Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro

Effect of PRL on PRLR/JAK2/STAT5 pathway in GT1-7

The viability of GT1-7 cells under different concentrations of PRL treatment was detected, and the results showed that PRL (400 ng/ml) has a significant inhibitory effect on the viability of GT1-7 cells, compared with the control group (P < 0.05). No effects have been found in any concentration of CFS on the viability of GT1-7 cells (Supplementary Figure 2). Hence, we used PRL at the concentration of 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, and CFS at a concentration of 10%. Then the phosphorylation levels of JAK, STAT5, and the levels of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot (Figure 1). Our data represented that compared with the Control group, the phosphorylation levels of JAK2 and STAT5, and the levels of PRLR and kisspeptin in the PRL (100 ng/ml and 200 ng/ml) group were significantly decreased (P < 0.05). Consistently, the mRNA expression of kisspeptin was significantly declined in the PRL (100 ng/ml and 200 ng/ml) group, but no apparent change was detected in PRLR mRNA expression (Supplementary Figure 3). Therefore, PRL was concentration-dependent. The higher PRL had a more significant effect, so we took 200 ng/mL PRL for the subsequent experiments.

The regulation of BSZY-D on PRLR/JAK2/STAT5 pathway via CSF in GT1-7

The phosphorylation levels of JAK, STAT5, and the levels of PRLR and kisspeptin in different groups were detected by western blot (Figure 2). Our data indicated that compared with the Control group, the phosphorylation levels of JAK2 and STAT5, and the levels of PRLR and kisspeptin in the HPRL group were decreased significantly (P < 0.05). Compared with the HPRL group, the phosphorylation levels of AK2 and STAT5, and the levels of PRLR and kisspeptin in the HPRL (BSZY-D/CSF) group were significantly elevated (P < 0.05), indicating that BSZY-D reversed the impact of HPRL on the downregulation of the PRLR/JAK/STAT5 pathway.

Similarly, the kisspeptin mRNA expression in the HPRL group was significantly decreased, however, the PRLR mRNA expression was not downregulated significantly; compared with the HPRL group, the kisspeptin mRNA expression in the HPRL (BSZY-D/CSF) group was significantly increased, suggesting that BSZY-D reversed the downregulation trend of kisspeptin through HPRL (Supplementary Figure 4).

The changes of PRLR expression in GT1-7 cells treated with different reagents

The level of PRLR in different GT1-7 cell groups were detected by western blot assay. As shown in Figure 3, compared with the Control group, the cytomembrane PRLR level in the HPRL group was significantly inhibited, but the total protein level of PRLR remained stable. Compared with the HPRL group, the protein level of PRLR in the HPRL (BSZY-D/CSF) group was significantly upregulated, which suggested that BSZY-D reversed the downregulation of PRLR protein level via HPRL (P < 0.05). Besides, we found that the PRLR ubiquitination level was increased in the HPRL group, however, there was deubiquitination of PRLR in the HPRL (BSZY-D/CSF) group (P < 0.05). It was indicated that HPRL caused the PRLR ubiquitination, but BSZY-D induced the deubiquitination of PRLR (Supplementary Figure 5).

Compared with the Control group, the PRLR protein level was downregulated in the HPRL group, but there was no difference in total protein level. Moreover, compared with the HPRL group, the PRLR protein level was augmented in the HPRL (BSZY-D/CSF) group, suggesting that CSF reversed the downregulation of PRLR protein level through HPRL (P < 0.05) (Figure 4).

The PRLR protein level of GT1-7 cells at different time points were detected using western blot. Compared with the Control + CHX group, the PRLR protein level was decreased significantly in the HPRL group. Compared with the HPRL + CHX group, the PRLR level in the HPRL (BSZY-D/CSF) + CHX group was significantly upregulated (P < 0.05). The results demonstrated that the PRLR degradation would be accelerated under the high PRL condition, and this degradation was inhibited after the addition of BSZY-D (Figure 5).

The changes of CSN5 expression in GT1-7 cells treated with different reagents

Co-IP was applied to test the expressions of β-Trcp, PRLR and CSN5 (Supplementary Figure 6). Compared with the Control group, the binding of PRLR and β-Trcp was significantly increased in the HPRL group, and the binding of PRLR and CSN5 was decreased; compared with the HPRL group, the binding of PRLR and CSN5 in the HPRL (BSZY-D/CSF) group was increased (P < 0.05). It was suggested that BSZY-D played a role by promoting the binding of PRLR and CSN5.

The levels of CSN5 and GATA1 in GT1-7 were detected by western blot (Figure 6). Our results represented that the CSN5 and GATA1 protein levels were significantly decreased in the HPRL group compared with the Control group. Compared with the HPRL group, the levels of CSN5 and GATA1 in the HPRL (BSZY-D/CSF) group were significantly elevated (P<0.05). In line with the results of western blot, the RT-PCR data showed that the CSN5 mRNA expression was significantly suppressed in the HPRL group compared with the Control group, and the GATA1 mRNA expression did not change significantly. Besides, compared with the HPRL group, the CSN5 mRNA expression was significantly increased in the HPRL (BSZY-D/CSF) group (P<0.05) (Supplementary Figure 7). It was indicated that BSZY-D reversed the downregulation effect of HPRL on CSN5.

Effect of lentivirus plasmids shRNA-CSN5 on PRLR/JAK/STAT5 pathway in GT1-7

Luciferase activity was detected by measuring the renilla luciferase activity of GT1-7 cells transiently transfected with the CSN5 luciferase promoter (Figure 7). Compared with the Control group, the CSN5 promoter activity in the HPRL group was significantly decreased; in turn, compared with the HPRL group, the CSN5 promoter activity in the HPRL (BSZY-D/CSF) group was significantly upregulated. Correspondingly, the binding of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but the binding of GATA1 and CSN5 promoter in the HPRL (BSZY-D/CSF) group was significantly elevated (P < 0.05) (Supplementary Figure 8). After the transfection of lentivirus plasmids, the CSN5 protein level was significantly inhibited in the shRNA-CSN5 group, suggesting that the shRNA-CSN5 plasmid reduced the expression of CSN5 in GT1-7 cells (P < 0.05) (Supplementary Figure 9).

After the confirmation of transfection efficiency, the impact of shRNA-CSN5 on PRLR/JAK/STAT5 signaling pathway in GT1-7 cells of different treatment groups was detected by western blot. As shown in Figure 8, compared with the Control group, the phosphorylation levels of JAK2 and STAT5, and the protein levels PRLR and kisspeptin in the HPRL (BSZY-D/CSF) + shRNA-NC group were significantly decreased (P < 0.05). After knocking down CSN5 gene, the above-mentioned protein levels could be reversed. This result indicated that BSZY-D enhanced the PRLR/JAK/STAT5 signaling pathway through CSN5.

Besides, there existed an increasing ubiquitination level of PRLR in the HPRL group, which could be reversed by knocking down shRNA-SCN5. It was suggested that BSZY-D played a role through targeting CSN5 (Supplementary Figure 10).

Cell culture and treatment

GT1-7 cell line was purchased from Millipore (Schwalbach, Germany). GT1-7 cells were cultured and maintained in DMEM medium (GIBCO, Grand Island, USA) containing penicillin (final concentration 100U/mL) (Sigma-Aldrich, St. Louis, MO, USA), streptomycin (final concentration 100μg/mL) (Sigma-Aldrich, St. Louis, MO, USA), 2mM L-Glutamine (Millipore, Schwalbach, Germany) and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). The cells were digested when reaching 90% confluence.

Medicine and extraction preparation

BSZY-D was concentrated to 2g/mL and stored at 4° C. In brief, the medicinal materials had been immersed in water for 30 min. Firstly, the deer horn slices were decocted and boiled for 20 min, and then added with the remaining medicinal materials. The decoction at a concentration of 2 g/mL was collected and stored at 4° C.

A total of 75 female SD rats of specific pathogen-free (SPF) grade (aged 8 weeks and weighing 220 ± 20 g) were purchased from Animal Model Center of Nanjing University [license No: SCXK (Su) 2018-0008], and raised in the SPF environment in the Experimental Animal Center of Nanjing Chinese Medicine University. The rats were reared at 40%-70% humidity and 21-25° C, maintained in a 12 h light/dark cycle. The experimental operations were approved by the Animal Ethics Committee of Nanjing Chinese Medicine University [approval No: SYXK (Su) 2014-0001].

Cerebrospinal fluid was prepared as follows: BSZY-D of 2 g/mL was given to normal female SD rats 1 mL/kg twice a day. After anesthesia, the neck skin of each rat was disinfected with 75% ethanol, and the foramen magnum was exposed. The micro-injector was pierced into the cistern magna and 100 uL cerebrospinal fluid (CSF) was slowly extracted. After centrifugation at 2000 r/min for 15 min, the supernatant was collected and stored at -20° C. The time point of CSF collection was after the estrus.

Experimental groups

Protocols in different experimental groups were shown as follows:

1) Experiment 1: 0% CSF (medicated cerebrospinal fluid), 5% CSF (medicated cerebrospinal fluid), 10% CSF (medicated cerebrospinal fluid) and 20% CSF (medicated cerebrospinal fluid).

2) Experiment 2: 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng /mL, 200 ng/mL and 400 ng/mL PRL.

3) Experiment 3: 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng /mL and 200 ng/mL PRL.

4) Experiment 4:

Normal group (Control group): 10% blank cerebrospinal fluid, cultured for 72 h.

PRL intervention group (HPRL group): 10% blank cerebrospinal fluid + 200 ng/mL PRL, cultured for 72 h.

Cerebrospinal fluid containing BSZY-D intervention group (BSZY-D/CSF group): 10% BSZY-D containing medicated cerebrospinal fluid, cultured for 72 h.

Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing cerebrospinal fluid culture + 10% blank cerebrospinal fluid + 200 ng/mL PRL, cultured for 72h.

5) Experiment 5:

Normal group + MG132 (Selleck Chemicals, Houston, Texas, USA) (Control + MG132 group): 10% blank cerebrospinal fluid + 10 uM MG132 inhibitor, cultured for 72h.

PRL intervention group + MG132 (HPRL + MG132 group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h.

Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% + MG132 [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing cerebrospinal fluid culture + 200ng/mL PRL + 10uM MG132 inhibitor, cultured for 72h.

6) Experiment 6:

Normal group + cycloheximide (CHX) (Seleck Chem, Shanghai, China) (Control + CHX group): 10% blank cerebrospinal fluid + 10uM CHX inhibitor.

PRL intervention group + cycloheximide CHX (HPRL + CHX group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10uM CHX inhibitor.

Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% + cycloheximide CHX [HPRL (BSZY-D/CSF) + CHX group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL + 10 uM CHX inhibitor.

The time points for detection were 0h, 2h, 4h, 12h, 24h, respectively.

7) Experiment 7:

Normal group + MG132 (Control + MG132 group): 10% blank cerebrospinal fluid + 10 uM MG132 inhibitor, cultured for 72h.

PRL intervention group + MG132 (HPRL + MG132 group): 10% blank cerebrospinal fluid + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h.

Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% + MG132 [HPRL (BSZY-D/CSF) +MG132 group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL + 10 uM MG132 inhibitor, cultured for 72h.

8) Experiment 8:

Normal group (Control group): 10% blank cerebrospinal fluid, cultured for 72 hours.

Prolactin intervention group (HPRL group): 10% blank cerebrospinal fluid + 200 ng/mL PRL, cultured for 72 h.

Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% intervention [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h.

9) Experiment 9:

Cerebrospinal fluid containing traditional Chinese medicine at a concentration of 10% intervention [HPRL (BSZY-D/CSF) group]: 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h.

Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% + ShRNA-CSN5 [HPRL (BSZY-D/CSF) + ShRNA-CSN5 group]: after the cells were stably transfected with CSN5-knockdown lentivirus, added 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h.

Cerebrospinal fluid containing traditional Chinese medicine a concentration of 10% + ShRNA- NC [HPRL (BSZY-D/CSF) + ShRNA-NC group]: after the cells were stably transfected with NC lentivirus, added 10% BSZY-D containing medicated cerebrospinal fluid culture + 200 ng/mL PRL, cultured for 72h.

CCK8 assay

The GT1-7 cell density was adjusted to 5000 cells/well, seeded into a 96-well plate, and cultured in the incubator at 37° C with 5% CO₂ for 24 h. Afterwards, the original culture solution was discarded and the cells were washed twice with phosphate-buffered saline (PBS). According to Experiment 1 or 2, GT1-7 cells were treated differently. Each experiment group had 3 parallel wells. After 72h of incubation, each well was added with 10 μL CCK-8 (Beyotime Biotechnology, Shanghai, China) solution and incubated for 2 h in an incubator. The optical density (OD) value of the liquid in each well was measured by a microreader (Berthold LB941) at the wavelength of 450 nm.

Western blot

Total protein was extracted from GT1-7 cells using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The tissue lysates were centrifuged at 12,000 rpm for 5 min at 4° C, and the supernatants were collected for further analysis. The bicinchoninic acid (BCA) assay was applied to detect the concentration of protein samples. Then, 60 μg protein samples per lane were separated using 8% SDS-PAGE and then transferred onto PVDF membranes (0.45 μm, Millipore, Schwalbach, Germany). The membranes were blocked in Tris-buffered saline Tween containing 5% skim milk for 1h at room temperature and then incubated overnight at 4° C with primary antibodies: Anti-phos-JAK2 (Tyr1007/1008) antibody (3771), Anti-JAK2 antibody (3230), Anti-phos-STAT5 (Tyr694) antibody (9314), Anti-STAT5 antibody (94205), Anti-NA/K ATP antibody (3010) (CST, Boston, USA); Anti-PRLR antibody (ab170935), Anti-kisspeptin antibody (ab19028) (Abcam, Cambridge, UK); Anti-PRLR antibody (ABIN152720) (IP), Anti-GATA1 antibody (ABIN2704828) (antibodies-online, Beijing, China). Next day, these membranes were washed with PBS and incubated with secondary antibodies for 1h at room temperature. After rinsing, the proteins were detected by enhanced chemiluminescence. The protein levels were quantified by densitometry using Image-Pro Plus 6.0 and normalized to the corresponding GAPDH level.

Real-time PCR

The total RNA was extracted from GT1-7 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA then underwent reverse transcription using the iScript cDNA kit (Bio-Rad, Hercules, CA, USA), followed by analysis using real-time PCR (RT-PCR) with the SsoFast Eva Green Super Mix (Bio-Rad, Hercules, CA, USA) on Roche Light Cycler 480 system.

Relative mRNA expressions of kisspeptin, PRLR, CSN5, GATA1, GAPDH were calculated by the comparative cycle threshold (CT) method, with expression of GAPDH as the internal reference. The 2^−∆∆Ct method was used for analysis. The specific primers applied for RT-PCR reaction are shown in Table 1.

Co-immunoprecipitation (Co-IP)

The GT1-7 cells were treated as described in Experiment 5, 7 and 9. Then the cell precipitation was added with RIPA lysis buffer. Protein A/G-agarose microspheres were washed twice with PBS, and prepared into 50% protein A/G-agarose working solution with PBS. Next, 50% protein A/G agarose working solution was added into the samples at a ratio of 100 μL per 1 mL. The protein A/G-agarose microspheres were discarded by centrifugation. BCA assay was applied to test the protein concentration. Afterwards, 5 μL antibody (PRLR150) (IgG antibody as the control group) was added for incubation at 4° C overnight and the final volume was 500 μL. Next day, 100 μL protein A/G-agarose microspheres was added to capture the antigen-antibody complex at room temperature for 1h. The protein A/G-agarose microsphere antigen-antibody complex was collected after centrifugation and washed with PBS. The supernatant was collected, and the loaded samples were boiled for 5 min to precipitate. The obtained samples were detected by western blot using antibodies (β-Trcp, 4394, Abcam, Cambridge, UK), as described in 3.4.

Immunofluorescence

The cells were fixed with 10% paraformaldehyde for 20 min at room temperature. The fixed cells were blocked with 1% bovine serum albumin (BSA) for 30 min and incubated with the corresponding primary antibody (PRLR 1:20) at 4° C overnight, followed by washing with PBST for 10 min. Then the secondary antibody was added for 2h-incubation at room temperature, followed by the 4’, 6-diamidino-2-phenylindole (DAPI) staining for 15 min, and observation under a laser confocal microscope (400 ×).

Construction of luciferase reporter gene plasmid

The full length of CSN5 (NM001277101) promoter was detected, combining with the target gene prediction software (Jaspar), to find the interaction site of CSN5 promoter and acting site of GATA1. Primer 5 was applied to design primers, making PCR product containing CSN5 promoter and GATA1 predicted binding site 1 (GGATGT).

According to the protocol of Experiment 8, each well of 96-well plate was added with 100 μL reporter gene cell lysate, followed by centrifugation at 15000rpm for 5 min to collect the supernatant. The sample was detected by a microplate reader with an interval of 2 s and a measurement time of 10 s. Then 100 μL luciferase detection reagent was added into 100 μL sample for RLU measurement.

Chromatin immunoprecipitation assay (ChIP)

The cells were treated with 1% paraformaldehyde and incubated at 37° C for 10min. The cells were resuspended in SDS lysis buffer and broken by ultrasonic, and the supernatant was collected. CHIP diluent was added with protease inhibitor, and this solution was used to dilute the cell supernatant. Then 200 μL cell supernatant was added into 1800 μL ChIP diluent, making the final volume as 2 mL. The 10 μL cell supernatant dilution was regard as input control. Protein A Agarose/salmon sperm DNA was added to the supernatant, followed by antibody (GATA1) incubation overnight. Then 200μL eluent was added to the protein A Agarose/antibody complex, and the supernatant was collected. Next, 20 μL 5MNaCl was added to each 500 μL eluent and subjected to water bath at 65° C for 4h, followed by 10 μL 0.5M EDTA, 20 μL 1M Tris-HC1 and 2 μL proteinase K at a 45° C-water bath for 1h. After filtered by DNA clean-up Column, the fluid was collected, and the qPCR was performed as described as 3.6.

Construction of lentivirus

Based on the construction of vector and identification of Colony PCR (Supplementary Figure 1), the bacterial solution with correct sequencing was seeded into 10 mL LB liquid medium containing Amp antibiotic and incubated, and then the plasmid was extracted. The GT1-7 cells were seeded into a 24-well plate and prepared for transfection. Then 0.8 μg plasmid was added to 50 μL Opti-MEM medium (GIBCO, Grand Island, USA) or 2.0 μL lipofectamine 2000 (Thermo Fisher Scientific, NY, USA) at room temperature for 5min, respectively. The two diluents were mixed for 5 min, and then 100 μL mixture was added into each well of the cell plate for 12 h-incubation at 37° C. After 72 h of transfection, the infective efficiency could be determined.

Statistical analysis

All data were expressed as means ± SEM. Assessment of statistical significance was done using statistical analysis software SPSS (IBM SPSS Statistics 21; SPSS Inc., Chicago, IL, USA). For comparisons between experimental groups, one-way ANOVA and Tukey’s test were applied. All the P values were two-sided and the differences were considered statistically significant at P < 0.05.

Ethical statement

The experimental operations were approved by the Animal Ethics Committee of Nanjing Chinese Medicine University (approval No: SYXK (Su) 2014-0001).