A total of 180 female patients (age, 25–45 years) diagnosed with BV at Yantai Hospital of Traditional Chinese Medicine between August 2017 and March 2018 were selected for this study. The experiment exclusion criteria were as follows: i) Patients at menstrual bleeding; ii) pregnant patients; iii) lactating patients; iv) patients who were treated with antibiotics, hormones or local drug treatments within 14 days; v) patients who participated in sexual activity within 24 h. Additionally, healthy individuals undergoing routine vaginal examination and 60 healthy female individuals (age, 25–45 years) were selected between August 2017 and March 2018 as the control group. Individuals were excluded from the current study if they were: i) Menstruating; ii) pregnant; iii) lactating; iv) treated with antibiotics, hormones or local drug treatments within 14 days; v) participated in sexual activity within 24 h. Each BV patient was administered berberine treatments by placing 0.3 g berberine HCl (Northeast Pharmaceutical Group Co., Ltd.) at the posterior vaginal fornix daily for 10 days per treatment for one month. Clinical observation was performed on all healthy subjects (control) and on patients before treatment (BT) and after treatment (AT) to monitor and compare the severity of symptoms. Healthy subjects were only evaluated once. This study was approved by the Ethics Committee of Yantai Hospital of Traditional Chinese Medicine. Written informed consent was obtained from all participants.

Observe the leucorrhea of the patient. Swabs of vaginal discharge and virginal epithelial cells were collected at the posterior vaginal fornix and the cervix from patients with BV and healthy subjects while the cervix was exposed using a vaginal speculum. Each swab was dissolved in 1 ml phosphate-buffered saline (PBS; 0.05 mol/l; pH 7.0) in centrifuge tubes. Samples were centrifuged at 11,180 × g under refrigeration at 4°C for 20 min and stored at −80°C until further use.

Human vaginal epithelial cells VK2/E6E7 (cat. no. BNCC340628) were purchased from Shanghai Cell Library (http://www.cobioer.com/index.html). Cells were incubated in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), at 37°C with 5% CO₂. Cells were treated with 0, 10, 50 and 100 µg/ml berberine for 24 h prior to the MTT assay.

VK2/E6E7 cells were divided into five groups: i) Control, samples incubated without FBS in the medium; ii) model, samples incubated without FBS in DMEM medium for 4 h, followed by the addition of 400 µM H₂O₂ for 24 h; iii) LT, samples incubated in DMEM medium with 10 µg/ml berberine HCl for 4 h, followed by the addition of 400 µM H₂O₂ for 24 h; iv) MT, samples incubated in EMDM medium with 50 µg/ml berberine HCl for 4 h, followed by the addition of 400 µM H₂O₂ for 24 h; and v) HT, samples incubated in DMEM medium with 100 µg/ml berberine HCl for 4 h, followed by the addition of 400 µM H₂O₂ for 24 h. Each group consisted of five replicates.

Spectrophotometry was used for the detection of SOD (cat. no. BC0170) and CAT (cat. no. BC0200) activity, as well as levels of MDA (cat. no. BC0020) and H₂O₂ (cat. no. BC3590), within the vaginal discharge samples. SOD (cat. no. BC0170), CAT (cat. no. BC0200), MDA (cat. no. BC0020) and H₂O₂ (cat. no. BC3590) kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. and used following the manufacturers' protocols.

VK2/E6E7 cells in the logarithmic growth phase were digested using pancreatic enzymes, and cell density was adjusted to 1×10⁵ cells/ml. The cells were transferred to a 96-well plate (100 µl/well) and incubated at 37°C with 5% CO₂ in an incubator until ~80% confluence was reached. The cells were grouped and treated as described above. At 24 h, supernatants were removed, 20 µl of 5 mg/ml MTT was added to each well and incubated for 4 h at 37°C. Subsequently, supernatants were removed again and 200 µl DMSO (Sigma-Aldrich; Merck KGaA) was added to each well and suspended evenly. Absorbance at 490 nm was detected using a photometric plate reader.

VK2/E6E7 cells in the logarithmic phase were digested using pancreatic enzymes, and cell density was adjusted to 1×10⁵ cells/ml. The cells were transferred to a six-well plate (1 ml/well) and incubated at 37°C with 5% CO₂ in an incubator until the cells covered the bottom of the wells. The cells were grouped and treated as described above. Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit and Reactive Oxygen Species Assay kit (cat. nos. CA1020 and CA1410, respectively; Beijing Solarbio Science & Technology Co., Ltd.) were used for the detection of apoptosis and ROS levels. For the detection of apoptosis level, cells from all groups were washed twice with 4°C sterile PBS and resuspended in 1 ml of 1X binding buffer to achieve the density of 1×10⁶ cells/ml. Subsequently, 100 µl of the suspension (1×10⁵ cells) was added to a tube with 5 µl Annexin V-FITC at room temperature and agitated gently for 10 min prior to the addition of 5 µl PI for 5 min at room temperature in light-sensitive environment. PBS (500 µl) was added to the tube and mixed evenly, and the mixture was analysed using a flow cytometer. For the detection of ROS levels, dichloro-dihydro-fluorescein diacetate (DCFH-DA) was diluted to 10 µmol/l with DMEM medium without FBS (1:1,000). Medium from each sample was removed and replaced with 1 ml diluted DCFH-DA. Each sample was incubated at room temperature for 20 min and washed 3 times with medium without FBS to remove excess DCFH-DA outside of the cells. Cells were dissociated with trypsin and detected using a flow cytometer (Gallios; Beckman Coulter, Inc.) with Cell Quest 5.1 software (BD Biosciences).

Western blotting was used for the detection of apoptosis-associated proteins, including Bax, Bcl-2, caspase-3, caspase-12 and cytochrome C from vaginal epithelial cells obtained from healthy subjects and patients before and after treatment, and for the detection of the expression levels of SOD and eNOS in vitro. Proteins were lysed with RIPA assay lysis buffer (Beyotime Institute of Biotechnology) and a PierceÔ BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.) was used to measure the total protein concentration according to manufacturer's instructions. Protein (40 µg/lane) was separated using 10% SDS-PAGE and transferred onto a PVDF transfer membrane. The membrane was then blocked in 5% non-fat dry milk for 1 h, followed by overnight incubation at 4°C with the following primary antibodies diluted in 5% BSA: Rabbit anti-Bax (cat. no. ab32124), rabbit anti-Bcl-2 (cat. no. ab32503), rabbit anti-caspase-3 (cat. no. ab13847), rabbit anti-cytochrome-C (cat. no. ab133504), rabbit anti-caspase-12 (cat. no. ab62484), rabbit anti-SOD (cat. no. ab13498) and rabbit anti-eNOS (cat. no. ab76198); all antibodies were purchased from Abcam and all were diluted to 1:1,000. Following incubation, the membrane was washed with TBS with 0.1% Tween-20 (TBST) three times for 10 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; cat. no. ab6721; Abcam) at room temperature for 2 h. The membrane was washed three times with TBST for 10 min, and the bands were detected using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). The resulting images were processed using ImageJ 1.46r (National Institutes of Health) for quantitation.

SPSS 19.0 software (IBM Corp.) was used for statistical analysis. All data are presented as mean ± standard deviation. One-way ANOVA with the least significant difference post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Clinical symptoms from all patients before and after berberine treatment were recorded and summarized (Table I). A total of 95% patients exhibited yellowish leukorrhea at the beginning of the study, which was markedly reduced following berberine treatment. The number of patients with vaginal itching decreased from 88.89 to 36.67%, and the number of patients with local pain decreased from 92.78 to 32.78% following berberine treatment. Additionally, the number of patients with yellowish leukorrhea decreased from 95 to 10.56%, and the number of patients with foul smell decreased from 36.11 to 5.56% following berberine treatment. According to the Nugent scores (2), all of the 180 patients were BV-positive (100%) at the beginning of the study; following berberine treatment, only 13 patients remained positive (7.22%), which corresponded to a 92.78% cure rate. These results indicated that berberine treatment significantly improved clinical symptoms and effectively cured BV.

SOD and CAT activity, as well as the concentrations of MDA and H₂O₂, in the vaginal discharge from control, BT and AT groups were evaluated. SOD and CAT activity levels were significantly lower in the BT group compared with the control group (P<0.05; Fig. 1A and B), whereas in the AT group, the activity levels were significantly higher compared with BT (P<0.05; Fig. 1A and B). By contrast, the levels of MDA and H₂O₂ were significantly increased in the BT group compared with the control group (P<0.05); however, following treatment, these levels decreased significantly compared with the BT group (P<0.05). These results indicated that oxidative stress in vaginal discharge may be reduced by treatment with berberine.

The effects of berberine on normal vaginal epithelial cells were investigated. VK2/E6E7 cells were treated with 10, 50 and 100 µg/ml berberine for 24 h, and the MTT assay was used for the evaluation of cell viability. Berberine treatment had no effect on cell viability (Fig. 2A). To specifically evaluate the effects of berberine on oxidative damage, the MTT assay was used in H₂O₂-treated cells. The results demonstrated that cell viability was significantly lower in the model group with H₂O₂ treatment compared with the control group (P<0.05; Fig. 2B). The berberine treatment groups exhibited a significant increase in cell viability compared with the model group in a dose-dependent manner (P<0.05; Fig. 2B). Furthermore, based on the flow cytometry results, ROS levels in the model group were significantly higher compared with those in the control group, whereas the berberine-treated groups exhibited significant decreases in ROS levels compared to the model group (P<0.05; Fig. 3). These results demonstrated that berberine may have an antioxidative effect.

Bcl2, Bax, caspase-3, cytochrome C and caspase-12 are proteins related to cell apoptosis. The expression levels of these proteins in the vaginal epithelial cell samples from patients with BV and healthy subjects are summarized in Fig. 4. The expression levels of Bcl-2 in the BT group were significantly lower compared with those in the control group (P<0.05), but exhibited a significant increase in the AT group compared with the BT group (P<0.05). In addition, the BT group exhibited significantly increased levels of Bax, caspase-3, cytochrome C and caspase-12 expression compared with the control group (P<0.05), whereas the AT group exhibited reduced levels of these proteins compared with BT (P<0.05). To further confirm that berberine treatment supressed apoptosis in vaginal epithelial cells, flow cytometry assay was used; the results indicated a higher level of apoptosis in the H₂O₂-treated model group compared with the control group (P<0.05; Fig. 5). Among the berberine-treated groups, apoptosis levels in the MT and HT groups were significantly lower compared with the model group (P<0.05; Fig. 5).

Expression levels of SOD and eNOS in the model group were significantly lower compared with those in the control group (P<0.05; Fig. 6). The Bax/Bcl2 ratio was significantly higher in the model group compared with that in the control group, which indicated a higher apoptotic rate. The Bax/Bcl2 ratio decreased significantly in the berberine-treated groups in a dose-dependent manner (P<0.05). Similarly, caspase-3, cytochrome C and caspase-12 exhibited significant increases in expression levels in the model group compared with the control group (P<0.05), whereas the berberine-treated groups exhibited decreased levels of expression of these proteins compared with the model group in a dose-dependent manner (P<0.05).