Efeito esofagoprotetor de um biopolímero, a goma do cajueiro, na doença do refluxo não-erosiva em murinos e em biópsias esofágicas humanas: perfil anti-inflamatório e efeito tópico

Abstract

Gastroesophageal reflux disease (GERD) can be defined as symptoms or mucosal damage produced by the abnormal reflux of gastric contents into the esophagus. Alternative ways of treatment must be considered since the standard therapy fails in all phenotypes of GERD, expressly in the most prevalent form (Non-Erosive Reflux Disease, NERD). We aimed to assess the esophageal mucosal protection effect of the biopolymer cashew gum (CG), a natural product obtained from bark of Brazilian northeastern tree (Anacardium occidentale L.), in mice (experimental model of NERD, non-erosive reflux disease) and in human esophageal biopsies. Experimental design was divided in “Pré-Clinical Studies: Murine NERD” and in “Human Oesophageal Biopsies Studies”. Firstly, mice were achieved by a surgical induction of NERD in which a pyloric sub-stenosis and ligation of forestomach were performed. After 7 days, animals were euthanized and samples of distal oesophagus taken to assess suggestive hallmarks of inflammation such as oedema, MPO activity and neutrophil infiltrate [histologic scores and keratinocyte-derived cytokine (KC)]. In functional studies, distal oesophageal samples of mice were mounted in Üssing chambers to evaluate Transepithelial Electrical Resistance (TER) and permeability to fluorescein. In human esophageal biopsies, it was evaluated TER and permeabiliy, but with pH adjustment to 2. As control in biopsies studies, alginate was used, besides sucralfate also was evaluated. In addition, an acute toxicity test was conducted in accordance with OECD guideline 423. Finally, we performed an epifluorescence microscopy to localize on the oesopageal epithelium the biopolymer previously labeled with FITC. In our study, animals with NERD established oesophageal oedema (augmented wet weight as an indirect oedema hallmark and altered histologic architecture as a direct oedema hallmark) and increased MPO activity (15.90±1.68 mg/cm and 23.14±9.3 U/mg tissue, respectively), while CG daily administration reduced (P<0.05) inflammatory parameters of oedema and neutrophil infiltration (10.52±0.5 mg/cm and 6.11±0.57 U/mg tissue, respectively). Mice with NERD had increased KC levels (40.82±11.71 pg/mg of tissue) in the oesophageal tissue while this phenomenon was (P<0.05) reduced by CG administration (4.46±1.3 pg/mg of tissue). The microscopic parameters (reduced intraepithelial neutrophils and oedema in the lamina propria) and reduced KC levels corroborate the anti-inflammatory profile of CG. Functional parameters studied demonstrated that CG protected the oesophageal mucosa. While NERD animals had a substantial drop in TER (-40±3.5%) and an augmented oesophageal mucosal permeability (13.9±1.51 RFI), CG 10% reverted partially (P<0.05) these functional markers (+0.3±0.82% and 3.99±1.13 RFI, respectively). It is worth mentioning that the CG did not induce any toxicity signs which ensured a safety biopolymer according to OECD guideline 423. Furthermore, our data in human biopsies showed that 30 minutes of mucosal exposure to the acidic solution caused TER drop (-27.3±2.4%) compared with 100% of basal resistance, that could be prevented (P<0.05) by alginate pre-coating (+3.7±6.1% over basal resistance). Similarly, pre-coating with CG solutions (10% and 5%) prevented the TER drop (P<0.05) when compared with basal resistance (+1.69±1.06% and -3.5±2.4%, respectively). CG at 2.5% and sucralfate did not protect the oesophageal mucosa against the challenge solution that mimics the gastric juice content. The mucosal protective effect was extended up to 1h in which unprotected biopsies had shown a drop of -36.79±5.5%, but alginate and CG 10% pre-coating prevented the resistance drop caused by acidification, pepsin and BA (+4.2±0.8% and +3.5±0.6%, respectively). The permeability to fluorescein increased in unprotected biopsies (135.7±37.43 RFI), whereas CG 10% prevented (P<0.05) this event (40.8±17.1 RFI). Epifluorescent microscope images showed mucosal luminal adherence of FITC-labeled CG 10% for up to 1h. Up to now, there is no therapeutic tools which could display in the same time a role of pre-epithelial barrier and anti-inflammatory framework to oesophageal mucosa injured by gastric contents such as CG did. Both in murine experimental NERD and in human oesophageal mucosal biopsies CG showed 1) anti-inflammatory potential to oesophagus by reducing key hallmarks of oesophageal inflammation in reflux oesophagitis, 2) topical protection against an “aggressive” acid, pepsin, BA solution, 3) in vitro adherence to human mucosa for at least 1 hour, and 4) nontoxic signs, i.e., well tolerable by mice. Then, it is the first time that a product displays not only a topical protection to oesophageal mucosa, but also an anti-inflammatory profile advantage that may be useful in GERD management. These benefits should be further explored in development of new topical and anti-inflammatory approach as add-on therapy for GERD.