Protective effect and mechanism of synagrantol A from Euphorbia pseudomollis on LPS-induced neuroinflammation

Species of Euphorbia (Euphorbiaceae) have traditionally been utilized worldwide for analgesic, anti-inflammatory, and anticancer applications. Latex and extracts from these plants have long been used in Brazilian indigenous medicine. The utilization of contemporary extraction and analytical methodologies to clarify the anti-inflammatory pharmacology of Euphorbia extracts establishes a translational connection between conventional knowledge and modern drug discovery.

Neuroinflammation plays a significant role in the pathogenesis of various neurological disorders. 3,4,12,13-Tetraacetyl-20-phenylacetylphorbol (synagrantol A, referred to as A3), a novel compound isolated from Euphorbia pseudomollis Bruyns (syn. Synadenium molle Pax), a shrub indigenous to East Africa, has been previously documented for its anti-inflammatory properties. The purpose of this research was to examine whether A3 could both in vitro and in vivo regulate LPS-induced neuroinflammation.

The chemical constituents of the plant extracts were characterized using UPLC, LC-MS, QTOF, and NMR technologies. In vitro, BV2 microglial cells were pretreated with A3 followed by LPS stimulation. Cell viability (CCK-8), nitric oxide (NO) production, inflammatory cytokine expression, and inducible nitric oxide synthase (iNOS) levels were assessed. RNA-seq was performed at 1 h and 4 h post-LPS stimulation. Direct compound-target interactions were evaluated through cellular thermal shift assays and molecular docking analyses. In vivo, mice received daily intraperitoneal LPS injections for 7 days, with or without A3 treatment (5 or 15 mg/kg). Behavioral assessments included the open field test (OFT). Histopathology, immunohistochemistry, and splenic metrics were analyzed.

The UPLC-QTOF-LC/MS-NMR analysis showed that 3,4,12,13-Tetraacetyl-20-phenylacetylphorbol was the essential component in the ethanol extracts of Euphorbia pseudomollis Bruyns. In vitro, A3 reduced LPS-induced NO production and iNOS expression in BV2 cells in a dose-dependent manner, while maintaining cell viability. RNA-seq revealed more differentially expressed genes at 4 h than at 1 h, with GSEA at both time points implicating the NF-κB signaling pathway. In vivo, A3 ameliorated LPS-induced weight loss, hypothermia, and physical deterioration. OFT showed reduced anxiety-like behavior and improved locomotor activity. Histological analyses demonstrated attenuated hippocampal neuronal loss, suppressed microglial activation, and reduced peripheral organ pathology, including splenomegaly.

A3 demonstrates significant anti-neuroinflammatory effects by inhibiting microglial activation, reducing inflammatory mediators, and downregulating NF-κB-associated gene expression, while also mitigating behavioral and pathological impairments in LPS-induced animals.