrease in lung index (D), and alleviation of viral load (E). (F) CDCA reverses the PEBL-mediated protection against pulmonary structural damage, as shown by H&E staining. Arrows indicate significant inflammatory cell exudate in the alveolar cavity. Scale bar = 50 μm. (G) CDCA inhibits the PEBL-mediated reduction in macrophage infiltration. Macrophage infiltration in lung tissue was evaluated by IHC, with CD68 as a marker for macrophages. Scale bar = 50 μm. (H-S) CDCA counteracts the PEBL-mediated reductions in IL-1β, IL-6, TNF-α, IFN-γ, IFN-α, and IP10 levels. Cytokine protein levels in serum were measured by ELISA, and cytokine mRNA levels in lung tissue were quantified by qPCR. (T-Y) CDCA reverses the PEBL-mediated decrease in FXR, ACE2, and SHP levels, as well as NF-κB-p65 nuclear translocation, as confirmed by qPCR and Western blot. (Z) CDCA inhibits PEBL-mediated suppression of NF-κB-p65 nuclear translocation, as demonstrated by IF in lung tissue. Representative images show the localization of NF-κB-p65 (green), and DAPI (blue), captured by confocal microscopy at 40 × magnification. Scale bar = 20 μm. OPC, positive control; PEBL-25, 25 mg/kg PEBL; PEBL-50, 50 mg/kg PEBL; Nuc, nucleus; Cyt, cytoplasm; Mem, membrane; weight loss calculated as the daily body weight divided by the initial weight on Day 1; dashed line represents 30 % weight loss; n = 6; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. PR8; ⁺P < 0.05 for PEBL-50 vs. CDCA + PEBL-50; *P < 0.05, ^**P < 0.01, ^***P < 0.001 for group comparisons; ns, non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

PEBL mitigates XBB infection-induced ALI by inhibiting FXR/ACE2 signaling. (A) Experimental protocol diagram to evaluate the effects of PEBL in XBB-infected ALI mice. CDCA reverses the PEBL-induced inhibition of weight loss (B) and viral titers (C). (D) H&E staining reveals that CDCA antagonizes the PEBL-mediated alleviation of pulmonary structural damage. Scale bar = 100 μm. (E-J) QPCR analysis shows that CDCA counteracts the PEBL-mediated decrease in IL-1β, IL-6, IL-8, TNF-α, IP10, and CCL2 levels. (K-L) Western blot analysis demonstrates that CDCA reverses the PEBL-mediated reduction in protein levels of FXR, ACE2, and SHP, as well as NF-κB-p65 nuclear translocation. PF, positive control; Nuc, nucleus; Cyt, cytoplasm; Mem, membrane; weight loss calculated as the daily body weight divided by the body weight on Day 0; dashed line represents 30 % weight loss; n = 5; *P < 0.05, ^**P < 0.01, ^***P < 0.001 for group comparisons.