**3. Discussion**

In our study, only 40% of our IC/BPS patients showed symptomatic improvement. IC/BPS patients with less inflammatory bladder conditions would likely have satisfactory outcomes with intravesical BoNT-A treatment. The levels of inflammatory and oxidative stress urinary biomarkers, such as MCP-1 and 8-isoprostane, were higher in IC/BPS patients with unsatisfactory treatment outcomes. Moreover, voided volume and MBC also affect the IC/BPS patients' treatment outcomes. IC/BPS symptom and problem severity and detrusor pressure are significant predictors of treatment outcomes in IC/BPS patients receiving intravesical BoNT-A injections. The IC/BPS patients with severe bladder inflammation might need more intravesical BoNT-A injections to attain a satisfactory outcome.

The urothelium is a stratified epithelium with three cell types (basal, intermediate, and superficial) and its functions include: forming a permeability barrier, acting as a sensory organ, and accommodating large volumes of urine [14]. The bladder urothelium is basically quiescent but regenerates readily upon injury [15]; the turnover rate of quiescent rodent urothelium is approximately once every 200 days [3]. Insufficient urothelial regeneration might decline the defense barrier and impact the barrier function whereby toxic substances or pathogens in the urine further stimulate local tissue inflammation [14], depolarize afferent nerve fibers, raise exposure to urinary toxins, incite chronic bladder inflammation, and aggravate sensory nerve activation, leading to chronic pain and insufficient or overabundant regeneration [3] which further results in higher levels of cytokine biomarkers in the urine.

The first documented therapeutic application of BoNT occurred in 1977. A purified BoNT (Oculinum©) was injected into the extra-ocular muscles to treat strabismus [16]. In 1988, Dykstra et al., first used BoNT in patients with lower urinary tract disorders to treat detrusor external sphincter dyssynergia [17]. Additionally, physicians also attempted to use intravesical BoNT-A injections as treatment for IC/BPS in 2003 [18]. The BoNT-A is one of the most powerful neurotoxins and inhibits the release of neurotransmitters from nerve fibers and urothelium [9]. The therapeutic effects might involve inhibiting the release of acetylcholine into the neuromuscular junctions of the detrusor muscle and anti-inflammatory responses [19]. Intravesical BoNT-A injections are listed in the AUA clinical guidelines as a fourth-line treatment option for IC/BPS [7].

In the present study, we reviewed the data of 220/521 IC/BPS patients receiving intravesical BoNT-A treatment over the past two decades, regardless of the number of BoNT-A injections each received. Only 40% of these patients showed symptomatic improvement, including those with long lasting disease durations and those who were refractory to medical treatments. Additionally, BoNT-A (100 U) is effective and safe as a treatment for IC/BPS [9]. Unfortunately, in the clinical practice, effective treatments for IC/BPS are lacking [1] and the pathophysiology of IC/BPS is also undetermined. However, previous guidelines believe the pathophysiology of NHIC might be multifactorial and include inflammation, post-infection autoimmune process, mast-cell activation, and urothelial dysfunction [1,7,9]. Furthermore, the bladder disorder could be first or secondary, which resulted from another cause [20]. However, bladder treatment combined with multimodal therapy is necessary [21].

Moreover, in the present study, we further explored the inflammatory and oxidative urinary biomarkers in these IC/BPS patients and found that patients without satisfactory treatment outcomes had higher MCP-1 and 8-isoprostane levels. In contrast, the patients reporting satisfactory treatment outcomes had significantly lower levels of inflammatory and oxidative stress urinary biomarkers. This leads us to presume that repeat BoNT-A injections not only reduce pain but also decrease bladder inflammation and improve IC symptoms and problems, resulting in better treatment outcomes. In the previous clinical trial using repeated BoNT-A injections, a higher success rate was found in patients receiving more than two injections over longer therapeutic durations [19]. In practice, when a patient reports no satisfactory outcome after receiving the BoNT-A injection, physicians might not recommend repeat injections and the patient probably would refuse such additional treatment without considering that the anti-inflammatory effect had not been attained during the initial treatment.

In summary, the etiology of IC/BPS might be affected by multiple factors, including a defective/damaged bladder urothelium, activation of C-fibers, neurogenic inflammation with mast cell activation, autoimmunity, occult infection, and pudendal nerve entrapment [1]. However, IC/BPS patients, with or without Hunner's ulcers, had significantly higher levels of urine cytokine biomarkers, including interleukin-8 (IL-8), C-X-C motif chemokine ligand 10 (CXCL-10), brain-derived neurotrophic factor (BDNF), eotaxin, and regulated upon activation/normal T cell expressed and secreted (RANTES) than the general population [13]. This revealed that chronic inflammation might be the fundamental pathophysiology of IC/BPS. Therefore, bladder tissue apoptosis among IC/PBS patients might result from inflammatory signal upregulation [22]. This study, based on the point of view analysis of IC/BPS patients' subjective treatment outcomes in correlation with objective factors, revealed that the unsatisfactory group has higher urine biomarker levels and lesser bladder capacities, indicating that they probably have more severe bladder inflammation and have not ye<sup>t</sup> achieved optimal treatment effects. In these patients, repeat intravesical BoNT-A injections are required to achieve a satisfactory outcome. Regardless of the therapeutic options used by patients with IC/BPS in the past, in the future, precision medicine such as urine biomarkers, are expected to be used during bespoke personal treatment courses irrespective of physiological or psychological treatments, and longer, more complete treatment periods are also essential.

The major limitation of this study is its retrospective study design and single-center. Thus, further research is needed before more definite recommendations can be made, and in addition, longer follow-ups are also needed. Future studies should investigate the correlation between urinary biomarkers before and after BoNT-A treatment. Moreover, a consistent bladder volume must be ensured before collecting a urine sample and any invasive examinations must not be performed on patients for at least 48 h to ensure nonstimulation of the urothelium cell. Furthermore, allocating IC/BPS phenotype to suitable treatment options is another direction for our future efforts.
