The effect of QiangGuYin on osteoporosis through the AKT/mTOR/autophagy signaling pathway mediated by CKIP-1

Experimental animal

Adult female Sprague-Dawley (SD) rats were purchased from the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine and were fed under environmental conditions without specific pathogens (temperature 20-25° C, relative humidity 55-65%, light/dark cycle 12 h) And maintain free access to water and food. All experimental procedures follow the Guidelines for the Care and Use of NIH Laboratory Animals (NIH Publication No. 80-23, revised in 1978) and have been approved by the Animal Care and Use Committee of Zhejiang University of Traditional Chinese Medicine (Approval Number: 20200622-09).

Preparation of QGY medicines and pharmacy-containing serums

QGY (No. Z20195155) is produced by the Pharmaceutical Preparation Center of the Second Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine. Each bottle of QGY contains 22.2mL concentrated soup, which were composed of 245g crude drugs, including honeysuckle stem 25 g, Cornu Cervi Degelatinatum 20 g, Caulis Spatholobi 25 g, Nidus Vespae 20 g, Gentiana macrophylla 15 g, Radix Sileris 15 g, cinnamon 10 g, Ligusticum wallichii 20 g, Eucommia ulmoides 15 g, Astragalus membranaceus 30g, Rhizoma Drynariae 20 g, and Dipsacus asperoides 30 g. The drug-containing serum was prepared as follows: 40 male SD rats weighed 280-300 g were randomly divided into control or drug groups. In drug group, rats were intragastrically administered QGY at a dosage of 10mL/kg twice a day. In control group, PBS was administered instead of QGY. After 3 days, rats were euthanized and blood was isolated by eyeball extirpating. Then, serum from drug group (QGY-serum) and control group (control-serum) were isolated and used in vitro experiments.

Animal osteoporosis model

After 12-week-old female SD rats (body weight 280-300 g) were adaptively fed for 3 days, 5 rats were subjected to a bilateral laparotomy group (Sham), and another 30 rats were subjected to bilateral ovariectomy according to the previous experimental method (OVX) [21]. OVX rats were randomly divided into 6 groups (n=5): vehicle group (OVX+PBS), low-dose QGY group (OVX+L-QGY, 2.5 mL/kg/day), medium-dose QGY group (OVX+M-QGY, 5 mL/kg/day), high-dose QGY group (OVX+H-QGY, 10 mL/kg/day), autophagy inhibitor group (OVX+3-MA), high-dose QGY + autophagy inhibitor group (OVX+H-QGY+3-MA). All drugs were treated orally for 6 weeks. After completing the experiment, samples of the right femur of each group of rats were taken for histological staining detection and micro-CT scan.

Histomorphometric analysis

A micro-CT imaging system (Skyscan 1176, Belgium) was used for morphological analysis of trabecular bone. The left femur sample was scanned at a resolution of 14.8 μm. Select the trabecular bone region of interest to quantify BMC. By measuring bone trabecular volume fraction (BV/TV, %), bone density (BMC/TV), bone trabecular number (Tb.N) and bone trabecular spacing (Tb.Sp, mm) and other related parameters, Reflects the internal microstructure of the femur.

The femur sample was fixed in 4 % paraformaldehyde, then the sample was embedded in paraffin and cut into 4 μm thick sections. Use hematoxylin-eosin (H&E) staining kit to observe the cell morphology of trabecular bone. In addition, according to the manufacturer’s instructions, slices were fixed in TRAP fixative solution at 4° C for 3 min, then incubated in TRAP working solution at 37° C in the dark for 60 min, stained with hematoxylin solution for 3-5 min, and then subjected to microscopic examination to confirm the bone cell differentiation.

Bone cell culture and differentiation

MC3T3-E1 osteoblasts and RAW264.7 cells were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). MC3T3-E1 osteoblasts were cultured in α-minimum essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. Cells were maintained in medium with ascorbic acid (50 μg/mL) and β-glycerol phosphatase (10 mM) for osteogenic differentiation for 21 days. In addition, RAW264.7 cells were cultured using a-MEM (10% FBS, 1%P/S) and maintained at 37° C with 5% CO₂ overnight, and were stimulated with M-CSF (20 ng/mL) and RANKL (50 ng/mL) in a-MEM to induce osteoclasts formation (1×10³ cells/well in 96-well plates).

Western blotting analysis

Lyse the tissue or cells in pre-cooled lysate (50 mM Tris-Cl, pH 7.4, 1 mM EDTA pH 8.0, 250 mM NaCl, and 1% Triton-X) for 30 minutes, and centrifuge at 12000 rpm at 4° C for 20 minutes. The supernatant was collected and the total protein concentration was determined by BCA protein analysis kit (Beyotime, Shanghai, China). The same amount of protein (40 μg) was separated by 10% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). The membrane was blocked with 4% skimmed milk for 2 h and incubated overnight at 4° C with the following primary antibodies: CKIP-1 (1:1000, Santa Cruz Biotechnology, SC-376355), LC3II/I (1:1000, Abcam, ab128065), p62 (1:1000, Abcam, ab91526), Atg5 (1:1000, Proteintech, 10181-2-AP), AKT (1:1000, Cell Signaling Technology, 9272), phospho-AKT (1:1000, CST, 4060), mTOR (1:1000, Cell Signaling Technology, 2983), phospho-mTOR (1:1000, Cell Signaling Technology, 2971), RUNX2 (1:1000, Abcam, ab236639), RANKL (1:1000, Santa Cruz Biotechnology, SC-377079), OPG (1:1000, Santa Cruz Biotechnology, SC-390518) and GAPDH (1:5000, Santa Cruz Biotechnology, United States, sc-25778). The membrane was incubated with HRP-conjugated antibody (Wuhan Boster Bioengineering Co., Ltd.) for 1 h, and then the enhanced ECL chemiluminescence reagent (Bio-Rad) and GelDoc imaging system (BioRad) were used to quantify immunoreactive bands.

Construction of CKIP-1 expression lentivirus and knockdown shRNA

CKIP-1 expression lentivirus and knockdown shRNA were constructed using standard methods. In detail, Taq Plus DNA Polymerase (ET105-02-01, TIANGEN), dNTP (CD117, TIANGEN), T4 DNA Ligase (FL101-02, TRANS), Xho I (1094A, TAKARA), Not I (1166A, TAKARA), DL15000 DNA Maker (MD110, TIANGEN), DL2000 DNA Maker (MD114, TIANGEN) were used according to the manufactures’ instructions. MC3T3-E1 cells and RAW 264.7 cells were then transfected with lentivirus and shRNA using polybrene (Sigma) for 24 h and passaged for following experiments.

Co-IP detection

Total cellular proteins of osteoblasts from different groups were extracted. Extracted proteins (500 μg/sample) were pretreated with 30 μL Agarose Protein A+G beads (CST, #9863 and 37478) for 2 h at 4° C. Then the beads were removed by centrifuge. After that, 3μg HA-Tag rabbit monoclonal antibody (IP-HA, 1:100, CST, #3724) or IgG (IP-IgG, 1:100, CST, #3900) was added into samples and incubated overnight at 4° C. Another 30μl Agarose Protein A+G beads (50%) were added into samples and incubated for 6h at 4° C. After centrifuge, the sediment was isolated and resuspended with 30μL loading buffer. Then, the proteins were measured by western blot as described above.

LC3 immunofluorescence assay

The cells were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) and blocked in 5% BSA for 1 h, and then incubated with rabbit monoclonal anti-LC3B antibody (Cell Signaling Technology, Danvers, MA, USA) overnight and with Alexa Fluor The 488-conjugated secondary antibody was incubated for 1h. Finally, it was counterstained with 4',6-dimidyl-2-phenylindole dye (DAPI) (Cell Signaling Technology, Danvers, MA, USA), and performed on a confocal laser scanning microscope (Leica, SP8, Germany) Capture images on.

ELISA analysis

7 days after osteoblast induction, ELISA was used to measure the protein expression levels of RANKL and OPG according to the manufacturer's instructions.

Quantitative real-time PCR

Total RNA was removed from the cells using Trizol reagent (Invitrogen, Carlsbad, NM, USA) according to the manufacturer’s instructions. cDNA was synthesized with ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Real-time PCR was performed on an ABI 7500 Fast Real-Time PCR System using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The optimal conditions for thermal cycling are as follows: initial denaturation at 95° C for 15 min, followed by denaturation at 95° C for 30 s, annealing at 60±3° C for 30 s, and extension at 60° C for 30 s for 40 cycles. The primers used for real-time PCR are as follows: CKIP-I (Forward): 5'-GCCGTGAGTCCTGAAGAGAAG-3', (Reverse): 5'-CGAGTAGGGTGGGCAAGATAG-3'; OPG (Forward): 5'-CCTGCCTGGGAAGAAGATCA-3', (Reverse): 5'-TTGTGAAGCTGTGCAGGAAC-3'; RANKL (Forward): 5'-GCACACCTCACCATCAATGCT-3', (Reverse): 5'-GGTACCAAGAGGACAGAGTGACTTTA-3'; GAPDH (Forward): 5'-ATGGGTGTGAACCACGAGA-3', (Reverse): 5'-CAGGGATGATGTTCTGGGCA-3'.

Alizarin Red Staining

The cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min at room temperature. According to the manufacturer's instructions, the cells were stained with Alizarin Red Staining Kit (Beyotime, Shanghai, China) for 60 min at room temperature. After washing 3 times, photograph the stained cells immediately.

Statistical analysis

All data were presented as mean ± standard deviation (mean ± SD). Differences among data from 3 or more groups were analyzed by one-way ANOVA with post hoc Tukey’s multiple comparison tests. Analyses were performed using Prism software (v. 8.0; GraphPad, San Diego, CA, USA). p<0.05 were recognized as statistically significant.

QGY improves bone condition in osteoporotic rats

To determine whether QGY restored bone loss caused by estrogen deficiency, OVX rats were treated with different doses of QGY for 6 weeks. The results of micro-CT three-dimensional imaging showed that compared with the Sham group, the OVX+PBS group had lower bone density and reduced bone trabeculae; QGY and autophagy inhibitor 3-MA treatments could improve bone conditions, and the effective effect of QGY was dose-dependent (Figure 1A). Further analysis of the image parameters found that compared with the Sham group, the BV/TV, BMC/TV, Tb.N index of the OVX+PBS group decreased, and the TB.Sp index increased; QGY treatment significantly increased BV/TV and bone density, and decreased Tb Sp, and is dose-dependent; autophagy inhibitor 3-MA treatment can significantly increase BV/TV and bone density. At the same time, high-dose QGY and 3-MA treatment can significantly improve bone microstructure (Figure 1B).

Figure 1. Micro-CT analysis after treating with QGY in animal model. Ovariectomy (OVX) was performed in rats, and then different doses of QGY were given to the rats. (A) Representative figures of three-dimensional micro-CT analysis. (B–E) Quantitative analysis of bone trabecular volume fraction (BV/TV, %) (B), bone density (BMC/TV) (C), bone trabecular number (Tb.N) (D) and bone trabecular spacing (Tb.Sp, mm) (E) in three-dimensional micro-CT analysis. The data are presented as the means ± SD (n=5). ^*p<0.05, ^**p<0.01. One-way ANOVA followed by Tukey's post hoc test.

To further evaluate the influence of QGY on bone structure, H&E staining was used to determine the thickness and number of bone trabeculae. Compared with the Sham group, the bone marrow cavity in the OVX+PBS group became larger, the bone trabeculae became thinner, and the structure was disordered and loose; QGY and 3-MA treatments thickened the bone trabeculae to varying degrees, the bone marrow cavity became smaller, and the structure was compact (Figure 2A). In order to evaluate the effect of QGY on osteoclasts, TRAP staining was used to determine the differentiation of osteoclasts. TRAP staining showed that the osteoclasts were wine-red, located at the edge of the trabecular bone. Compared with the Sham group, the number of osteoclasts in the OVX+PBS group was significantly increased; QGY and 3-MA treatments significantly reduced the number of osteoclasts (Figure 2B). These results indicate that QGY can improve the bone condition of osteoporotic rats in a dose-dependent manner, possibly by regulating autophagy.

Figure 2. Staining analysis after treating with QGY in animal model. (A) H&E staining of the trabecular bone. (B) TRAP staining and quantitative analysis of the trabecular bone. The data are presented as the means ± SD (n=5). ^*p<0.05, ^**p<0.01. One-way ANOVA followed by Tukey's post hoc test.

QGY reduces the expression of CKIP-1 in the femoral tissue of osteoporotic rats and mediates autophagy through the AKT/mTOR pathway

In order to study the molecular mechanism of QGY improving osteoporosis, the expression of related proteins in the autophagy pathway mediated by CKIP-1 and AKT/mTOR was evaluated. Western blotting analysis showed that, compared with the Sham group, the expressions of CKIP-1, LC3II/I and RANKL were up-regulated in the femoral tissues of the OVX+PBS group, while the expressions of p62, p-AKT, p-mTOR, RUNX2, and OPG were down-regulated, suggesting that the increase of autophagy levels inhibit the AKT/mTOR signaling pathway; QGY and 3-MA treatment reverses the expression level of related proteins, and the effect of QGY shows a dose-dependent characteristic (Figure 3A, 3B). The gene expression levels of CKIP-1, RANKL and OPG were further checked, and it was found that QGY and 3-MA treatments also reduced the gene expression levels of CKIP-1 and RANKL and promoted the gene expression level of OPG (Figure 3C). It is speculated that QGY may inhibit autophagy, activate the AKT/mTOR pathway, reduce the expression of CKIP-1 and RANKL, and promote the expression of RUNX2 and OPG, thereby improving osteoporosis.

Figure 3. The effect of QGY on the protein expression of femoral tissue in osteoporotic rats. (A, B) Representative figures (A) and quantitative analysis (B) of protein expression of CKIP-1, LC3II/I, p62, p-AKT/AKT, p-mTOR/mTOR, RUNX2, RANKL and OPG. (C) Gene expression levels of CKIP-1, RANKL and OPG. The data are presented as the means ± SD (n=5). ^*p<0.05, ^**p<0.01. One-way ANOVA followed by Tukey's post hoc test.

QGY mediates the effect of CKIP-1 on autophagy of osteoblasts and osteoclasts

In order to study the mechanism of QGY-mediated CKIP-1 in improving osteoporosis, the interaction between CKIP-1 and AKT was first evaluated. The results showed that in osteoblasts, compared with the HA-CKIP-1-OE group, the binding content of CKIP-1 and AKT decreased significantly after QGY treatment, indicating that CKIP-1 and AKT interact, and QGY treatment can reduce its binding capacity; the opposite trend appears in osteoclasts (Figure 4A). These results indicate that there is an interaction between CKIP-1 and AKT, and QGY inhibits the binding of CKIP-1 and AKT in osteoblasts and promotes the binding of CKIP-1 and AKT in osteoclasts.

Figure 4. QGY mediates the effect of CKIP-1 on autophagy through the AKT/mTOR pathway. (A) Co-IP assay in the osteoblasts and osteoclasts. (B, C) Representative figures and quantitative analysis of protein expression of Atg5 and p62 in the osteoblasts (B) and osteoclasts (C). (D) LC3 immunofluorescence assay in the osteoblasts and osteoclasts. (E) RANKL and OPG content analysis in the osteoblasts. (F) Gene expression levels of CKIP-1, RANKL and OPG in the osteoblasts. The data are presented as the means ± SD (B, C, E, F: n=6; D: n=3). ^*p<0.05, ^**p<0.01. One-way ANOVA followed by Tukey's post hoc test.

Further analyze the effect of QGY on autophagy. First, it was determined that QGY could reduce the mRNA level after CKIP-1 overexpression (Figure 4F). In osteoblasts, compared with the control group, the Atg5 protein expression level of the CKIP-1 OE group increased, the p62 protein level decreased (Figure 4B), and the LC3 punctate aggregates (autophagosomes) increased (Figure 4D), indicating that CKIP-1 mediates autophagy activation of osteoblasts. On the contrary, in osteoclasts, compared with the control group, the Atg5 protein expression level of the CKIP-1 OE group decreased, the p62 protein level increased (Figure 4C), and the LC3 punctate aggregates decreased (Figure 4D). This indicates that CKIP-1 mediated autophagy inhibition, and QGY treatment reversed CKIP-1 mediated osteoblasts and osteoclast autophagy. Next, the regulation of autophagy was tested. In osteoblasts, compared with the CKIP-1 OE infection group, after the addition of autophagy inhibitor 3-MA, the intracellular LC3 II/I level decreased, and the p62 protein expression level increased; after the addition of the autophagy activator rapamycin, LC3 II/I level increased, but p62 protein expression level decreased; after adding QGY treatment, LC3 II/I level decreased, p62 protein expression level increased; autophagy inhibitor 3-MA treatment can increase the autophagy inhibitory effect of QGY on cell autophagy, and autophagy activator rapamycin can also increase the activation of QGY on cell autophagy (Figure 5A). In osteoclasts, this trend is reversed (Figure 5B). These results indicate that QGY inhibits the combination of CKIP-1 and AKT, activates the AKT/mTOR pathway, inhibits autophagy in osteoblasts, thereby improving osteoporosis, which is consistent with the results of in vivo experiments.

Figure 5. QGY mediates the effect of CKIP-1 on autophagy of osteoblasts and osteoclasts. (A, B) Representative figures and quantitative analysis of protein expression of LC3II/I and p62 in the osteoblasts (A) and osteoclasts (B). The data are presented as the means ± SD (n=6). ^*p<0.05, ^**p<0.01. One-way ANOVA followed by Tukey's post hoc test.

The effect of CKIP-1 mediated autophagy on the differentiation and function of osteoblasts and osteoclasts

The differentiation ability of osteoblasts was evaluated by Alizarin Red staining. The results showed that 3-MA treatment can significantly increase the calcified nodules in osteoblasts infected with CKIP-1 OE, and the osteogenic capacity is enhanced; after rapamycin treatment, the calcified nodules are reduced and the osteogenic capacity is reduced; after QGY treatment, the calcified nodules are obvious, and the osteogenic ability is enhanced; 3-MA can enhance the deposition of QGY on the calcified nodules of osteoblasts, and rapamycin can inhibit the promotion of QGY on the calcified nodules of osteoblasts (Figure 6A). These results indicate that QGY promotes the differentiation ability of osteoblasts and enhances the osteogenic ability by inhibiting the level of autophagy.

Figure 6. CKIP-1 mediated autophagy on the differentiation and function. (A) Alizarin Red Staining in the osteoblasts. (B) RANKL and OPG content analysis in the osteoblasts. (C) Representative figures and quantitative analysis of protein expression of RUNX2 in the osteoblasts. (D) TRACP activity analysis in the osteoblasts. (E) Gene expression levels of CKIP-1, RANKL and OPG in the osteoblasts. The data are presented as the means ± S.D (n=6). ^*p<0.05, ^**p<0.01. One-way ANOVA followed by Tukey's post hoc test.

Further detect the content of RANKL and OPG in the supernatant of osteoblasts. The results showed that compared with the control group, the RANKL content in the supernatant of CKIP-1 OE group was significantly increased, and the OPG content was significantly reduced; QGY treatment could reduce RANKL content and increased OPG content, indicating that QGY inhibits osteoblast secretion of RANKL and promotes OPG secretion (Figure 4E). Treatment with autophagy inhibitor 3-MA can significantly reduce RANKL secretion and increase OPG secretion in osteoblast, and increase the regulation of QGY on RANKL and OPG secretion; treatment with autophagy inducer rapamycin can increase RANKL secretes and inhibits OPG secretion, and inhibits the regulation of QGY (Figure 6B). Similarly, the mRNA levels of RANKL and OPG showed a consistent trend (Figures 4F, 6E). These results indicate that QGY inhibits the secretion of RANKL by osteoblasts and promotes the secretion of OPG by regulating autophagy, which is consistent with the results of in vivo experiments.

Western blotting analysis was used to detect the expression level of osteoblast marker RUNX2 protein. The results show that the autophagy inhibitor 3-MA can promote the protein expression of RUNX2 in the osteoblasts CKIP-1 OE group; the autophagy activator rapamycin can inhibit the protein expression of RUNX2; QGY treatment can increase the protein expression of RUNX2. In addition, 3-MA can enhance the promotion of QGY on RUNX2 expression, while rapamycin can block the induction of QGY on RUNX2 expression in osteoblast (Figure 6C). To evaluate the anti-tartrate acid phosphatase (TRACP) activity of osteoclasts. The results showed that treatment with 3-MA can inhibit TRACP activity of osteoclasts infected by CKIP-1 OE; treatment with rapamycin can increase TRACP activity. In addition, QGY treatment can inhibit TRACP activity; 3-MA can enhance the inhibitory effect of QGY on osteoclast TRACP activity, and rapamycin can inhibit the inhibitory effect of QGY (Figure 6D). These results indicate that QGY inhibits TRACP activity through autophagy regulation on osteoclast.