This study explored the mechanism of Integrin αvβ6 regulation related macrophage-to-myofibroblast transition (MMT) in neurogenic bladder fibrosis (NBF) by using rat spinal cord half-transection model and in vitro cell experiments (Figure 1).

Neurogenic bladder (NB) refers to the dysfunction of lower urinary tract storage and urination due to damage to the central or peripheral nervous system, resulting in a series of lower urinary tract symptoms (LUTS) and complications [1]. It is necessary to further understand the mechanism of NB bladder fibrosis in order to effectively delay or block it. Recently, the role of MMT in promoting multiple organ fibrosis has attracted attention. Studies have showed that MMT can be found in progression of renal fibrosis, myocardial fibrosis et al and play a key role in fibrosis [2-3]. However, there is no reports on the MMT changes in bladder, especially in severe NBF. Therefore, this study explores the mechanism of Integrin αvβ6 regulation related MMT in NBF. Forty 1-week-old SD rats were randomly divided into sham-operated group, sham+ MORF-627 (10 mg/kg), NBF and NBF + MORF-627 (10 mg/kg, Intraperitoneal Injections) groups. Cystometry and bladder tissue samples were collected in four week and eight weeks for Single-Cell RNA Sequencing(scRNA-seq), Transcriptome Sequencing, Hematoxylin and Eosin (HE), Masson staining, and WB and qPCR detection. Primary rat bone marrow mononuclear cells were isolated, extracted, and cultured. After co-stimulation with TGF-β1 (10 ng/mL) for 24h, cells were collected. Cell marker was detected using WB, immunofluorescence, and annexin/PI assays. The key activating protein of Rho-ROCK pathway is phosphorylated p-MLC2.

Bladder tissue can detect the following cell types, including immune cells, myeloid cells, fibroblasts, epithelial cells, NK cells and endothelial cells et al by using scRNA-seq. It presented M2 macrophages (Mø) found more expression of myofibroblast markers like ACTA2 in NBF group. Meanwhile, drug treatment can alleviate Mø ACTA2 expression in sham+MORF-627 and NBF+MORF-627 group. This preliminarily speculated that Integrin αvβ6 inhibitor MORF-627 influence the MMT in NBF. Transcriptome Sequencing GO results showed that differentially expressed genes were enriched in the integrin related pathways like HYPOXIA and INTEGRIN(Figure 2). Compared with the different groups, HE/Masson staining/WB results showed differences in the expression of Integrin αvβ6/Rho-ROCK/TGF-β signaling pathway and fibrosis markers. Immunofluorescence/WB experiments suggest that co-cultivation of Mø and TGF-β affects the expression of ACTA2 in Mø(Figure 2).

Integrin αvβ6 is highly expressed in spinal cord injury-induced neurogenic bladder, correlating with bladder pressure and fibrosis severity. Animal experimental results using Integrin αvβ6 inhibitors in animal models with bladder hypertension demonstrate that Integrin αvβ6 is a key initiating marker in the MMT and Rho-ROCK/TGF-β pathways. TGF-β stimulation of Mø can induce MMT conversion. It provides new molecular therapy intervention targets which will help develop drugs to alleviate fibrosis.

Upregulation of Integrin αvβ6 through Rho-ROCK/TGF-β pathway can exacerbate bladder fibrosis progression and influence the MMT.

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