Gut microbiota from nCAL patients promotes colon anastomotic healing by inducing collagen synthesis in epithelial cells

Background and Aims Colon anastomotic leak (CAL) is considered one of the most feared and serious postoperative complications in colorectal cancer (CRC) patients, with no effective prevention strategies to date. Based on previous studies, gut microbiota is associated with anastomotic healing, but its ability to effectively promote anastomotic healing remains largely unknown. Methods We performed a clinical study to analyze the gut microbiota profiling in CRC patients who developed CAL and those who did not (nCAL) using 16S-rRNA-based next-generation sequencing (NGS). To investigate these changes in an in vivo model, we performed fecal microbiota transplantation in a colon anastomosis rat experimental model to elucidate the causal effect between gut microbiota and anastomotic healing. Notably, RNA-seq in the anastomotic tissue of the latter experimental model was utilized to discover the potential molecular mechanism. Results Our analysis implicated that gut microbiota profiling was profoundly different between CAL and nCAL patients. Strikingly, the rat experimental model transplanted with fecal microbiota derived from nCAL patients demonstrated enhanced anastomotic healing properties. Moreover, collagen synthesis, EMT, and TGF-beta/Smad signaling pathways were upregulated in the same rats. Concordantly, we discovered that the better anastomotic healing profiling displayed in gut microbiota derived from nCAL patients is dependent on the TGF-beta/Smad-induced EMT in vitro and in vivo. Conclusions Collectively, our clinical study identified the postoperative gut microbiota profile is associated with CAL in CRC patients. On the contrary, fecal microbiota from nCAL patients promotes anastomotic healing via TGF-beta/Smad-induced EMT, with subsequent collagen synthesis and enhanced anastomosis healing.