A single oral administration of resveratrol cocrystals enables potent antisepsis therapy via interfering neutrophil extracellular traps formation

Abstract

Sepsis is a life-threatening syndrome characterized by organ dysfunction resulting from host response disorders and is a common complication in patients suffering from clinical trauma, burns, and infections. Resveratrol, a natural polyphenol compound, possesses anti-inflammatory, antiviral, antibacterial, and antifungal properties, as well as cardiovascular and anti-tumor protective effects. However, its therapeutic applications have been limited due to low bioavailability, poor aqueous solubility, and rapid metabolism. To address these limitations, this study focused on the preparation of resveratrol (RES) cocrystal with nicotinamide (NCA) and RES polymorphs through recrystallization for oral application. The identity of the new crystalline forms was confirmed using powder X-ray diffraction (PXRD), Fourier transform-infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and single-crystal X-ray diffraction (SCXRD). Physicochemical evaluations revealed that the RES polymorphs and RES-NCA cocrystal exhibited higher solubility and dissolution rates compared to pristine RES. In vitro experiments with LPS-stimulated HL-60 cells demonstrated that the RES-NCA cocrystal effectively inhibited neutrophil extracellular traps (NETs) formation by targeting peptidylarginine deiminase 4 (PADI4) and reducing inflammatory markers. Additionally, in vivo studies using a cecal ligation and puncture (CLP)-induced sepsis model in mice showed that oral administration of the RES-NCA cocrystal significantly reduced NETs formation, organ injury, and mortality. These findings suggest that orally administered RES-NCA cocrystal holds promise as a therapeutic agent for sepsis treatment, offering enhanced solubility and bioavailability, along with potent effects in inhibiting NETs formation and reducing sepsis-induced organ injury.

Keywords: Cocrystal engineering; Neutrophil extracellular traps; Oral administration; Resveratrol; Sepsis.

Graphical abstract

Fig. 1

The crystal structure analysis of RES polymorphs (A) ORTEP diagram with 50 % probability and (B) hydrogen bonding network leading to the packing structure along with bc direction. Hydrogen atoms are excluded for clarity. Colour code, Red: Oxygen; Gray: Carbon. Represents (C). Hirschfeld surface, D. relative contribution of every type of contacts and (E) Two-dimensional fingerprints plots of RES polymorphs showing the contribution of each type of contact within the molecule. The solid-state characterizations where (F) SEM images of RES polymorphs and (G) RES-NCA cocrystal with 1 μm scale bar, (H) Powder XRD, (I) FT-IR and (J) DSC comparison of RES, coformer NCA and newly fabricated RES polymorphs and RES-NCA cocrystal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Comparative effects of RES polymorphs and RES-NCA cocrystal on NETs formation in LPS-stimulated HL-60 cells. A. Western blotting showing the expression levels of PADI4, NE, and MPO proteins in HL-60 cells with different treatments. B. Quantification of protein band intensities normalized to GAPDH, presented as fold changes relative to the control group. The graph compares the expression levels of NET markers PADI4, MPO, and NE (n = 3). ^#P < 0.05 Sham vs CLP group, *P < 0.05. CLP + RES-NCA cocrystal vs CLP group. C. Confocal microscope images of CitH3 (red) and DAPI (blue) showing NETs formation. D. Confocal microscope images of for NE (red) and DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Biosafety study. A. In vivo evaluation of systemic toxicity after treatment. Variations of ALT (Alanine aminotransferase), AST (Aspartate aminotransferase), LDH (Lactate dehydrogenase), creatinine, and CK-MB (Creatine kinase-MB) in blood samples were analyzed. B. H&E staining of major organs (heart, liver, spleen, lung, and kidney) of mice treated with Saline, RES polymorphs, and RES-NCA cocrystal.

Fig. 4

Comparative analysis of renal function, survival, and TUNEL assay in sepsis models treated with RES polymorphs and RES-NCA cocrystal. A. Therapeutic diagram of RES-NCA cocrystal and RES polymorphs in the mouse model of CLP-induced sepsis. B. Representative TUNEL staining images at 20× and 40× magnification, illustrating apoptotic cells in kidney. Insets highlight areas with TUNEL-positive cells. C–D. BUN and creatinine levels in serum for assessing kidney injury and renal function (n = 5). E. Kaplan-Meier survival curves (n = 10). F. Quantification of TUNEL-positive cells, indicating apoptosis levels in kidney tissues (n = 5). Data are shown as the means±SD, ^#P < 0.05 Sham vs CLP group, *P < 0.05. CLP + RES-NCA cocrystal vs CLP group, ^&P < 0.05 CLP + RES-NCA cocrystal vs CLP group.

Fig. 5

RES-NCA cocrystal and RES polymorphs mitigate intestinal damage and microvascular impairment under septic conditions. A-B. HE staining and TUNEL staining of ileal tissues near the cecal ligation site. Images are shown at 20× and 40× magnification, with insets highlighting regions of interest. C. Quantification of Chiu's score (n = 7), indicating the degree of intestinal damage. D. Quantification of TUNEL-positive cells (n = 6), showing the number of apoptotic cells in the ileal tissues. ^#P < 0.05 Sham vs CLP group, *P < 0.05. CLP + RES-NCA cocrystal vs CLP group.

Fig. 6

Therapeutic effects of RES-NCA cocrystal and RES polymorphs on intestinal and renal NET formation and inflammatory responses in septic conditions. A-B. Immunofluorescent staining of ileal tissues near the cecal ligation site and renal tissues. Staining shows CD31 (red), VCAM-1 (magenta), MPO (green), and DAPI (blue) to assess endothelial and microvascular injury and NET formation. Immunofluorescent colocalization was performed to assess NET formation (MPO) in microvascular (CD31) and endothelial (VCAM-1) injury. C. Relative expression of serum NET biomarkers MPO-DNA and H3cit measured by ELISA(n = 5). D-E. Quantification of TNF-α and IL-6 levels in serum (pg/mL) by ELISA (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

The schematic graph illustrates the preparation of resveratrol cocrystal (RES-NCA) and the anti-inflammatory mechanism of sepsis-induced neutrophil extracellular traps (NETs) inhibition. (Created with BioRender.com). A. Formation of a cocrystal of resveratrol (RES) with nicotinamide (NCA) and RES polymorphs through a recrystallization method. B. The experimental scheme of the treatment of CLP-induced sepsis using resveratrol cocrystal. C. Mechanism of NETs formation and inhibition by the RES-NCA cocrystal during sepsis. (a). In normal neutrophils, peptidylarginine deiminase 4 (PAD4) and granules filled with enzymes such as myeloperoxidase (MPO) and neutrophil elastase (NE) are retained in the cytoplasm. (b). In activated neutrophils during sepsis, excessive ROS stimulates the release of multiple enzymes from granules and promotes the translocation of PAD4 into the nucleus. The nuclear PAD4 citrullinates certain histone arginines, particularly histones H3, which interrupt the electrostatic interactions between histones and DNA in nucleosomes and cause the dissociation of nuclear and granule membranes. (c). The overproduction of NETs exacerbates inflammation during sepsis. (d). Upon treatment of the RES-NCA cocrystal, histone H3 citrullination was blocked in activated neutrophils due to the antagonistic binding of the RES-NCA cocrystal. (e). After treatment with RES-NCA cocrystal, the citrullinated histone H3, the release of DNA and enzymes are reduced, thereby mitigating neutrophil-driven inflammation of sepsis.