Supramolecular prodrug inspiried by the Rhizoma Coptidis - Fructus Mume herbal pair alleviated inflammatory diseases by inhibiting pyroptosis

Abstract

Sustained inflammatory responses are closely related to various severe diseases, and inhibiting the excessive activation of inflammasomes and pyroptosis has significant implications for clinical treatment. Natural products have garnered considerable concern for the treatment of inflammation. Huanglian-Wumei decoction (HLWMD) is a classic prescription used for treating inflammatory diseases, but the necessity of their combination and the exact underlying anti-inflammatory mechanism have not yet been elucidated. Inspired by the supramolecular self-assembly strategy and natural drug compatibility theory, we successfully obtained berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS), which is an herbal pair from HLWMD. Using a series of characterization methods, we confirmed the self-assembly mechanism of BCS. BBR and CGA were self-assembled and stacked into amphiphilic spherical supramolecules in a 2:1 molar ratio, driven by electrostatic interactions, hydrophobic interactions, and π-π stacking; the hydrophilic fragments of CGA were outside, and the hydrophobic fragments of BBR were inside. This stacking pattern significantly improved the anti-inflammatory performance of BCS compared with that of single free molecules. Compared with free molecules, BCS significantly attenuated the release of multiple inflammatory mediators and lipopolysaccharide (LPS)-induced pyroptosis. Its anti-inflammatory mechanism is closely related to the inhibition of intracellular nuclear factor-kappaB (NF-κB) p65 phosphorylation and the noncanonical pyroptosis signalling pathway mediated by caspase-11.

Keywords: Anti-inflammation; Pyroptosis; Self-assembly; Supramolecular; Traditional Chinese medicine.

Graphical abstract

Scheme 1

Schematic representation of (A) the self-assembly and (B) the anti-inflammatory mechanisms of the berberine (BBR) and chlorogenic acid (CGA) supramolecular (BCS). NPs: nanoparticles; LPS: lipopolysaccharide; TLR4: Toll-like receptor 4; IκBα: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; iNOS: inducible nitric oxide (NO) synthase; IL: interleukin; TNF-α: tumor necrosis factor alpha; NLRP3: nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3; GSDMD: gasdermin D.

Fig. 1

Micromorphological properties and spectroscopic characterization of berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS), BBR, and CGA. (A, B) Transmission electron microscopy (TEM) images of BCS: BCS with scale bar of 500 nm (A) and 1 µm (B). (C) Size distribution of BCS determined by nanoparticle (NP) tracking analysis (NTA). (D) Ultraviolet-visible (UV-vis) absorption spectrograms of BCS, BBR, and CGA with a scan range from 200 to 500 nm. (E) Fourier transform-infrared (FT-IR) spectroscopy of BCS, BBR, and CGA with a scan range of 4000 to 400 cm⁻¹. (F) Fluorescence emission spectrum of BCS, BBR, and CGA with Ex = 350 nm and Em from 400 to 800 nm.

Fig. 2

Characterization of the self-assembly mechanism of berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS). (A) 500 MHz ¹H nuclear magnetic resonance (NMR) spectrum of BBR, CGA, and BCS in dimethyl sulfoxide (DMSO)-d6 solution. (B, C) The corrected data of isothermal titration calorimetry (ITC) (B) and fitting curves (C), including the thermodynamic parameters of BBR-CGA titration. (D) Illustration of the self-assembly mechanism and stability of BCS. RC: Rhizoma Coptidis; FM: Fructus Mume.

Fig. 3

Evaluation of the cellular uptake and biocompatibility of berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS). (A) Drug release profile of BCS and BBR. (B) Hemolytic activities of BCS ranging from 10 to 100 μM. In-vitro biosecurity characterization of BCS, BBR, and CGA. Inset shows photographs of corresponding solutions after blood centrifugation. (C, D). In-vitro cell viability assays of BCS, BBR, and CGA after 24 h (C) and 48 h (D). (E) Fluorescence images of celluar uptake of fluorescein isothiocyanate (FITC)-labelled BCS (10 μM) after treatment at various time intervals (3, 6, 9, and 12 h). DAPI: 4′,6-diamidino-2-phenylindole.

Fig. 4

Investigation of the anti-inflammatory properties of berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS). (A) Fluorescence imaging of intracellular reactive oxygen species (ROS) with BCS, BBR, and CGA at 10 μM. (B) Quantitative assessment of intracellular ROS levels following treatment with varying concentrations of BBR, CGA, BCS, and control, with or without lipopolysaccharide (LPS) induction (1 μg/mL). (C, D) Enzyme-linked immunosorbent assay (ELISA) measurements of interleukin-1β (IL-1β) (C) and IL-18 (D) secretions into the cell culture media. (E) Nitric oxide (NO) levels were determined by Griess assay. All data are expressed as means ± standard deviation (SD). Statistical differences were analyzed using one-way analysis of variance (ANOVA). ^####P < 0.0001, compared with the control group; ^∗∗∗∗P < 0.0001, compared with the LPS-induced model group.

Fig. 5

Anti-inflammatory effect of berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS) mediated through the inhibition of cell pyroptosis. (A) The death of pyroptotic cells were detected by propidium iodide (PI) staining (red) and visualized in conjunction with Hoechst 33342 (blue). (B) Scanning electron micrographs (SEM) of RAW264.7 cells post-treatment with lipopolysaccharide (LPS) (1 μg/mL) and/or BCS (10 μM) for 24 h. (C) Quantification of PI-positive cells using ImageJ software from five random fields, and the percentage of cell death was calculated as the ratio of PI-positive cells to all cells (labelled by Hoechst 33342). (D) Lactic acid dehydrogenase (LDH) release as an indicator of cell death. Data are expressed as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA). ^####P < 0.0001, compared with the control group; ^∗∗∗∗P < 0.0001, compared with the LPS-induced model group.

Fig. 6

BCS modulation of nuclear factor-kappaB (NF-κB) and caspase-4/5/11-regulated non-canonical proptosis pathways in RAW264.7 cells. (A) Differential gene expression and functional annotations induced by berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS) were represented by enrichment circle plots. (B) Top15 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis shown in a scatterplot. (C) Heatmap representation of microarray analysis in control, lipopolysaccharide (LPS)-induced model (1 μg/mL), and BCS-treated RAW264.7 cells (10 μM), with three biological replicates per group. (D–K) Real-time polymerase chain reaction (PCR) assay of messenger RNA (mRNA) levels for tumor necrosis factor alpha (TNF-α) (D), interleukin-6 (IL-6) (E), inducible nitric oxide (NO) synthase (iNOS) (F), caspase 11 (G), caspase-1 (H), nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLPR3) (I), IL-1β (J), and IL-18 (K), respectively. Data are expressed as means ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA). ^####P < 0.0001, compared with the control group; ^∗P < 0.05, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001, compared with the LPS-induced model group. RIG-I: retinoic acid-inducible gene I.

Fig. 7

Downregulation of the nuclear factor-kappaB (NF-κB) and caspase-4/5/11-associated protein expression by berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS) in the noncanonical pyroptosis pathway. (A) Western blot analysis of protein levels in cell lysates, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as a loading control. (B) Quantitative evaluation of relative NF-κB p65 phosphorylation levels. (C) Normalized caspase-11 expression relative to the control group. Data are expressed as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA). ^##P < 0.01 and ^####P < 0.0001, compared with the control group; ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001, compared with the lipopolysaccharide (LPS)-induced model group.