Strengthening causal inference and analytical rigor in the Wumei Pills-Lactobacillus reuteri-intestinal stem cell axis for chemotherapy-induced mucositis

Abstract

A recent preclinical study reported that Wumei Pills (WMP) and Lactobacillus reuteri (L. reuteri) mitigate 5-fluorouracil-induced intestinal mucositis by promoting intestinal stem cell (ISC)-mediated repair via Wnt/β-catenin signaling. The mechanistic interpretation rests largely on systemic inflammation readouts, correlative microbiota changes, and immunohistochemistry of pathway markers. From a clinical standpoint, chemotherapy-induced mucositis remains a common and burdensome toxicity that leads to dose reductions, treatment delays, and infection risk; current care is largely supportive and does not directly restore ISC-mediated repair. This unmet need motivates rigorous appraisal of the proposed “WMP → L. reuteri → ISC/Wnt” axis. To highlight key methodological considerations that may affect causal inference and analytical rigor in the proposed “WMP → L. reuteri → ISC/Wnt” pathway. This letter critically appraises the study’s design, endpoints, and analyses against current best practices in mucositis biology, microbiome causality testing, Wnt/β-catenin pathway validation, and preclinical statistics, and synthesizes concrete, literature-grounded remedies. Six issues with potential impact on interpretation were identified: (1) Reliance on serum cytokines/lipopolysaccharide to infer local mucosal inflammation, with limited tissue-level indices (e.g., myeloperoxidase, interleukin-1β, immune-cell infiltration); (2) Absence of necessity/sufficiency tests to verify microbiota mediation (e.g., L. reuteri depletion, WMP-donor fecal microbiota transplantation, probiotic add-back); (3) Pathway evidence tiering - Wnt/β-catenin activation not confirmed by β-catenin nuclear translocation or downstream targets (Axin2, c-Myc, cyclin D1), and Lgr5 quantification/specificity insufficient; (4) Statistical design under-specified (power justification, blinded assessment, control of multiple comparisons) and potential cage effects unmodeled; (5) Limited dose-response and safety profiling for WMP/L. reuteri; and (6) Constrained generalizability (single sex/strain/age, lack of ABX-only controls, single time-point). The reported benefits of WMP and L. reuteri in chemotherapy-induced mucositis are promising, but stronger causal and analytical foundations are needed. Incorporating tissue-level inflammation readouts, microbiota loss-/gain-of-function designs, definitive Wnt/β-catenin activation assays, rigorous statistical practices (including mixed-effects models for cage clustering and multiplicity control), dose-response/safety evaluation, and broader experimental scope (sex/age/strain, ABX-only controls, time-course) will yield more robust and translationally relevant conclusions.

Keywords: Chemotherapy-induced mucositis; Wumei Pills; Lactobacillus reuteri; Intestinal stem cells; Wnt/β-catenin

Core Tip: In this methodological critique, we highlight six key areas to bolster the evidence that Wumei Pills (WMP) and Lactobacillus reuteri mitigate chemotherapy-induced mucositis via intestinal stem cell activation. We urge the authors to incorporate tissue-level inflammation indices (myeloperoxidase, interleukin-1β, immune cell infiltration) rather than relying solely on serum cytokines and lipopolysaccharide. We also suggest causality-verification experiments (probiotic depletion or fecal microbiota transplantation) to confirm the WMP-microbiota-intestinal stem cell linkage. The letter recommends more rigorous validation of Wnt/β-catenin signaling (e.g., demonstrating β-catenin nuclear translocation and downstream target gene activation), stronger study design practices (sample size calculation, blinded outcome assessment, accounting for cage effects, and false-discovery rate adjustments), dose-response and safety evaluations for WMP/Lactobacillus reuteri, and expanded experimental conditions to test the robustness of the therapeutic effect across different biological contexts. These improvements will help substantiate the mechanistic claims and translational potential of this promising gut microbiota-mediated therapy.