Khairul Najmi Muhammad Nawawi1,4, Nurainina Ayob2, Raja Affendi Raja Ali1,4, Mohamad Hizami Mohamad Nor1, Tan Geok Chin3,4, Zhiqin Wong1,4, Yin Ping Wong3, Muaatamarulain Mustangin3, Norfilza Mohd Mokhtar2,4

1Gastroenterology Unit, Department of Medicine, Faculty of Medicine, The National University of Malaysia, Cheras, Kuala Lumpur, Malaysia, 2Department of Physiology, Faculty of Medicine, The National University of Malaysia, Cheras, Kuala Lumpur, Malaysia, 3Department of Pathology, Faculty of Medicine, The National University of Malaysia, Cheras, Kuala Lumpur, Malaysia, 4GUT Research group, Faculty of Medicine, The National University of Malaysia, 56000, Cheras, Kuala Lumpur, Malaysia

Background/Aims: Malfunction of gut-liver axis begins with the dysbiosis of gut microbiota, causing impaired mucosal immune function and decreased intestinal permeability. This study aimed to assess the expression change of CD4+, CD8+ and zona occludens-1 (ZO-1) in duodenal mucosa of NAFLD patients, after supplementation with probiotics.

Methods: Forty NAFLD patients were randomized into probiotics and placebo groups. Multi-strain probiotics containing six different Lactobacillus and Bifidobacterium species at the concentration of 30 billion CFU was used. Duodenal mucosa was obtained by performing oesophagogastroduodenoscopy. Nuclear staining of intraepithelial lymphocytes was evaluated quantitatively from three different villi, and was later analysed as mean of labelled nuclei over a total of 100 enterocytes, while cytoplasmic staining was evaluated semi-quantitatively. Both scores were then verified by two pathologists. Analysis was performed with either paired-t test or Wilcoxon signed rank test.

Results: The mean age of patients was 52 ± 12 years, with mean BMI of 28.2 ± 4.1. Immunohistochemistry (IHC) analysis showed a significant decrease in CD8+ cytoplasmic staining in villi area for placebo group after the 6-month study period (Z=-2.673, p=0.008). However, there were no significant change in CD8+ staining intensity for probiotics group, as well as CD4+ for both groups. For the tight junction protein assessment, IHC showed significant decrease in ZO-1 staining intensity in placebo group post study (mean -0.94, Z=-2.859, p=0.04). On the other hand, there was no difference in ZO-1 staining for probiotics group.

Conclusion: Without supplementation of probiotics, small intestine mucosal immune function and intestinal permeability in NAFLD patients appears to deteriorate over a period of time. This was shown by the decrease expression in small intestinal CD8+ and ZO-1. Although we failed to demonstrate a significant positive change in probiotics group, probiotics might have a complementary role in stabilizing the mucosal immune function and intestinal permeability in NAFLD patients.

Keywords: Probiotics, NAFLD, mucosal immune function, intestinal permeability