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

Osteoporosis is a systemic bone disease characterized by decreased bone mass and deterioration of bone microstructure, which leads to increased bone fragility and increased risk of fractures. Casein kinase 2 interacting protein 1 (CKIP-1, also known as PLEKHO1) is involved in the biological process of bone formation, differentiation and apoptosis, and is a negative regulator of bone formation. QiangGuYin (QGY) is a famous TCM formula that has been widely used in China for the clinical treatment of postmenopausal osteoporosis for decades, but the effect in regulating CKIP-1 on osteoporosis is not fully understood. This study aimed to explore the potential mechanism of CKIP-1 participating in autophagy in bone cells through the AKT/mTOR signaling pathway and the regulatory effect of QGY. The results in vivo showed that QGY treatment can significantly improve the bone quality of osteoporotic rats, down-regulate the expression of CKIP-1, LC3II/I and RANKL, and up-regulated the expression of p62, p-AKT/AKT, p-mTOR/mTOR, RUNX2 and OPG. It is worth noting that the results in vitro confirmed that CKIP-1 interacts with AKT. By up-regulating the expression of Atg5 and down-regulating the p62, the level of LC3 (autophagosome) is increased, and the cells osteogenesis and differentiation are inhibited. QGY inhibits the combination of CKIP-1 and AKT in osteoblasts, activates the AKT/mTOR signaling pathway, inhibits autophagy, and promotes cell differentiation, thereby exerting an anti-osteoporosis effect. Therefore, QGY targeting CKIP-1 to regulate the AKT/mTOR-autophagy signaling pathway may represent a promising drug candidate for the treatment of osteoporosis.


INTRODUCTION
Osteoporosis (OP) is a systemic multifactorial skeletal disorder characterized by decreased bone mass, deterioration of bone microarchitecture and increased bone fragility, resulting in a tendency of fracture and leading to possible lifelong disability or death [1,2]. With the expanding ageing population, the prevalence of OP is increasing, and it has become an important public health concern in many countries [3]. Researches have shown that the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which leads to OP [4]. Among them, the imbalance in the number and activity of osteoblasts and osteoclasts plays a major role [5]. Considering that the adverse reactions of the present used drugs in clinical with accurate efficacy are also obvious, the development of possible novel therapeutic strategies has become of real importance.
AGING CKIP-1 mediates cell growth, differentiation, apoptosis, cytoskeleton [6,7], and bone formation, and has been found to be a promising target for OP therapy. Studies have shown that CKIP-1 knockout (KO) mice increase bone density and bone mass with age, and the activity of osteoblasts is significantly higher than those of wildtype mice [8], while the small interfering RNA of CKIP (siRNA) significantly promoted bone formation in vivo and in vitro [9]. Autophagy is the main catabolic process for eukaryotic cells to degrade and recycle damaged macromolecules or organelles. As a cell survival pathway, it plays a vital role in maintaining bone homeostasis, and the changes in this pathway are to some extent associated with osteoporosis [10][11][12][13].
Herbal medicines, especially traditional Chinese medicines (TCMs), which are based on natural compounds, have been clinically used in Asian countries for more than 2,500 years, for the treatment of various diseases including osteoporosis [14][15][16][17]. For example, Bu-Shen-Ning-Xin decoction was reported to suppress osteoclastogenesis by regulating RANKL/OPG ratio and protected mice from osteoporosis [17]. Another TCM formula, Fufang Zhenshu Tiaozhi (FTZ), also processed anti-osteoporotic activities against aginginduced osteoporosis in mice [14]. Wang et al. reported that another TCM formula Gushukang granules protected mice from osteoporosis by inhibiting osteoclastogenesis and stimulating osteoblastogenesis [16]. Based on the widely published TCM-related studies, few adverse effects were reported. Thus, growing attentions were attracted for the clinical usage and research application of TCMs. QiangGuYin (QGY) is a famous TCM formula that has been widely used in China for the clinical treatment of postmenopausal osteoporosis for almost 20 years [18][19][20]. In 2017, we published our multi-center clinical trial involving 240 participants. Based on the results of our study, 1-Year treatment with QGY effectively and safely increased Bone Mineral Density (BMD) and reversed osteoporotic bone turn over [20]. The underlying mechanism, however, remained to be uncovered. Therefore, this study tried to explore the potential mechanism of CKIP-1 involved in autophagy in bone cells through the AKT/mTOR signaling pathway and the regulation of QGY.

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. 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.

Histomorphometric analysis
A micro-CT imaging system (Skyscan 1176, Belgium) was used for morphological analysis of trabecular bone.

AGING
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.

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

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.

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.

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).
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.

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, AGING 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 dosedependent 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.

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.
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 AGING 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.

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.
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 AGING 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.

DISCUSSION
CKIP-1 is a transcription factor that acts as a scaffold linker to mediate interactions with various signals and cellular proteins, and is considered to be an important gene responsible for the development and progression of osteoporosis. Previous studies have shown that CKIP-1 is a regulator of cell viability, apoptosis, cytoskeleton formation and cell differentiation. Recent studies have also confirmed that CKIP-1 is a negative regulator of bone formation, leading to excessive activation of osteoclasts and bone loss. Drugs targeting osteoblasts CKIP-1 can reverse bone formation and prevent osteoporosis [22]. In CKIP-1 KO mice, osteoblast activity and BMD were significantly increased [23]. SiRNA knockout targeting CKIP-1 improved bone formation and osseointegration in a rat AGING model of osteoporosis [24]. In another study using a rat mandibular distraction osteogenesis (DO) model, CKIP-1 silencing inhibited BMSC apoptosis and promoted osteogenic differentiation [25]. Consistent with previous studies, our research shows that CKIP-1 mediates the autophagy activation of osteoblasts and inhibits differentiation, increases the expression ratio of RANKL/OPG in osteoblasts, and then inhibits cell osteogenesis; CKIP-1 also mediates the autophagy inhibition of osteoclast. Importantly, QGY treatment promotes osteoblast differentiation and enhances osteogenic capacity by targeting CKIP-1 mediated AKT/mTOR pathway activation and autophagy inhibition (rather than osteoclasts), thereby playing a role in improving osteoporosis (Figure 7).
The AKT/mTOR pathway is a key regulator of bone formation [26]. Previous studies have shown that AKT knockout mice have delayed ossification [27]. AKT is also a key factor in the differentiation process of AGING osteoblasts [28]. Xi et al. reported that the AKT/mTOR signaling pathway participates in the inhibition of osteoporosis by promoting osteoblast proliferation, differentiation and bone formation in vitro and in vivo models [29]. In this study, we found that overexpression of CKIP-1 significantly inhibits the AKT/mTOR pathway, while OGY treatment reactivates the inhibited AKT/mTOR pathway, which is consistent with previous studies [9,[30][31][32][33][34][35]. Therefore, CKIP-1 acts as an intermediary between QGY and AKT/mTOR signaling crosstalk. After treating osteoblasts with AKT/mTOR pathway inhibitors, it is further proved that QGY exerts anti-osteoporosis effects through AKT/mTOR pathway. In addition, the AKT/mTOR signaling pathway is also one of the most important ways to regulate autophagy. The autophagy pathway is considered to play a supporting role in the development of osteoporosis [36][37][38][39]. In this study, autophagy was activated during osteoblast differentiation. Among them, ATG5 and p62 have been shown to regulate autophagy degradation [40], and the lipidated form of LC3 converted from LC3-I to LC3-II is considered as an autophagosome marker because it locates and aggregates on autophagosomes [41]. QGY treatment targets CKIP-1 mediated AKT/mTOR pathway activation and autophagy inhibition.
RANKL (receptor activator of nuclear factor kappa B ligand) is a key osteoclast differentiation factor, which is secreted in large quantities by fibroblasts, osteoblasts and stromal cells around the prosthesis. It combines RANK and cooperates with NF-κB, promote osteoclast differentiation and survival, and osteoclast bone resorption; OPG, as an osteoclast differentiation inhibitor, usually competes with RANK to integrate RANKL, blocking the promotion effect of RANKL/RANK on osteoclast and osteoclast bone resorption, thereby inhibiting osteolysis [42]. The imbalance between osteoblast bone formation and osteoclast bone resorption ultimately leads to osteolysis around the prosthesis, where the OPG/RANKL ratio is one of the best characterizing biomarkers related to the pathology of bone destruction [43]. As expected, our results indicate that QGY treatment also promotes osteoblast differentiation and enhances osteogenic capacity, thereby playing a role in improving osteoporosis.
Our data is also supported by other reports. For example, NUPR1 is considered to be a new type of bone formation regulator, and its deficiency will reduce the production of early osteoclasts and enhance the production of osteoblasts, resulting in increased bone formation [44]. In addition, resveratrol regulates the expression of autophagy-related proteins in a dosedependent manner, such as p62, LC3-II, Atg5, Atg7 and Atg12, etc., inhibits autophagy in osteoblasts and activates osteoclast autophagy, and regulates autophagy. AGING It promotes osteoblast differentiation and inhibits osteoclast differentiation, thereby improving postmenopausal osteoporosis [45]. Consistently, our study also showed the opposite autophagy expression and osteogenic ability in osteoblasts and osteoclasts, which may be related to the restoration of the balance between bone formation and bone resorption by QGY regulating the AKT/mTOR signaling pathway mediated by CKIP-1.
To sum up, consistent with our previously published clinical trial [20], this study provides new information that QGY treatment promotes growth by targeting CKIP-1 mediated AKT/mTOR pathway activation and autophagy inhibition. Osteocytes differentiate and enhance the ability of bone formation, thereby playing a role in improving osteoporosis. Importantly, activation of autophagy with rapamycin not only reversed the inhibition of QGY on the RANKL/OPG ratio and osteoclast differentiation, but also further enhanced this inhibition by using 3-MA to inhibit autophagy. The synergistic in vivo anti-osteoporosis effect of QGY and 3-MA confirms the results we found in cell research. As far as we know, the current research reveals for the first time the CKIP-1 regulatory mechanism of this popular herbal formula to treat osteoporosis.

AUTHOR CONTRIBUTIONS
LK and XS designed the study. YY, HZ, SW, JS, CH, TC, MF, SL, SK and HX performed the experiments. BT, BL, YM and JL analyzed data. YY, HZ, SW, JS and CH wrote the manuscript. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

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