Benign prostatic hyperplasia (BPH) is a progressive disease in elderly men characterized by the enlargement of prostate periurethral region (Bushman, 2009; Chapple, 2010). Although the precise mechanism of BPH is still unclear, inflammation has been shown to play a critical role in BPH progression (Ileri et al., 2018; Kramer et al., 2007; Trumble et al., 2015). Clinically, almost all the prostates surgically isolated from BPH patients were observed with increased immune cell infiltration (Robert et al., 2009), and degree of inflammation in the prostate is closely correlated with prostate enlargement (Bostanci et al., 2013). Basically, it has been well accepted that during BPH development, inflammatory injury leads to cytokines and growth factors production, which subsequently promotes prostatic cell proliferation and prostate enlargement (Giri and Ittmann, 2001). However, 90% of inflammation in prostate is nonbacterial (Wang et al., 2018), and most of BPH patients neither have clinical signs of infection nor any correlation with bacterial or other foreign antigens (Kramer et al., 2007), which indicates an autoimmune abnormality occurs and may be involved in BPH development nodules (De Nunzio et al., 2016).

Experimental autoimmune prostatitis (EAP) model in rats has been established as a chronic non-bacterial prostatitis animal model that rats injected with prostate extract exhibited inflammatory cytokine overexpression and immune cells infiltration (Rivero et al., 2002). However, recently, it has been found the prostates from EAP rats were highly enlarged as well, which indicates not only a potential role of inflammation in BPH progress, but also provides a tool to investigate the regulatory effect of anti-inflammatory drugs for BPH therapies (Zhang et al., 2020).

Macrophage is one of the main immune cell types observed in BPH specimens, and it promotes prostatic cell proliferation directly within an inflammatory microenvironment (De Nunzio et al., 2011). There are two major types of macrophages named M1 and M2 that are differentiated from M0 and then activated during BPH development (Murray et al., 2014). M1 macrophages mainly produce pro-inflammatory cytokines to resist pathogen and tissue damage, while M2 macrophages produce anti-inflammatory cytokines and a series of growth factors to promote wound healing (Deng et al., 2019). Although both M1 and M2 macrophages are detected in BPH patients, studies have illustrated that M1 macrophages failed to contribute to prostatic cell proliferation (Deng et al., 2019; Sheng et al., 2018; Tong et al., 2022), which were consistent with our pre-experimental findings (data not shown). In contrast, M2 macrophages promote prostatic cell proliferation (Dang and Liou, 2018), and induce myofibroblast transformation and stromal tissue reconstruction in the early stage of BPH (Sheng et al., 2018). Altogether, macrophage M2, instead of M1, may directly promote the development of BPH.

Considering the ages of BPH patients, medication is usually the primary therapeutic option instead of surgery. 5α-reductase inhibitors and α-1 blockers (Ventura et al., 2011) have been widely applied while long-term use may result in several potential side effects such as sexual dysfunction, higher incidence of orthostasis and absence of emission (Fullhase et al., 2013; Ventura et al., 2011). Traditional Chinese medicines (TCMs) have been shown to their advantages as multi-targets and fewer side effects in BPH treatment (Xue et al., 2019). Danzhi Qing'e (DZQE) decoction is a TCM prescription composed of four herbs including Salvia miltiorrhiza Bunge, Psoralea corylifolia Linn, Eucommia ulmoides Oliver and Anemarrhena asphodeloides Bunge. According to the theory of TCM, DZQE has its ability to tonify the “yang”, regulate the “yin”, and activate blood circulation by dissolving the stasis. In the clinic, previous reports showed that DZQE effectively relieved menopausal symptoms such as hot flushes and night sweats in menopausal stages through modulation of hormone balance (Fu et al., 2016). Tanshinone IIA (Tan IIA) and bakuchiol (Ba) are two compounds isolated from Salvia miltiorrhiza Bunge and Psoralea corylifolia Linn, both of which were abundant in DZQE (supplementary material). Various mechanisms have been proposed to account how Tan IIA and Ba exhibit their anti-inflammatory activities in human myeloid leukemia cell line THP-1 or mouse macrophage cell line RAW 264.7 (Fan et al., 2009; Gao et al., 2019; Lim et al., 2019). In BPH-related studies, Tan IIA suppressed the expressions of proliferating cell nuclear antigen (PCNA) and androgen and estrogen signaling mediators androgen receptor (AR), estrogen receptor (ER) α in Estradiol/Testosterone (E2/T)-induced BPH rats in vivo, and it also inhibited the proliferation of prostatic epithelial RWPE-1 cells and stromal WPMY-1 cells by arresting their cell cycles in vitro (Wang et al., 2015). Bakuchiol also suppressed E2/T-induced rat prostate enlargement through down-regulation of PCNA and smooth muscle cell marker α-SMA expressions (Miao et al., 2019). Moreover, bakuchiol has been confirmed active in the balance of prostate estrogenic and androgenic signaling by down-regulation of stromal aromatase and up-regulation of epithelial ERβ (Miao et al., 2019). However, the mechanism of Tan IIA and Ba for inflammation-induced BPH remains unclear.

Here, our study was to explore whether DZQE inhibited EAP-induced BPH in rats, and further uncover its mechanism and active compounds involved.