2.1 Animals and Drugs
Sixty SPF 6-week-old female Sprague-Dawley (SD) rats (weighing 180 ± 20 g) were purchased from Hebei Experimental Animal Center (Shijiazhuang, Hebei, China, licence number: 1705351). The animal experiment was approved by the medical ethics committee of Hebei University of Traditional Chinese Medicine and was in compliance with all regulatory guidelines. All methods were carried out in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All the rats were allowed food and water ad libitum. and they entered the experiment after 1 week of adaptive feeding. XHP (Zhejiang Tianyitang Pharmaceutical Co., Ltd. Division, lot No.: 1703011). DMBA (7,12-Dimethylbenz[a]anthracene, TCI, CAS57-97-6). Oestradiol benzoate injection (Ningbo No. 2 Hormone Factory, lot No. 110252511). Tamoxifen citrate tablets (Yangzijiang Pharmaceutical Group Co., Ltd., lot No.: 17041311). Progesterone injection (Ningbo No. 2 Hormone Factory, lot No. 110251670).
Preparation of DMBA: accurately weigh DMBA, dissolve it in sesame oil at a ratio of 7 mg·ml-1, and put it in a water bath at 60℃ for dissolving by ultrasonic oscillation as a standby.
Preparation of XHP aqueous solution: according to the needs of the experiment, soak the XHP in a little distilled water the day before the experiment, and the drug particles can be soaked exactly. On the day of the experiment, soft soaked XHP is crushed, and the concentrations are prepared with distilled water. XHP aqueous solutions of 270, 550, and 1370 mg·kg-1 are reserved for use.
Tamoxifen aqueous solution preparation: According to the experimental needs, a tamoxifen aqueous solution of 4 m·kg-1 was prepared with distilled water.
2.2 Model and Drug Use
2.2.1 Animal Model Establishment
We established a rat model of precancerous breast lesions as previously reported in the literature [9-11]. On the 1st day, DMBA dissolved in sesame oil was given to rats by gavage at a ratio of 1 ml·100 g-1. From the second day, 5 days as a cycle (on days 1-3, rats were injected with oestradiol benzoate at 0.5 mg·kg-1·d-1 into the inner side of the hind legs). On the 4th day, rats were injected with progesterone at 4 mg·kg-1·d-1 into the inner side of the hind legs. Observation on day 5). After 12 consecutive cycles, we established a rat model of precancerous breast lesions.
2.2.2 Grouping and Administration Methods
According to weight stratification and the random grouping method, 60 rats were divided into a normal control group (n = 10) and a disease model group (n = 50). The administration methods were as follows: Normal control group: After disposable gavage of 1 mL·100 g-1 sesame oil without DMBA, the rats were routinely fed. Disease model group: The model was reproduced in accordance with 2.2.1 Model Establishment method and fed routinely. After 10 weeks of successful modelling, rats in the disease model group were stochastically divided into 5 groups, with 10 rats in each group: disease model group, tamoxifen group and XHP low-, middle- and high-dose groups. The administration methods were as follows: Disease model group: fed routinely. In the tamoxifen group, 1 ml·100 g-1 tamoxifen (4 mg·kg-1) was given by gavage once a day for 28 days and the rats were fed routinely. XHP low-, middle- and high-dose groups: Xihuang pills were given at low (270 mg·kg-1), middle (550 mg·kg-1) and high-dose (1370 mg·kg-1) 1 ml·100 g-1 by gavage once a day for 28 days and fed routinely. At the end of the 14th week of the animal experiment, the materials were collected. Before taking the material, the rats were fasted for 24 h, drank freely, and were euthanized by anaesthetic overdose of sodium pentobarbital (200 mg·kg-1, i.p.). Six pairs of mammary glands and surrounding skin and subcutaneous tissue of the rat chest and abdomen were collected under sterile conditions, approximately 1.0 cm × 1.0 cm. Some tissue samples were fixed with 10% neutral buffered formalin and embedded in paraffin for HE and immunohistochemical and TUNEL staining. Some tissue samples were frozen in fluid nitrogen and kept at -80 °C for immunoblotting and later RT-qPCR.
2.3 HE Staining
The tissue was embedded in paraffin, and the sections were deparaffinized. Then, haematoxylin stain, 10 minutes, tap water for 1 minute; 1% hydrochloric acid ethanol (70% ethanol (99 ml) + concentrated hydrochloric acid (1 ml)), 20 seconds, tap water for 10 minutes; eosin stain, 10 minutes, tap water for 1 minute. Finally, the pathological changes in breast tissue were observed by transparent and sealed pieces.
2.4 Immunohistochemical Detection
Paraffin-embedded blocks were cut into 3-μm sections. The sections were then routinely dewaxed and rehydrated, followed by antigen retrieval for 5 min by sodium citrate buffer (pH 6.0) at 100°C and cooling naturally to room temperature. Then, each section was treated with 100 μL 3% H2O2 for 10 min to reduce endogenous peroxidase activity, washed using PBS (at pH 7.4, 3 times for 3 min), and blocked with 100 μL of 2.5% normal goat serum for 30 min at room temperature prior to aspiration of the blocking solution. Then, 100 μL of diluted primary antibody was added and incubated at room temperature for 1 h, washed using PBS (at pH 7.4, 3 times for 3 min), added to 100 μL secondary antibody and incubated at room temperature for 30 min, washed using PBS (at pH 7.4, 3 times for 3 min), treated with 100 μL diaminobenzidine (DAB) substrate–chromogen solution for 5 min and counterstained with Harris haematoxylin. Finally, the sections were differentiated in 1% acid alcohol, dehydrated and sealed with neutral gum, and then observed and photographed under a microscope. The above primary antibodies were as follows: phospho-S6 ribosomal protein (Ser235/236) XP® rabbit mAb (cat. no. 4858S; 1:400 dilution; CST); VEGF antibody (cat. no. NB100-664; 1:50 dilution; Novus); antiphospho-p70 S6 kinase (pThr389) antibody (cat. no. SAB4503957; 1:50 dilution; Sigma).
2.5 TdT-mediated dUTP Nick End Labelling (TUNEL)
First, paraffin-embedded breast tissue blocks were cut into 3-μm sections, and each section was stained, parched chip, dewaxed and rinsed with PBS. Then, 50 μL TUNEL reaction mixed liquids (in situ cell death detection kit-POD, cat. no. 11684817910; Roche) was added to each section, and the reaction time was 1 h in a dark wet box at 37°C, followed by rinsing with PBS (3 min × 3 times) protected from light. (After staining with TUNEL reaction mixed liquids, the tissue showed green fluorescence under a 488 nm wavelength light fluorescence microscope). PBS was removed, and 50μL DAPI (cat. no. AR1177; BOSTER) was added to each section, incubated for 10 min at room temperature and rinsed 3 times in PBS, each time for 3 min. (After DAPI staining, the tissue showed blue fluorescence under 405 nm wavelength light of fluorescence microscope, i.e., IF single staining). Finally, the fluorescent antiquencher (cat. no. P0126; Biyuntian, China) was added and observed after sealing. TUNEL-positive cells showed green labelling under a fluorescence microscope, while TUNEL-negative nuclei appeared blue with DAPI.
2.6 Reverse Transcription Real-Time qPCR (RT-qPCR)
Breast tissues (20-50 mg) were split and centrifuged by RNA-Solv Reagent (Omega), total RNA was isolated, and the RNA concentration and quality were assessed at 260/280 nm and 260/230 nm using a UV-Visible spectrophotometer (756 MC, Shanghai Precision Scientific Instrument Corp., Shanghai, China). The ratios of OD260/OD280 were between 1.8 and 2.1. RNA concentration (µg/µl) = (OD260-OD320) × dilution ratio × 0.04.c). Total RNA (2 µg) was reverse-transcribed using the PrimeScript® RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara, Dalian, China) according to the manufacturer’s protocol. Then, quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems 7500Fast Real-Time PCR System and SYBR® Premix Ex Taq™ (Tli RNaseH Plus; TaKaRa, Dalian, China). The following primers were used: GAPDH (internal control), forward primer sequence: 5′-CAGGAAATGATGACCTCCTGAAC-3′, reverse primer sequence: 5′-TGTTT TTGTAAGTATCTTGGTGCC-3′, amplicon length was 80 bp; VEGF, forward primer sequence: 5′-GCAGATCATGCGGATCAAACC-3′, reverse primer sequence: 5′-GCTCACAGTGAATGTGGTCACTTA-3′, amplicon length was 136 bp; and PTEN, forward primer sequence: 5′-GCGTGCGGATAATGACAAGG-3′, reverse primer sequence: 5′-AGCCTCTGGATTTGATGGCTC-3′, amplicon length was 157 bp. The PCR conditions were as follows. Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The specificity of the PCR product was confirmed by melting-curve analysis. Relative mRNA expression levels were calculated according to the 2-ΔΔCt method, and the formula was as follows[12, 13]: ΔCt = CT (target gene) - CT (internal reference); ΔΔCT = ΔCt experiment group – ΔCt control group.
2.7 Western blot
Rat breast tissue proteins were extracted with RIPA lysis buffer (R0020, Solarbio)), and the protein concentration was measured by a BCA protein assay kit (PC0020; Solarbio). Then, each lysed sample was mixed with SDS-PAGE loading buffer at a 4:1 ratio, boiled in a 100°C water bath for 5 min and centrifuged at 14,000 rpm for 5 min. Twenty microlitres of the protein samples were subjected to SDS-PAGE analysis and then transferred onto PVDF membranes (Millipore) at 100 V for 90 min. After transfer, membranes were blocked with 5% milk in TBST (T1081, Solarbio) for 1 h and then incubated with primary antibodies at 4°C overnight. The above primary antibodies were as follows: P-PI3K(SAB4503957, Absin), P-AKT(SER473)(4060S, CST), P-AKT(Thr308) (13038S, CST), P-mTOR(5536S, CST), PTEN(9188S, CST), P-tuberin/TSC2(3617S, CST), tuberin(p-s939)(ab52962, abcam), P-4E-BP1 (2855S,CST) and β-actin(Sc-47778, Santa). All primary antibodies were used at a concentration of 1:1000. Then, the membranes were washed with TBST 3 times for 10 min each, incubated with secondary antibody (goat antirabbit IgG-HRP or goat antimouse IgG-HRP, Santa) at a 1:5000 dilution for 1 h at room temperature, and then washed with TBST 3 times for 10 min each. Then, the protein bands were detected using enhanced chemiluminescence (ECL).
2.8 Statistical Methods
All data are expressed as the mean ± standard deviation and were analysed using the SPSS 20.0 statistical software package. Comparisons between groups were performed by one-way ANOVA. Differences in pathology data among the groups were analysed with the Wilcoxon rank-sum test. P < 0.05 was statistically significant.