**6. Etiology of Prostatic Inflammation**

The etiology of prostatic inflammatory process remains unclear. It is believed that several possible mechanisms are responsible for triggering a prostatic inflammatory response. Firstly, infection-induced inflammation hypothesis is evidenced by the presence of bacterial and viral strains in BPH tissue specimens [49]. Toll-like receptors (TLRs) expressed by BPH cells recognize structurally conserved molecules derived from pathogens. TLRs-mediated production of proinflammatory cytokines (IL-6) and chemokines (IL-8 and CXCL10) initiate and enhance the inflammatory process [50]. Secondly, autoimmune responses could be involved in prostatic inflammation. Epidemiologic studies showed that the prevalence of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is eight times more than bacterial prostatitis [51]. Prostate, as well as testes, are considered as immunologically privileged organs [52]; however, self-antigens release following a tissue injury results in autoimmunity [53]. Autoantibodies against prostate-specific antigen (PSA) or prostate acid phosphatase (PAP) were shown to be involved in the prostatic inflammatory process [54]. PSA has been demonstrated to be able to activate CD4+ T cells [55]. In addition, BPH cells, as well as other APCs, produce high levels of IL-12 and IL-23, promoting CD4+ T cell activation and differentiation [47], which in turn, differentiate into interferon gamma-secreting T-helper (Th) type 1 and IL-17-secreting Th17 cells [48]. These downstream pro-inflammatory cytokines manifest a positive feedback signal to inflammatory interaction between BPH and immune cells. Thirdly, neurogenic inflammation plays an important role in chronic prostatic inflammation. Neurotrophin, a nerve growth factor (NGF), is responsible for mediating prostatic neurogenic inflammation. The serum NGF level was found to be correlated with the severity of pain in CP/CPPS [56]. Moreover, damaged tissue has been found to contain higher NGF level [57], which can cause mast cells degranulation [58]. Infiltrating mast cells in BPH tissues can promote BPH development via activation of IL-6/Signal transducer and activator of transcription 3/Cyclin D1 signaling pathway [59]. NGF can also sensitize sensory nerve and induce production of neuropeptides, including substance P and calcitonin gene-related peptide (CGRP) [60]. Further, substance P can stimulate reactive oxygen species generation via its proinflammatory activity [61]. Prostatitis following intraprostatic formalin injection has been reported to induce prostate-to-bladder afferent cross-sensitization, increased urothelial NGF expression, and subsequent bladder overactivity [62]. Fourthly, oxidative stress can be one of the causes of inflammation. Interestingly, the ARR2PB-Nox4(ARR2PB-NADPH oxidase 4) transgenic mice showed increased prostate weight, increased epithelial proliferation, and histological changes, including epithelial proliferation, stromal thickening, and fibrosis through Nox4 promoting oxidative stress [63].

Furthermore, it is believed that chronic pelvic ischemia can generate oxidative stress [64]. Elderly patients with LUTS showed decreased prostate perfusion on transrectal color Doppler ultrasonography [7]. Serum glutathione peroxidase and superoxide dismutase levels, which have antioxidant effects, reportedly declined in dogs with BPH [65]. Oxidative stress also triggers prostate cells proliferation through the activation of cyclooxygenase (COX) pathways [66,67], whereas COX-2 inhibition can induce significant apoptosis in the prostate cell [67]. Fifthly, the autonomic nervous system (ANS) also contributes to prostatic inflammation and growth. Adrenergic innervation plays a role in prostate growth. ANS hyperactivity is significantly associated with LUTS, and serum norepinephrine level increased after tilt predicted prostate size [68]. Moreover, chronic administration of α1-adrenergic agonists induces proliferation of prostatic cells in a rat model [69]. Besides, norepinephrine can stimulate the proliferation of human non-epithelial prostatic cells [70]. α1-adrenoceptors have been linked with inflammatory pathways through activation of transforming growth factor β signaling cascade, regulating various events associated with the BPH development [69,71]. In summary, the major etiology of chronic prostatic inflammation includes prostatic infection, autoimmune responses, neurogenic inflammation, oxidative stress, and autonomic dysfunction.

#### **7. Anti-Inflammatory E**ff**ects of Botulinum Toxin A (BoNT-A)**

BoNT-A is well-recognized for its ability to block acetylcholine release at the neuromuscular junction by cleaving synaptosomal-associated proteins [72]. Intraprostatic injection of BoNT-A has been shown to induce relaxation of prostatic muscle through downregulation of α-adrenergic receptor expression and reducing smooth muscle contractility [73,74]. On the other hand, BoNT-A also causes morphological atrophy of the glands via chemodenervation and anti-inflammatory effects [75,76]. Clinically, BoNT-A has demonstrated therapeutic anti-inflammatory effects, including the reduction of pain, edema, erythema, and heat emission [77]. A study in a complete Freund's adjuvant-induced arthritic rat model revealed the anti-inflammatory effect of BoNT-A by attenuating anti-ionized

calcium-binding adaptor molecule 1 and IL-1β immune-reactive cells [78]. Moreover, BoNT-A reduces rosacea-associated skin inflammation by directly inhibiting mast cell degranulation [79]. Intravesical BoNT-A injections plus hydrodistension reduce bladder pain and NGF levels in patients with interstitial cystitis [80], probably through blocking bladder pain responses and CGRP release from afferent nerve terminals, as depicted in a rat model [81]. BoNT-A may also inhibit peripheral and subsequent central sensitizations via suppressing substance P, glutamate, and adenosine triphosphate, showing reduction of somatic and visceral pain [82]. Interestingly, BoNT-A pretreatment could inhibit intraprostatic capsaicin injection-induced COX-2 expression in prostate and spinal cord [83]. Furthermore, BoNT-A also significantly prevented oxidative stress in vascular endothelial cells in cutaneous ischemia/reperfusion injured mouse model [84]. It has been shown that BoNT-A inhibits IL-8/CXCR1 signaling cascade in endothelial cells through inhibiting Rho signaling pathways [85]. In summary, BoNT-A exhibits anti-inflammatory effect by suppressing cytokine generation, mast cell activation, neurogenic inflammation, and oxidative stress in different organs. These reports from the basic research of BoNT-A potentially strengthened evidence for its therapeutic effects in patients with BPH; however, further studies are warranted for some of the proven anti-inflammatory effects on prostatic growth and inflammation.

#### **8. Clinical Perspectives**

Although previous single-arm studies showed promising results in improving international prostate symptom score (IPSS), maximal flow rate (Qmax), PV, and post-void residual urine volume (PVR) following intraprostatic injection of BoNT-A, two recent large scale randomized control trials failed to show significant efficacy of BoNT-A on all outcomes [76,86–92]. Moreover, a systematic review including three large randomized placebo-control studies (experimental group, *n* = 260; control group, *n* = 262) showed only marginal benefits to IPSS (−1.02; 95% confidence interval: −1.97, −0.07) for the BTX-A versus placebo groups. There were no significant differences in Qmax, PV, and PVR between the two groups, which was attributed to the placebo effect [93]. The EAU guidelines on the management of non-neurogenic male LUTS documented that "Results from clinical trials have shown no clinical benefits for BoNT-A compared to placebo for the management of LUTS due to BPO," and strongly recommended not to offer intraprostatic BoNT-A injection treatment to patients with male LUTS [1]. Intraprostatic BoNT-A injection is also not listed as one of the treatment choices in the AUA guidelines [2]. However, it is difficult to explain how intraprostatic BoNT-A injection significantly decrease PV, as reported by other meta-analyses [94–96].

Although limited reports are available for the therapeutic effects of BoNT-A on CP/CPPS, several beneficial effects on LUTS have been noted. In an uncontrolled randomized clinical trial conducted in men with refractory CP/CPPS, the patients were classified into two groups according to the route of BoNT-A injection, transurethral or transrectal. After intraprostatic injection of BoNT-A (100 U), Qmax, voiding score, and quality of life (QoL) were significantly improved in both groups during the follow-up period [97], with mean initial PV ranging from 36.4 to 37.9 ml among the groups. Another randomized, controlled study of transurethral intraprostatic injection of BTX-A (100 or 200 U depending on PV) showed significant improvement not only in pain score and QoL but also in the urinary domain of chronic prostatitis symptom index 1-month post-injection [98]. Other outcomes, including IPSS, frequency of diurnal, and nocturnal urination, showed significant reduction compared to baseline at 1, 3 and 6-month after BoNT-A injection. The mean initial PV in the experimental group was 22.27 mL. Chuang et al. reported that 16 men with symptomatic BPH and PV <30 mL were successfully treated with transperineal intraprostatic BoNT-A injection [89], with significant improvement in PV and IPSS following BoNT-A injection. However, the mean PV of men included in the two largest randomized trials failed to show beneficial effects on PV (range, 43.8–48.8 mL) [91,92]. Therefore, these results imply that intraprostatic injection of BoNT-A might be effective in relieving LUTS in patients with small prostate and refractory CP/CPPS. As mentioned above, patients suffering from BPH and CP/CPPS might show overlapping symptoms due to similar etiologies shared by BPH and prostatitis.
