Study of gastric inflammation

The gastrointestinal tract represents an important barrier between humans and the microbial population. For this reason, the stomach is continuously exposed to the risk of developing inflammation of the mucosa, which leads to the onset of gastritis and ulcers, of which Helicobacter pylori, a Gram-negative pathogen that colonizes the stomach of humans and primates, is the main culprit. H. pylori infection in humans is a public health issue: not only is more than 50% of the world’s population affected, but the WHO has also classified this microorganism as a carcinogenic bacterium. The clinical course of H. pylori gastritis is highly variable and influenced by various factors depending both on the microorganism itself and on the host; however, it is widely documented that the presence of gastritis is strongly correlated with the onset of duodenal or gastric ulcers, mucosal atrophy, carcinoma and gastric lymphoma.

H. pylori-infected gastric epithelial cells have been shown to express high levels of cytokines such as IL-8, a potent neutrophil-activating chemokine that plays a key role in gastric pathologies. Cag-PAI (Cag Islands of Pathogenicity Islands) H. pylori strains induce a much stronger IL-8-mediated response than Cag-PAI negative strains; this response is favored by the activation of transcription factors such as NF-κB (Nuclear Factor kappaB) and Activator Protein-1 (AP-1). IL-6 and IL-21 are also produced by gastric mucosal cells and are found in high quantities in biopsies from patients with H. pylori. As amply demonstrated, in H. pylori-associated gastritis and peptic ulcers, most of these cytokines induce over-expression of several metalloproteases (MMPs), including MMP-9. Several studies have identified an increased production of MMP-9 in gastritis induced by H. pylori and increased plasma concentration of MMP-9 in patients with gastric cancer. IL-8 and total MMP-9 levels decrease significantly after eradication of H. pylori from the mucosa of the antrum and from the body of the stomach, while they remain unchanged if the treatment fails. Elastase, a serine protease secreted by neutrophils, is another enzyme involved in the degradation of the extracellular matrix during acute and chronic gastric inflammation processes.

The research currently underway aims to identify new extracts of plant origin useful in the treatment of inflammation of the gastro-intestinal tract, bearing in mind that the most successful drug therapy currently involves the use of drugs such as proton pump inhibitors and H2 receptor antagonists2of histamine, molecules that can present various side effects, such as nausea, abdominal pain, constipation, flatulence and diarrhea for pump inhibitors and headache, fatigue, asthenia, muscle pain and constipation for H antagonists2.

Methods used in the laboratory

In vitro assays

The assays involve the use of cell cultures of human gastric epithelium, both of tumor origin (AGS) and non-pathological (GES-1). These cells represent the main in vitro models described in the literature to study the anti-inflammatory activity at the gastric level.

  1. Measurement of NF-κB and AP-1-driven transcription following pro-inflammatory stimuli that mimic the immune response to infection (TNF-α, IL-1β) or the infection itself (co-culture with Helicobacter pylori).
  2. Measurement of the translocation of NF-κB from the cytoplasm to the nucleus, an event capable of triggering the pro-inflammatory cascade at the gastric level, following a pro-inflammatory stimulus with TNF-α, IL-1β, Helicobacter pylori.
  3. Measurement of nuclear translocation of NRF2, a transcription factor involved in the anti-oxidant response.
  4. Evaluation of the release and gene transcription of IL-8, IL-6, MMP-9 by the gastric epithelium and analysis of the respective gene expression.
  5. Measurement of the activity of elastase, a protease released by neutrophils in the gastric lumen following gastritis and ulcers.
  6. Effect on the production of reactive oxygen species (ROS) and antioxidant enzymes.
  7. In vitro digestion with gastric enzymes, in order to establish how the stomach modifies products of vegetable origin.

In vivo assays

The assay involves the use of rats with ethanol-induced ulcer 1 hour before sacrifice. In this model it is also possible to evaluate the preventive effect of the substances under study. We proceed with the sampling of the gastric mucosa on which we are able to evaluate the following parameters:

  1. Effect on CINC-1, the IL-8 analogue in the rat.
  2. Nuclear translocation of NF-κB.
  3. Nuclear translocation of NRF2.
  4. Antioxidant activity by ORAC test.
  5. Superoxide dismutase (SOD) and catalase activity.
  6. Evaluation of lipid peroxidation by measurement of MDA.
  7. Effect on the release of reactive oxygen species (ROS).