A preliminary study of the pathological characteristics of the internal orifice and the local mucosal microbial community in complex anal fistula

Abstract

[Objectives] To observe the histological morphology and Mucoprotein 2 (MUC2) expression in the internal orifice of complex anal fistula, as well as to compare the difference of the composition and structure of the microbial community in the mucosal tissue of the internal orifice of patients with complex anal fistula with the anal sinus mucosa of the healthy population. [Methods] (1) Part 1: Forty patients with complex anal fistula were included, who underwent surgery at the Department of Anorectal Surgery, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from October to December 2020. During the operation, the tissues of internal orifice and fistula, surrounding normal intestinal gland, anal sinus, and anal gland were collected. The histological morphology of internal orifice, fistula, anal sinus, anal gland and anal gland tube was observed under a microscope. The expression of MUC2 protein in the intestinal gland and anal gland was observed and compared under a microscope. (2) Part 2: Ten patients with complex anal fistula and 10 healthy volunteers were recruited from February to March 2022. They were divided into the anal fistula group and the control group. Baseline data were collected and mucosal tissue at the anal gland (or internal orifice) was collected for 16s rDNA high-throughput sequencing. [Results] (1) Part 1: The internal orifice of anal fistula is formed by anal gland infection, showing glandular structure. A large number of inflammatory cells and plasma cells were infiltrated around the fistula. The anal gland opened at the distal side of the anal sinus, and its surface contained a small number of goblet cells and a large number of columnar epithelial cells (or stratified flat epithelial cells). A small amount of inflammatory cells infiltration was observed around the anal gland tube. The expression of MUC2 protein was positive in intestinal gland, but weakly positive or negative in normal anal gland. (2) Part 2: Through Alpha and Beta diversity analyses, significant difference in species composition was found between the two groups, and the evenness of the anal fistula group was significantly lower than that of the control group, while the richness was significantly higher than that of the control group (P<0.05). Through species classification annotation, it was found that there was a significant difference between the two groups (P<0.05). The abundance of Fusobacterium varium in the anal fistula group was significantly higher than that in the control group. Among the top 10 species in the two groups, the abundance of Acinetobacter junii in the control group was significantly higher than that in the anal fistula group (P=0.012). Through the search of KEGG database, there was also significant difference in the predicted functions between the two groups. Ethylbenzene degradation, ECM-receptor interaction, and Pentose phosphate pathway were mainly enriched in the anal fistula group (P<0.05), and the relative abundance of Pentose phosphate pathway in anal fistula group was significantly higher than that in the control group (P<0.05). [Conclusion] The occurrence and development of complex anal fistula may be caused by the lack of MUC2 protection of the anal gland and the change in the local microbial community of anal gland mucosa.

Keywords

• complex anal fistula
• intestinal flora
• mucoprotein 2