*4.2. Animals*

The 72 male pathogen-free Sprague Dawley rats at 6–7 weeks of age were purchased from the Shanghai Sippr- BK Laboratory Animal Co., Ltd (Shanghai, China). Rats were housed 4 per cage with ad libitum access to water and feed in an environmentally controlled animal room (temperature 24 ± 2 ◦C, relative humidity 50 ± 10%, 12 h light-dark cycle) at the Shanghai Institute for Biomedical and Pharmaceutical Technologies. All experimental procedures were approved by the Animal Care and Use Committee of Shanghai Institute for Biomedical and Pharmaceutical Technologies (certificate no. 2020-29), and performed in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

#### *4.3. Study Design and Treatment*

After 5 days of acclimatization, the rats, except for twelve in the sham operation group, were castrated under pentobarbital sodium anesthesia before the experiment to prevent the influence of intrinsic testosterone. Except for the sham-operated rats which were just submitted to anesthesia and testicles exposure, castration was performed by removing testicles with epididymal fat via the scrotal route, as published previously [30].

Twelve normal rats, administrated olive oil orally and injected olive oil subcutaneously daily, served as the sham operation group. The other sixty castrated rats were randomly assigned into five groups (*n* = 12/group): (A) BPH model control group administrated olive oil orally and testosterone (5 mg/kg/day, s.c.), (B–D) AST groups treated with AST (20, 40 and 80 mg/kg /day, respectively) dissolved in olive oil orally and testosterone (5 mg/kg/day, s.c.), (E) EPR group (positive control group) given EPR (2 mg/kg/day) by oral gavage and testosterone (5 mg/kg/day, s.c.). EPR is used to treat benign prostatic hyperplasia to decrease prostate size and relieve the symptoms [31]. All rats in this experiment were treated once a day for four consecutive weeks. All animals' body weights were measured once a week. At the end of experiment, under pentobarbital sodium

anesthesia, prostates of all the rats were removed and weighed. One part of each ventral prostate lobe was fixed in 10% neutral buffered formalin for histopathological examination, while the remainder of each prostate was flash frozen in liquid nitrogen, and then stored at −80 ◦C for other analyses.

## *4.4. Prostate Index*

The prostate index was calculated as the ratio of the prostate weight to the rat's body weight.

## *4.5. Histopathological Examination*

Following dehydration, the fixed prostates were embedded in paraffin, cut into 4 μm sections, and then stained with hematoxylin and eosin (H&E). Coverslips were mounted on the microscope slides containing tissue sections using mounting medium. Then, morphological changes were investigated and epithelium thicknesses of prostates were measured under a light microscope.
