A neurogenic bladder (NB) is a common complication of spinal cord injury [1]. Bladder dysfunction caused by complete injury of the T10 and above spinal cord segment is often characterized by simultaneous non-inhibitory contraction of the detrusor, bladder sphincter (internal urethral sphincter) and/or external urethral sphincter, resulting in increased internal bladder pressure [2], it manifests as urine retention, which may cause renal reflux and, over time, renal damage, eventually leading to renal failure in severe cases [3].

Previous research has revealed that the bladder sphincter, external urethral sphincter, and urethral mucosa each account for a third of micturition control [4].

The bladder sphincter is located at the bladder neck and is doubly innervated by sympathetic (T11-L2) and parasympathetic (S2-S4) nerves, which have the function of controlling urination. Under normal conditions, parasympathetic excitation of the bladder during urination and inhibition of the sympathetic nerves cause contraction of the detrusor muscle and diastole of the bladder sphincter, producing urination. After an injury above the mid-thoracic spinal cord, the sympathetic and parasympathetic regions of the spinal cord, which innervate the bladder sphincter, lose control of the higher centers and become synergistically impaired, with continued sympathetic excitation and increased release of norepinephrine in the bladder sphincter, causing continuous contraction of the bladder sphincter, resulting in increased pressure at the urethral outlet during urination and impaired urination [5, 6]. Therefore, the idea of clinical treatment for neurogenic bladder with synergistic dysfunction of the detrusor-vesical sphincter (bladder neck) should be to suppress overactivity of the bladder sphincter. Current studies have focused on the bladder forced urinary muscle and the external urethral sphincter, and relatively little research has been done on the bladder sphincter. Clinical treatment of bladder neck dysfunction by cystotomy [7] and $\alpha$-blockers [8] has also been reported, and although some efficacy has been achieved, there are still drawbacks such as postoperative complications and drug side effects. Therefore, the search for other potential targets of intervention to guide the treatment of bladder neck dysfunction after spinal cord injury is particularly important in clinical aspects.

Bioinformatics analysis focuses on exploring potential therapeutic targets; it can evaluate the pathological mechanism of a disease from multiple angles [9]. Tandem mass tag (TMT)-based quantitative labeling technology can comprehensively detect the proteins expressed in tissues, and this technology also has high sensitivity and high-depth proteome coverage for the application of proteomics [10].

In the present study, a T10 spinal cord injury model was prepared by modifying the Hassan Shaker spinal cord transection method [11]. Differentially expressed proteins (DEPs) in the bladder sphincter were then detected using TMT-based quantitative labeling, and bioinformatics analysis was conducted on these DEPs to identify new biomarkers and targets to guide the treatment of NB.

After spinal cord injury above T10, the bladder sphincter, which is innervated by the T10-L2 sympathetic nerves, loses the control from the higher nerve center and has non-inhibitory contraction, resulting in bladder urination dysfunction [16]. The greatest clinical risk is the sudden increase in bladder pressure and large residual urine volume during voiding due to the inability of the bladder sphincter to relax at the same time as the contraction of the detrusor muscle, which can lead to recurrent urinary retention, urinary reflux, hydronephrosis and urinary tract infection, and even to renal failure. Because of the important role of the bladder sphincter in the voiding process, it is clinically useful to target it as a target site in the treatment of the detrusor-sphincter synergistic bladder disorder.

Therefore, we prepared a rat model of detrusor-sphincter synergistic dysfunctional bladder after transection injury of the T10 spinal cord segment, and the bladder sphincter (bladder neck) was selected as the object of observation in this study. The histomorphological results showed that, compared with the blank group, the mucosal epithelial layer of the bladder sphincter was thicker in the model group, with mucosal epithelial detachment in some areas; the lamina propria was heavily infiltrated with inflammatory cells and the elastic fibers were reduced; the smooth muscle cell nuclei in the muscle layer were enlarged, the muscle fibers were hypertrophied, and the muscle fibers were disorganized. In addition, urodynamics showed that both LPP and MCC were significantly higher in the bladder of the model group compared with the blank group, which suggested that the bladder sphincter and/or external urethral sphincter were excessively contracted after modeling, resulting in obstruction of urinary drainage, and thus LPP and MCC were significantly increased. Thus, a rat NB model of sphincter overactivity can be successfully caused by transection of the T10 spinal cord segment. We selected bladder sphincter (bladder neck) tissue and performed proteomic identification using TMT quantitative labeling technology to screen for differentially expressed proteins in bladder sphincter tissue, and the results showed that 250 DEPs were identified in the model/blank group of bladder sphincter. To explore potential clinical intervention targets for the disease, bioinformatic analysis was next performed on the differentially expressed proteins, and the results showed that these 250 DEPs were significantly enriched in 15 KEGG pathways. We focused on pathways closely related to the regulation of smooth muscle contraction, and the relevant DEPs were significantly enriched in extracellular matrix (ECM)-receptor interactions, adherent plaques, PI3K-Akt signaling pathway, cytosolic relaxin signaling pathway, and vascular smooth muscle contraction signaling pathway.

The three signaling pathways, extracellular matrix (ECM) receptor interactions, PI3K-Akt signaling pathway and adherent plaques, are very closely linked (Fig. 7A). Extracellular matrix (ECM) is a macromolecular material that is synthesized and secreted into the cell membrane or interstitial matrix, including collagen fibers, non-collagen fibers, elastic fibers, proteoglycans and aminoglycans. The complex network of ECMs not only has physical roles such as support, protection, and connectivity, but also exerts a full range of biological effects on cellular activities through signal transduction pathways in vivo. It has been shown that in bladder smooth muscle, alterations in collagen fibers, elastic fibers, and adhesion proteins in the ECM seriously affect the function of bladder smooth muscle cells. We found that in cluster 1 Lama2, Lama4, Fn1, Fbn1, Lamb1, and Col3a1 are all ECM components (Fig. 6), and by analyzing the KEGG pathway of DEPs in cluster 1, we found that these DEPs mainly focus on ECM-receptor interaction, Focal adhesion, PI3K-Akt signaling pathway, relaxin signaling pathway and etc (Fig. 6). Previous studies have also found significant alterations in ECM components in bladder smooth muscle bundles after spinal cord injury, which remains consistent with our study. In normal bladder tissue, the contractility of smooth muscle cells is closely related to ECM, which may be related to the fact that ECM connects to the smooth muscle cell intracellular actin filaments through the corresponding receptors on the smooth muscle cell membrane, converting extracellular mechanical signals into chemical signals that ultimately affect the contractile properties of smooth muscle cells.

Fig. 7.

KEGG signaling pathway diagram. The red boxes marked are the DEPs screened in this study. (A) is the relationship between ECM-receptor interaction, Focal adhesion and PI3K-Akt signaling pathway, (B) is the Vascular smooth muscle contraction pathway in which DEPs are involved, (C) is the relaxin signaling pathway in which DEPs are involved. Relaxin signaling pathway.

ECM has been found to activate focal adhesion kinase (FAK) in situ after binding to the integrin receptor on the cell membrane; FAK further binds and activates many signal proteins to regulate physiological activities, such as actin polymerization and cell migration, proliferation, and apoptosis [17]. Previous studies have revealed that, in the gastric fundus smooth muscle, the phosphorylation of FAK can activate the calcium-sensitive signaling pathway and promote the contraction of the gastric fundus smooth muscle cells [18]; furthermore, in physiologically stretched smooth muscle in the bladder, the activation of FAK can enhance the contraction and proliferation of the smooth muscle cells [19]. Phosphorylated FAK can further activate the PI3K/Akt signaling pathway, which is related to biological processes such as cell proliferation, differentiation, protein synthesis, and actin polymerization [20]. Activated PI3K generates a second messenger, phosphatidylinositol-3,4,5-triphosphate (PIP3), on the cell membrane, which further activates p21-activated protein kinases (PAKs). PAK can inhibit the phosphorylation of myosin light chain kinase (MLCK) stimulated by acetylcholine to inhibit the contraction of smooth muscle cells [21]. Yiming Wang et al. [22] found that PAK regulates contraction mediated by $\alpha$1-adrenoceptors in order to regulate the contraction of smooth muscle cells through adrenergic nerve fibers. In addition, the PIP3 complex can transport synaptic vesicles containing PIP3 from the cell body to axon terminals, and its directional transport function contributes to the local enhancement of the signal intensity of nerve terminals [23].

Synaptic vesicle glycoprotein 2A (Sv2A) in DEPs is involved in the signaling pathway of ECM-receptor interaction (Fig. 7A). Sv2A is a glycoprotein, specifically expressed in synaptic vesicles, that can regulate the release of neurotransmitters dependent on action potential. Recently, a study has reported that Sv2A has many potential functions, including vesicle transport, stabilizing the vesicle load of neurotransmitters, and regulating calcium sensitivity [24]. A previous study found that Sv2A-deficient mice had persistent seizures; levetiracetam, an anti-epileptic drug that targets Sv2A, has been widely used in clinical practice [25]. Another previous study identified that Sv2A overexpression can reduce the release of neurotransmitters [26]. This indicates that the abnormal increase or decrease of Sv2A expression may affect the release of neurotransmitters in synapses. Sv2A is not only expressed in the central nervous system but also controls the release of neurotransmitters by regulating the size of the easy-release pool in peripheral sympathetic synapses [27]. However, the bladder sphincter is mainly innervated by sympathetic nerves. In the present study, the expression of Sv2A in the bladder sphincters in the model group was significantly lower than in the blank group, which may be one of the reasons for the involuntary contraction of the bladder sphincter after T10 spinal cord injury.

The results of the present study found that the vascular smooth muscle contraction signaling pathway was significantly enriched, and we identified Calm2, Cald1 as the main DEPs involved in this signaling pathway (Fig. 6). In the study results, Calm2, a gene encoding calmodulin (CaM), and Cald1, a gene encoding calmodulin binding protein (CaD), were significantly elevated in the bladder sphincter of the model group. The change in Ca${}^{2+}$ concentration in the cytoplasm was the main factor affecting the contraction of the vascular smooth muscle cells [28]. The bladder sphincter is a smooth muscle, and its contraction depends on the concentration of Ca${}^{2+}$ in cytoplasm. Increased Ca${}^{2+}$ concentration activates the Ca${}^{2+}$/calmodulin (CaM)/MLCK pathway and stimulates myosin light chain (MLC20) phosphorylation, resulting in myosin–myosin interaction, which leads to smooth muscle bundle contraction (see Fig. 7B). Therefore, the high expression of CaM can enhance the contractility of the smooth muscle bundle [29]. The CALM2 gene, which encodes calmodulin, and the CALD1 gene, which encodes calmodulin binding protein (CaD) in DEPs, participate in this signaling pathway and, in the present study, increased significantly in the bladder sphincters of the model group.

The abnormal expression of the CALM2 gene leads to the abnormal opening of the voltage-gated Ca${}^{2+}$ channel, sarcoplasmic reticulum Ca${}^{2+}$ release channel, and lanidine receptor subtype 2 plasma channel. CALM2 has also been classified as a gene that affects calcium treatment [30]. The CaD encoded by the CALD1 gene is a component of microfilaments in smooth muscle cells, and it can bind with actin, tropomyosin, calmodulin, and myosin to regulate the contraction of smooth muscle cells and the formation of a cytoskeleton [31]. In addition, Ca${}^{2+}$ plays a role in the contraction of smooth muscle cells and is a key ionic signal regulating the release of neurotransmitters through pre-synaptic membrane exocytosis. CaM and CaD together transmit the change in concentration of Ca${}^{2+}$ in cells to regulate the transport and exocytosis process of synaptic vesicles [32]. A recent study found that CaD affected the contraction of the bladder smooth muscle by acting as a molecular brake and maintaining the integrity of the contraction mechanism [33]; furthermore, in pathological bladder smooth muscle, the expression of contractile proteins (such as CaD) has been found to increase significantly [34]. Therefore, CaM and CaD may be one of the pathological mechanisms of non-inhibitory contraction of the bladder sphincter after SSCI.

In the relaxin signaling pathway, G-protein coupled receptor (GPCR) and relaxin family peptide receptor 1 (RXFP1) play an important role in promoting vasodilation and antifibrosis, activating nitrogenous nerve fibers and protecting the heart [35, 36]. Edward et al. [37] found that RXFP1 was expressed in the smooth muscle cell layer of the human bladder triangle (where the bladder sphincter is located) and fornix, and expression of transforming growth factor $\beta$1 could be affected when the human bladder smooth muscle is stimulated with relaxin in vitro. Another study stated that relaxin-related receptors have become a drug target for the treatment of lower urinary tract fibrosis [38]. In addition to its antifibrotic effect, relaxin also produces a second messenger, nitric oxide (NO), to activate the NO/cyclic guanosine monophosphate/protein kinase G signaling pathway, thereby inhibiting the contraction of smooth muscle [39] (see Fig. 7C).

Arrestin $\beta$2 was first found in photoreceptors on the bovine retina. It participates in the signal transduction function of GPCR by mediating receptor desensitization and re-sensitization, competitively inhibiting the binding phosphorylated GPCR receptors to ligands. It also transports the corresponding receptor into the cell body after binding to the GPCR receptors, thus preventing the binding of the receptors to ligands [40]. A previous study revealed that GPCR is also involved in the activities of sympathetic and parasympathetic nerves: it binds to dopamine D1 receptors, muscarinic M2 receptors, $\alpha$-adrenergic receptors, and $\beta$-adrenergic receptors, thus participating in more physiological processes regulated by the autonomic nervous system [41]. Another study found that the significant expression of arrestin $\beta$2 increases the internalization of $\alpha$-adrenergic receptors on the cell membrane, preventing the binding of neurotransmitters to their corresponding receptors [42]. There are many $\alpha$-adrenergic receptors on the cell membrane of the bladder sphincter; in sympathetic nerves the neurotransmitters released by $\alpha$-adrenergic fibers can control the contraction of the bladder sphincter by binding $\alpha$-adrenergic receptors to the cell membrane [43]. In the present study, the arrestin $\beta$2 in the bladder sphincters of the model group decreased significantly, which hindered the internalization of the $\alpha$-adrenergic receptors. This may be one of the pathological mechanisms of non-inhibitory contraction of the bladder sphincter after SSCI.

It is of great significance to explore the pathological mechanism of non-inhibitory contraction of the bladder sphincter caused by spinal cord injury above the T10 segment from the perspective of ECM-receptor interaction, the focal adhesion-activated PI3K-Akt signaling pathway, the smooth muscle contraction signaling pathway, and the cell-relaxation signaling pathway. Sv2A, which is involved in the release of neurotransmitters from synaptic vesicles, arrestin $\beta$2, an inhibitory protein involved in the internalization of $\alpha$-adrenergic receptors and GPCR receptors, and CaM and CaD, which are involved in the calcium-sensitive signaling pathway, may be potential targets for developing new ways to treat bladder sphincter overactivity caused by spinal cord injury.

It has been found that in patients with incomplete suprasacral spinal cord injuries (such as ASIA B, C spinal cord injuries) there is still a dysfunction of the detrusor-vesical sphincter synergy, and overactivity of the bladder sphincter may occur. For these conditions, clinical management can include inhibition of excessive bladder sphincter contraction as an important therapeutic idea. Therefore, incomplete spinal cord injury with overactive bladder sphincter can also consider the above-mentioned targets for intervention.

In this study, we screened for DEPs in the bladder sphincter after T10 spinal segment injury by TMT quantitative proteomics, however, we have not yet validated those DEPs that potentially modulate bladder sphincter contraction. We will continue to validate the screened DEPs to further explore these potential therapeutic targets and pathways for bladder sphincter overactivity.

The data have been uploaded to ProteomeXchange with identifier PXD034595 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD034595).

Study design—KA, LZ. Project administration—KA, MX. Experiment implementation—QRQ, QL, XW. Data analysis—LYT, YYL, FQ. Paper writing—QRQ, LYT, KA. Paper review & editing—HZ. Experimental support—KA, LZ. All authors approved the final version of the manuscript.

Experimental animal ethics committee of Hunan University of Chinese Medcine. Ethical approval number: LL2019092303.

Not applicable.

General program of National Natural Science Foundation of China (81874510); University Student Innovation and Entrepreneurship Training Program Project of Hunan Province (S202110541019).

The authors declare no conflict of interest.