Trimethylamine N‐oxide, a Gut Microbiota Metabolite, Promotes Diabetes‐Mediated Erectile Dysfunction via a PANoptotic‐Like Process Involving Pyroptosis, Apoptosis, and Necroptosis in CCSMCs

ABSTRACT Background Trimethylamine N‐oxide (TMAO), a key metabolite derived from gut microbiota dysbiosis, is known to influence various diabetic vascular complications. However, its specific role and the programmed cell death mechanisms underlying the development of diabetes‐mediated erectile dysfunction (DMED) remain to be fully elucidated. Objective To investigate the mechanism by which TMAO mediates corpus cavernosum smooth muscle cell (CCSMC) dysfunction and promotes DMED in type 1 diabetic rats, with a focus on the involvement of the PI3K/Akt‐regulated PANoptotic‐like process. Methods A type 1 diabetes‐mediated erectile dysfunction (T1DMED) rat model was established. Fecal samples were collected for gut microbiota analysis, and serum TMAO levels were measured. Primary CCSMCs were isolated, and cell purity was confirmed via immunofluorescence and flow cytometry. An in vitro TMAO‐induced CCSMC injury model was constructed, followed by transcriptome sequencing to identify differentially expressed genes (DEGs) and enriched KEGG signaling pathways. Western blotting and immunohistochemistry (IHC) were performed to detect markers of a PANoptotic‐like process (encompassing pyroptosis, apoptosis, and necroptosis) and pathway proteins in penile tissues. Human single‐cell RNA sequencing (scRNA‐seq) data from penile cavernous tissues were analyzed to compare the PI3K/Akt axis and the PANoptotic‐like process‐related genes in CCSMCs between DMED patients and healthy controls. TMAO‐injured CCSMCs were treated with the ROS scavenger N‐acetylcysteine (NAC) and the PI3K/Akt inhibitor LY294002. Flow cytometry and Western blotting were used to quantify markers of this death program and pathway activity. Results T1DMED rats exhibited reduced gut microbiota diversity, with Dorea as the dominant genus. Serum TMAO levels were significantly elevated in the ED group compared with the normal and diabetic non‐ED groups. Parabacteroides was positively correlated with TMAO levels, whereas Oscillospira showed a negative correlation. Transcriptome sequencing of TMAO‐treated CCSMCs revealed significant enrichment of the PI3K/Akt signaling pathway. IHC and Western blotting confirmed the marked activation of the PANoptotic‐like process in penile tissues of T1DMED rats. Analysis of human scRNA‐seq data revealed significant upregulation of PI3K/Akt signaling and genes associated with the PANoptotic‐like program (e.g., CASP1 , CASP3 , CASP8 , and MLKL ) in CCSMCs from DMED patients. Flow cytometry demonstrated increased Caspase‐1 and Caspase‐3 positivity in TMAO‐treated CCSMCs. NAC significantly reduced both readouts, whereas LY294002 significantly reduced Caspase‐3 positivity but not Caspase‐1 positivity. Western blotting confirmed that PI3K and PANoptotic‐like markers were upregulated in TMAO‐treated CCSMCs, while α‐SMA and eNOS were downregulated. NAC treatment attenuated the TMAO‐induced oxidative stress cascade, leading to a reduction in these death markers and restoration of α‐SMA/eNOS expression. Similarly, LY294002 attenuated several TMAO‐induced molecular changes associated with the PANoptotic‐like process and partially restored the expression of CCSMC‐associated functional markers. Conclusion Gut microbiota dysbiosis in T1DMED rats was associated with elevated serum TMAO, and TMAO triggered a PANoptotic‐like process in CCSMCs via PI3K/Akt activation, thereby contributing to the progression of DMED.