Antimuscarinic drugs are well-known agents that have earned their place in the nowadays management of overactive bladder (OAB). One should not forget about their dark side, i.e., side effects leading to the phenomenon of the low proportion of patients still on drugs at 1-year observation (Lua et al., 2017). According to the authors of the National Overactive Bladder Evaluation (NOBLE) study, OAB exerts a clinically significant impact on quality of life, such as sleep, and other realms of mental health (Stewart et al., 2003). Thus, having in mind that OAB is a debilitating condition with a great and long-term impact on social activity, it seems natural that new substances receive growing attention (Chughtai et al., 2013). It is coherent with the observation that nearly 75% of individuals with OAB reach for complementary medicine (Chughtai et al., 2013).

The incorporation of herbal drugs into the current management of OAB has been shown to be an ambiguous attempt though (Chughtai et al., 2013). The state-of-the-art summary is that no competitive agents to the existing pharmacological treatment have been found and even promising candidates lack well-designed studies to confirm their efficacy (Zhou et al., 2019). Among several herbal agents for OAB, the most studied were Gosha-jinki-gan (Kajiwara and Mutaguchi, 2008), Hachi-mi-jio-gan (Ito et al., 2009), resiniferatoxin, or capsaicin (Heng et al., 2011). Little has been done in phytomedicine for OAB since the last decade though (Betschart et al., 2013; Chughtai et al., 2013). In general, herbal drugs were reported to have a lower efficacy but definitely fewer adverse effects than cholinolytics (Xiao et al., 2016) and have been perceived as rather complementary treatment than monotherapy (Xiao et al., 2016; Chen et al., 2021). The results of few clinical trials have been published, e.g., Betschart et al. (2013) analyzed Bryophyllum pinnatum for its safety and effectiveness, Xiao et al. (2016) focused on Weng-li-tong, Chen et al. (2021) used cinnamon patch for alleviating OAB symptoms, and finally, Schloss et al. (2019, 2021) considered Urox^® for the treatment of nocturnal enuresis and Schoendorfer et al. (2018) for alleviating OAB symptoms and urinary incontinence. The latter is a herbal mixture of Crateva nurvala, Equisetum arvense, and Lindera aggregata that has been recognized in traditional medicines for years (Schoendorfer et al., 2018). Since 2011, Urox^® has been also enlisted in the Therapeutic Goods Administration in Australia, with below 0.1% of adverse events and no reports on interference with known medications (Schoendorfer et al., 2018). The true nature of the mechanisms involved in its potential efficacy in OAB remains undetermined though.

In our previous studies, we had confirmed that detrusor overactivity was induced by retinyl acetate (RA) in rats (Wróbel et al., 2015; Wróbel et al., 2020) and described that Potentilla chinensis extract (PCE) has the potential to reverse RA–induced detrusor overactivity (DO) (Wróbel et al., 2020). The aim of the current study was to investigate whether phytomedicine extracts from Lindera aggregata root, Equisetum arvense stem, and Crateva nurvala stem bark (Urox^®) would reverse RA-induced changes in several cystometric and biochemical parameters characteristic of bladder overactivity and to thus check if this herbal supplement could be a reasonable strategy, as a future pharmacological treatment for patients with OAB.

All applied experimental procedures were approved by the local ethics committee and were conducted in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU).

In the present study, we shed some light on the potential mechanisms involved in OAB symptoms reduction after Urox^® administration. Firstly, in the established, RA-induced model of OAB, we observed typical urodynamic parameters, indicative of increased voiding frequency, with crucial changes in the central micturition areas for c-Fos expression and shifts in the levels of biomarkers detected in the bladder urothelium/detrusor muscle or urine. Then, the abovementioned phenomena were successfully reversed due to the 14-day oral administration of a herbal drug, Urox^®.

Previous studies have revealed the potency of Urox^® components in alleviating symptoms of OAB. A randomized double-blind placebo-controlled trial demonstrated that Urox^® reduces the frequency of urinary per day and episodes of nocturia, and alleviates urgency and total incontinence with no severe adverse events reported (Schoendorfer et al., 2018). Schauss and Spiller (2006) reported that UroLogic™ a herbal drug containing Equisetum arvense and Crateva nurvala, was effective in OAB symptoms reduction, together with inducing relief for bladder discomfort. Furthermore, in another study, they found no interference with P450 cytochromes, which is indicative of its safety when being used with other medications (Schauss and Elizabeth, 2006). In turn, Lindera aggregata was used for frequent voiding or incontinence (Schoendorfer et al., 2018). Based on the available clinical studies (the dosages between 420 and 840 mg, administered for 2–6 weeks), we included 840 mg of Urox^® for 14 consecutive days (Ito et al., 2009; Heng et al., 2011). What is more, we confirmed the value in pretests (data not presented) as a minimally effective dose with no behavioral changes in rats, including rat activity.

Recently, we established a novel model of OAB using RA-induced changes in rat bladder, in which cystometric parameters evoked by RA were reversed by the administration of oxybutynin chloride (Wróbel et al., 2015). In our present experiments, we observed attenuation of the changes caused by RA after administration of Urox^®. The most significant storage phase parameters were improved, i.e., BP, DOI, FNVC, and ANVC were lowered, while TP, VTNC, and BC were increased. These parameters greatly resemble the most important features of OAB to be observed in the urodynamic study. This leads to the conclusion that Urox^® is potent in resolving OAB symptoms in our model. The normalization of the storage phase parameters may be due to the desensitization of the afferent fibers reaching the urinary bladder, as described previously (Wróbel et al., 2020). What is worth emphasizing is that no influence on MVP and PVR were observed, which suggests that Urox^® does not compromise normal voiding. The direct proof for the action of Urox^® on detrusor stability was the ability to lower DOI, as traditionally in in vivo studies, unstable detrusor contractions appear in the urine storage period as a standard finding to diagnose OAB (Pan et al., 2012).

Here, we focused on determining the mechanisms that may play a role in Urox^® action. We observed no influence on bladder epithelial thickness both in the RA-induced DO model and Urox^® or RA plus Urox^® groups. Furthermore, we did not recognize any statistically important changes in bladder blood supply. Blood flow in the bladder is not well understood (Andersson et al., 2017). Andersson et al. (1985) found in an animal model that vasodilation increased with bladder capacity and was lowered in an empty bladder. However, some authors indicate that there is a decrease in mucosal rather than muscle layer blood supply during the filling phase (Andersson et al., 2017). Still, our experiments did not indicate any connection with both OAB pathophysiology and the possible effect of Urox^® on these parameters.

When focusing on the safety of the drug, it should be emphasized that no cardiovascular effects were noted, including changes in blood pressure or heart rate in normal rats. Furthermore, no diuretic effect was observed as the urine output remained the same after Urox^® administration. These observations should be confirmed in human studies but may be allowed to remain optimistic as for the administration of the drug in patients with cardiovascular diseases. On the other hand, the elevated urine production would impose detrusor instability and exacerbate the filling-phase lower urinary tract symptoms.

Most importantly, in the present study, we performed experiments to assess the effects of Urox^® on c-Fos expression in the neuronal voiding centers (MPA, PMC, and vlPAG). c-Fos expression is thought to be a marker of neuronal activity (Lee et al., 2003), which becomes highly increased due to bladder sensation in PAG and PMC (Dinis et al., 2004). The central micturition regions are activated by bladder stimuli in OAB, which in turn leads to an increase of c-Fos (Kim et al., 2012). Kim et al. (2012) reported that the expressions of c-Fos in the neuronal voiding centers were significantly elevated in the cyclophosphamide-induced OAB model. Here, we have found that RA induced significant c-Fos expression in all the analyzed centers. Additionally, the effect was attenuated by Urox^®. Thus, the lowered levels of c-Fos may be a significant sign announcing symptoms relief due to OAB and may partly explain one of the possible mechanisms of Urox^® activity.

In biochemical analyses, we also found that Urox^® did not reveal any activity for the induction of the proinflammatory cytokines (TNF-α, IL-1β, and IL-6). In our model of DO induction based on RA, no such mechanisms were determined to play a role, as well. Furthermore, urinary BDNF and NGF were examined as potential biomarkers for overactive bladder (OAB) (Wang et al., 2014). Additionally, the NGF levels were positively correlated with the severity of OAB (Tian et al., 2019). In our experiments, these were urine BDNF and NGF levels that were elevated in the RA group and normalized in the combination group due to the positive effect of Urox^®. Another novel biomarker in OAB diagnostics is malondialdehyde (Rada et al., 2020), and increased values of this substance together with the concentration of 3-nitrotyrosine were detected both in OAB patients and in vivo studies on OAB and bladder inflammation (Kim et al., 2012; Barut et al., 2019; Tian et al., 2019). Similar to the abovementioned growth factors, we observed a significant fall in both malondialdehyde and 3-nitrotyrosine levels in the combination group when compared to RA-treated animals.

Increased density of suburothelial nerve fibers in OAB individuals that are immunoreactive for CGRP are reported (Fowler et al., 2008). On the other hand, onabotulinumtoxinA is thought to be successful in the treatment of OAB due to the inhibition of CGPR release in the bladder (Rapp et al., 2006). In our experiments, we proved that similar mechanisms can be involved in the efficacy of Urox^® in the animal model of OAB. Furthermore, the expression of multipotent enzymes that play a role at various steps of molecular metabolism, i.e., ATP and Rho kinase, were also elevated due to RA action. The possible multifactorial mechanisms of Urox^® may be the reason for its potency to induce normalization of their levels in our experiments.

An increased expression of TRPV1 in bladder mucosa of OAB patients was proved to be directly correlated to OAB occurrence (Zhang et al., 2015). Additionally, there is a significant elevation in molecules responsible for acetylcholine transportation: VAChT and OCT-3 in patients with OAB (Wolf-Johnston et al., 2012). All these possible additional molecular pathological pathways may be attenuated with Urox^®, as we have shown above. The decreased levels of VAChT in detrusor after Urox^® treatment restrict detrusor muscle contractility which corresponds with improved cystometric results described in the respective sections.

The increased permeability of urothelium lies at the origins of bladder inflammation (Hurst et al., 2015). Here, we included Z01 and E-cadherin in the experiments to investigate this idea and revealed that again Urox^® increased the expression of these two biomarkers. It strongly suggests that Urox^® limits the pathological sensitization of the neurons located beneath the urothelium. As a consequence, we strongly suggest the incorporation of several biomarkers of OAB into clinical practice as only future studies may reveal the optimal set.

We are aware of some of the limitations of our study. We included only female rats, and it can lead to potential bias in the interpretation of some of the results. It involves the necessity for further experiments in male rats. The properties of Urox^® found in vitro and in vivo cannot be directly generalized and is expected in unchanged form in the human population, which entails clinical studies. Both dosage and treatment period were selected based on preliminary unpublished data as no exact analyses are available in the literature.

In conclusion, phytomedicine extracts (Urox^®) were found to be potent to reverse RA-induced changes in both several cystometric and biochemical parameters that are determinants of OAB. We observed no effects on basic cardiovascular parameters and daily urine output, which may promote the initiation of the analysis of its safety in humans. The actions of Urox^® were proved to be dependent on several factors, such as growth factors and several OAB biomarkers but not pro-inflammatory cytokines.