2023 International immunopharmacology Mechanism / Preclinical Saline / IV

2023 · Dumbuya — Hydrogen-rich saline regulates NLRP3 inflammasome activation in sepsis-associated encephalopathy rat model

Original title: Hydrogen-rich saline regulates NLRP3 inflammasome activation in sepsis-associated encephalopathy rat model.

Super-Abstract

In a juvenile rat model of sepsis-associated encephalopathy (SAE), hydrogen-rich saline (HRS) reduced brain inflammation, suppressed NLRP3 inflammasome activation, decreased neuronal apoptosis, and lowered oxidative stress markers. The study provides mechanistic insight into how H₂ may protect the brain during sepsis — but it is a rodent experiment and does not constitute evidence in humans.

Classified as a Mechanism / Preclinical study using Saline / IV. See Methodology for how we grade evidence.

Commentary

Sepsis-associated encephalopathy is a severe complication of sepsis characterised by acute brain dysfunction and long-term cognitive deficits; effective treatments are lacking. The NLRP3 inflammasome is a key intracellular danger sensor whose uncontrolled activation drives neuroinflammation. This study shows that intraperitoneal HRS administered after LPS-induced sepsis in juvenile rats attenuates NLRP3/ASC/caspase-1 signalling, reduces microglial and astrocyte activation (IBA-1, GFAP), and shifts macrophage polarisation toward the anti-inflammatory M2 phenotype (CD206 up, CD86 down). Oxidative damage (8-OHdG, MDA, MPO) was also reduced. The mechanistic detail is notable, though the juvenile rat model may not fully represent adult human SAE. IV saline as a delivery vehicle is not the same as drinking hydrogen-rich water.

Key quotes

„Rats treated with HRS showed decreased TNF-α and IL-1β expression levels.“ — key pro-inflammatory cytokines reduced by HRS
„Western blot analysis showed decreased NLRP3, ASC, caspase-1, MMP-2/9, TLR4 and Bax protein levels after HRS treatment, while Bcl-2 expression increased after HRS treatment.“ — molecular pathway evidence for reduced inflammasome activity and apoptosis
„HRS attenuated neuroinflammation, NLRP3 inflammasome activation, neuronal injury, and mitochondrial damage via NLRP3/Caspase-1/TLR4 signalling in the juvenile rat model, making it a potential therapeutic agent in the treatment of paediatric SAE.“ — authors' mechanistic conclusion — juvenile rat model

Our assessment

This is a mechanistic animal study in juvenile rats. It maps a plausible molecular pathway (NLRP3/Caspase-1/TLR4) through which H₂ might protect the brain during sepsis. The breadth of biomarkers measured is a strength. Limitations include the use of a juvenile rodent model, intraperitoneal delivery (not oral), and the absence of functional cognitive outcome measurements. Clinical relevance for human SAE patients remains to be established.

Study design

Type: controlled animal experiment · Model: juvenile rat, LPS-induced sepsis (5 mg/kg i.p.) · H₂ delivery: hydrogen-rich saline 5 ml/kg i.p., 1 h post-LPS
Result: reduced TNF-α, IL-1β; enhanced SOD, CAT, GPX; decreased MDA, MPO; suppressed NLRP3/ASC/caspase-1/TLR4/MMP-2/9/Bax; increased Bcl-2; reduced neuronal apoptosis and glial activation

Abstract

Sepsis-associated encephalopathy (SAE) is characterised by long-term cognitive impairment and psychiatric illness in sepsis survivors, associated with increased morbidity and mortality. There is a lack of effective therapeutics for SAE. Molecular hydrogen (H2) plays multiple roles in septic diseases by regulating neuroinflammation, reducing oxidative stress parameters, regulating signalling pathways, improving mitochondrial dysfunction, and regulating astrocyte and microglia activation. Here we report the protective effect of hydrogen-rich saline in the juvenile SAE rat model and its possible underlying mechanisms. Rats were injected intraperitoneally with lipopolysaccharide at a dose of 5 mg/kg to induce sepsis; Hydrogen-rich saline (HRS) was administered 1 h after LPS induction at a dose of 5 ml/kg and nigericin at 1 mg/kg 1 h before LPS injection. H&E staining for neuronal damage, TUNEL assay for detection of apoptotic cells, immunofluorescence, ELISA protocol for inflammatory cytokines and 8-OHdG determination and western blot analysis to determine the effect of HRS in LPS-induced septic rats. Rats treated with HRS showed decreased TNF-α and IL-1β expression levels. HRS treatment enhanced the activities of antioxidant enzymes (SOD, CAT and GPX) and decreased MDA and MPO activities. The number of MMP-9 and NLRP3 positive immunoreactivity cells decreased in the HRS-treated group. Subsequently, GFAP, IBA-1 and CD86 immunoreactivity were reduced, and CD206 increased after HRS treatment. 8-OHdG expression was decreased in the HRS-treated rats. Western blot analysis showed decreased NLRP3, ASC, caspase-1, MMP-2/9, TLR4 and Bax protein levels after HRS treatment, while Bcl-2 expression increased after HRS treatment. These data demonstrated that HRS attenuated neuroinflammation, NLRP3 inflammasome activation, neuronal injury, and mitochondrial damage via NLRP3/Caspase-1/TLR4 signalling in the juvenile rat model, making it a potential therapeutic agent in the treatment of paediatric SAE.

Source & links

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