Kynurenic acid alleviated endothelial injury through GPR35 in fluid resuscitation of sepsis
Authors: Chen Cheng , Chen Changxing , Huang Qiuping , Meng Xiaoxiao , Zhu Yong , Zhang Jiaxiang , Zhang Yu , Lu Jian , Wang Ruilan 
Published: November 2023

Abstract
Background
Capillary leakage caused by endothelial injury is the core pathological mechanism of sepsis, and fluid resuscitation is an important treatment. The mechanism of metabolic changes during fluid resuscitation in sepsis is unclear. The aim of this study was to expound the metabolic changes in fluid resuscitation of sepsis and further find ways to alleviate endothelial injury in sepsis.
Methods
General characteristics of patients and blood cell analysis results were collected. C-reactive protein (CRP), Interleukin (IL)-1β, IL-6 and tumor necrosis factor(TNF-α) were detected using enzyme linked immunosorbent assay (ELISA). Circulation indicators of patients including mean arterial pressure (MAP), central venous pressure (CVP), urine output, central venous blood oxygen saturation (ScvO2), and lactic acid of patients was collected. Differential metabolites from fluid resuscitation were detected by non-targeted gas chromatography-mass spectrometry (GC-MS) and enriched in relevant metabolic pathways through the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway. Differential metabolites and clinical indicators was detected through correlation analysis. The effects of the metabolites on endothelial cells were confirmed using in vitro and in vivo experiments. In vitro experiments, the effect of kynurenic acid (KYNA) on proliferation of human umbilical vein endothelial cells (HUVECs) through cell counting kit-8 (CCK8) assay. The effect of KYNA on cell migration was detected by scratch test. The effect of KYNA on the expression of junction protein (claudin-1, claudin-2, claudin-5, ZO-1, cx-43) was detected by western blot. Cecum ligation and puncture (CLP) was constructed. In vivo experiments, the effect of KYNA on vascular leakage was conducted using H&E and evans blue staining. The effect of KYNA on junction protein claudin-5 was detected by immunohistochemistry. The expression of KYNA receptor GPR (G-protein coupled receptor) 35 was detected by western blot and immunofluorescence. The shRNA vector of GPR35 was constructed, the downstream target genes of GPR35 were detected by transcriptome sequencing. The molecular binding relationship between GPR35 and mitochondrial solute carrier family 25A41 (SCL25A41) was further clarified through molecular docking. The effect of KYNA on endothelial cell was confirmed by ATP content.
Results
The tryptophan metabolism pathway was enriched during the first three periods of fluid resuscitation. A significant negative correlation was observed between KYNA and IL-1β. KYNA was negatively correlated with the content of lactic acid (r = -0.6253, p = 0.0096), the results indicated that KYNA plays an anti-inflammatory role and could improve microcirculation in fluid resuscitation. High level of expression of both KYNA and IL-1β indicates the optimization period. The stable period is defined by a high level of KYNA but a low level of IL-1β. In vivo and in vitro experiments confirmed that KYNA had anti-inflammatory effects on lipopolysaccharide (LPS)-treated HUVECs and reduced IL-1β and TNF-α in septic mice caused CLP. H&E staining indicated that KYNA could reduce the infiltration of inflammatory cells in the lung, alveolar collapse, and pulmonary vascular congestion in CLP-induced sepsis. Evans blue staining showed that KYNA could alleviate fluid leakage from lung tissue. The expression of KYNA receptor GPR35 was increased. Transcriptome sequencing showed that inhibiting GPR35 significantly reduced the expression of SCL25A41. GPR35 and SLC25A41 can be tightly bound through molecular docking experiment. ATP content was increased which stimulated by KYNA.
Conclusion
KYNA is a metabolite whose effect of improving endothelial injury in fluid resuscitation in sepsis. Administration of KYNA warrants investigation as a potential therapeutic agent for capillary leakage in sepsis.