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Wuda granules in the inflammation-estrogen-microbiota axis Dina Johar* Department of Biochemistry and Nutrition, Faculty of Women for Arts, Sciences and Education, Heliopolis, Cairo, Egypt *Dina Johar, PhD. Department of Biochemistry and Nutrition Faculty of Women for Arts, Sciences, and Education Ain Shams University, Cairo, Egypt Phone:+2 01060782045 Email: dinajohar@gu.edu.eg *To whom correspondence should be addressed Abstract The inflammation-estrogen-microbiota axis dynamically remodel one another in a two-way, body-wide control loop. In Chinses Traditional Medicine, Wuda granules (WDG), which are derived from simo decoction, have been used clinically, and modified to enhance intestinal motility, restore bowel activity, relieve obstruction-related discomfort, and promote stool and gas elimination. Given the high recurrence rates of post-surgical functional dyspepsia and related GI disorders such as paralytic ileus and mechanical intestinal obstruction, it remains a significant clinical challenge to determine whether targeted treatments with WDG alter gut bacteria to raise body estrogen levels. In this commentary, we highlight recent findings from Wang et al.(2026)[1]. We draw attention to the potential benefits of studying experimental models in parallel with multi-omics approaches. The commentary offers insights for clinical decision-making and pre-clinical trials. Keywords Wuda granules; Post-operative ileus; Proteomics; Gastroenterology; Probiotics; Isovaleric acid. Core Tip The study of Wang et al, (2026)[1], presents an interesting, rich, multi-omics evaluation of WDG in a mouse model of POI, suggesting involvement of estrogen signaling and gut microbiota. However, additional experimental controls and mechanistic evidence is necessary to be fully supported. In multi-omics approaches, addressing the statistical test descriptions, sample sizes for high-throughput assays, multiple testing corrections, and computational pipeline parameters is necessary to ensure the robustness of the conclusions. Further contextualization regarding the functional validation of the proposed estrogen-mediated mechanisms and the physiological interpretation of the metabolite data are insightful. Addressing these limitations will significantly strengthen the study's mechanistic conclusions. Background The WDG-based therapies have shown potential to treat intestinal motility, relieve obstruction-related discomfort and promote elimination of stools and gases. However, their success is limited by difficulty in ensuring survival and targeting in the harsh gastrointestinal environment, especially post-surgery. The paper by Wang et al., 2026[1], provides an interesting and rich multi-omics evaluation of WDG in a mouse model of Post-Operative Ileus (POI), suggesting involvement of estrogen signaling and gut microbiota activity. In the proteomics dataset, sham controls are used to distinguish biological changes caused by a specific disease or treatment from changes due to the experimental procedure itself, such as surgery or anesthesia[2]. Including sham analyses, helps research to identify proteins that are only dysregulated because of the condition being studied rather than the stress of the intervention, as this reduces bias and improves accuracy[3]. Although the experimental design by Wang et al.(2026)[1] included a sham group, the resultant proteomics analysis compared the WDG-treated and POI model groups to identify the differentially expressed proteins without indicating the inclusion of sham/healthy baseline controls in the clustering or Venn diagram analysis. Without a baseline comparator, their claim that "WDG substantially reverses POI-induced protein dysregulation." is not logically supported, since the data only show that WDG creates a different expression profile from the POI state. Including sham control in the proteomics data set to definitively determine whether WDG normalizes protein expression or induces a novel state is key to exclude causality claims. Further, the study relies heavily on high-dimensional data (proteomics, 16S rRNA sequencing and Short-Chain Fatty Acid (SCFA) profiling), yet there is no indication that multiple testing correction was applied. Evaluating thousands of proteins or microbial taxa using standard P values thresholds greatly increases the false-positive rate, while applying and reporting appropriate multiple testing corrections (i.e., False Discovery Rate/ Benjamini-Hochberg) for these analyses helps to clarify the method-derived conclusion. I.e., in the description of the proteomic Venn diagram, the study states that there are "348 proteins uniquely present in each group." It is highly improbable that the WDG-treated group and the POI model group contain exactly 348 unique proteins each. Estrogen receptors (ERs) are nuclear proteins that bind estrogens, acting as transcription factors. The two primary intracellular receptors, ERα and ERβ, often play distinct or opposing roles in cell proliferation[4], regulate the synthesis of specific RNAs and proteins, regulate complex metabolic processes, and influence inflammation. Beyond reproduction studies, controversial findings on ERβ expression levels in disease progression, prediction, prognosis, and therapy remain paradoxical. This may be partly due to the ERβ- compensatory expression associated with some antisera available for research[5]. The hormone effects on the brain are particularly interesting, as ERβ is involved in anxiety and aggression[6]. Various antiestrogen strategies have been used in research, such as the high-affinity ERβ antagonist PHTPP: (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol), are ERβ-specific. PHTPP blocks ERβ signaling in vivo and in vitro, reducing the expression of target genes like the Minichromosome Maintenance Complex Component 5 (mcm5), which regulates cell growth. In the ERβ-KO mouse model, the gene coding for Erβ is permanently lost. Knocking down ERβ using ERβ-shRNA in ERβ-positive human bladder cancer BCa J82, 647v, and T24 Erβ-positive cell lines led to suppressed cell growth and invasion[7]. Further, pharmacological or genetic suppression of ERβ alter mitochondrial metabolism, resulting in metabolic dysregulation[8]. Systems‐based approaches have focused almost exclusively on the use of data‐driven approaches based on ever‐expanding ‘omics’ data sets, which had a profound impact on clinical immunology. However, they are neither determined nor restricted by previous biological knowledge. Specifically, the hypotheses and predictions generated by data‐driven models are generally abstract, and relate to patterns or correlations rather than to biological mechanisms. The Wang et al. study[1] states that the therapeutic effects of WDG are "mediated, at least in part, via ERβ-dependent signaling pathways." based on the restoration of ERβ levels. However, this observation is correlative. To establish a causal mechanistic link, performing in vivo experiment using an ERβ antagonist- as aforementioned- to determine whether blocking this receptor abrogates the anti-inflammatory and motility-restoring effects of WDG is an inevitable approach. Further, molecular docking data are used to suggest that WDG constituents (i.e. linderane and quercetin) "act as key ligands modulating estrogenic signaling.". While docking provides theoretical binding affinity, it cannot distinguish between receptor agonists and antagonists, nor does it prove functional modulation. Including in vitro functional assay (i.e. an ER transactivation/luciferase reporter assay) to confirm that these compounds actively stimulate ER signaling is informative. The conclusion proposes a unified "inflammation-estrogen-microbiota axis" as the key mechanism of WDG-mediated regulation of POI. The study investigates estrogen receptor expression, inflammatory markers, and microbial shifts simultaneously, without demonstrating a precise directional or causal relationship between these three compartments. Because it is fundamentally unclear whether ER activation induces microbial shifts, or if microbial metabolites induce ER activation and subsequent anti-inflammatory responses, emphasizing that WDG concurrently modulates these pathways is necessary, unless additional experiments (e.g., evaluating the microbiome in ER-antagonized mice) are performed to establish the sequence of the axis. Postoperative ileus is characterized by impaired or absent gastrointestinal motility. Isovaleric acid (IVA) is a BCFA produced in the gut as a microbial metabolite, specifically by fermentation of branched-chain amino acid (leucine) by the gut microbiota. The authors state that IVA promotes relaxation of colonic smooth-muscle cells and "thereby improves GI motility". The physiological context of this claim in the literature is unclear, as smooth muscle relaxation typically inhibits contractions and delays transit. Explicitly addressing how a mechanism of muscle relaxation resolves a hypomotility disorder, or if it instead plays a role in resolving localized spasms to allow for coordinated peristalsis is insightful. The authors appropriately acknowledged the lack of Fecal Microbiota Transplantation (FMT) as a limitation for verifying the role of the gut microbiota. To provide more actionable future directions, the authors should also propose experiments to isolate the direct versus indirect effects of WDG, such as treating POI mice with broad-spectrum antibiotics before WDG administration. This would help determine if the therapeutic efficacy of WDG is strictly dependent on microbial metabolites (i.e., IVA) or if direct pharmacological effects (i.e., direct ERβ agonism by absorbed WDG compounds) independently drive recovery. DECLARATIONS Funding This commentary did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The author declares that no honorarium, grant, or other form of payment was given to anyone to produce the manuscript. Conflict of interest The author declares no conflict of interest exists. Consent to publish Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article. Acknowledgment N/A REFERENCES 1 Wang T, Xu Y, Ou Y, Xiong W, Luo L, Li J, et al. Wuda granules target estrogen receptors and modulate gut microbiota to alleviate postoperative ileus: A multi-omics perspective. World J Gastroenterol. 2026;32:115995. [DOI: 10.3748/wjg.v32.i15.115995] 2 Candamo-Lourido M, Dopico-López A, López-Arias E, López-Amoedo S, Correa-Paz C, Chantada-Vázquez MP, et al. Comparative brain proteomic analysis between sham and cerebral ischemia experimental groups. Int J Mol Sci. 2024. 25;14:7538. 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