Ganoderic Acid A Prevented Osteoporosis by Modulating the PIK3CA/p‐Akt/TWIST1 Signaling Pathway (2025)

2.1 Reagents

Ganoderic acid A (batch number 20180327, and purity [HPLC 256 nm] > 99.9%) was purchased from Baoji Chenguang Biotechnology Co., LTD. Dimethyl sulfoxide (DMSO) was purchased from Sigma. Tween80 and Tween20 were purchased from Solarbio Biotech Co., LTD (Beijing, China). LY294002 (purity [HPLC] > 99.92%) from MedChemExpress(MCE), and MK2206 (purity [HPLC] > 99.59%) were purchased from Shanghai Taoshu Biotechnology Co., LTD. Serum detection kits (LDH, MDA, SOD, AKP) were purchased from Nanjing Jiancheng Biotechnology Co., LTD. Antibodies against OPN, RUNX2, β-catenin, TWIST1, AKT, and p-Akt (ser473) were purchased from PROTEINTECH GROUP, INC., and the antibody against PIK3CA was purchased from Beijing Bioss Biotechnology Co., LTD. Horseradish enzyme-labeled goat anti-rabbit and anti-mouse IgG secondary antibodies were purchased from PROTEINTECH GROUP, INC. DMEM medium was purchased from Seven Innovative Biotechnology Co., LTD., and fetal bovine serum was purchased from Shanghai Yamei Biotechnology Co., LTD.

2.2 Bioinformatics Analysis

The GA-A-related targets were obtained through BATMAN-TCM (Score cut off ≥ 20, http://bionet.ncpsb.org/batman-tcm/), Super Pred (http://prediction.charite.de/), and Swiss Target Prediction (p ≤ 0.5, http://www.swisstargetprediction.ch/) websites; The OP related targets were obtained through the Gene—Cards (score ≥ 1, https://www.genecards.org/), Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/), and DisGe—NET (score ≥ 0.4, https://www.disgenet.org/) web sites. The Venn diagram tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to obtain the GA/OP-related targets. The obtained drug disease target proteins were imported into the online database STRING (https://string-db.org/) to construct the PPI (protein–protein interaction) network, and Cytoscape software (version 3.7.2) was used to further visualize the PPI network to find the central targets.

2.3 Molecular Docking

The structure of the target protein (PIK3CA) was extracted from the RCSB bank database (PDB, http://www.rcsb.org/pdb/home/home.do. PDB ID: 7R9V), which is a database of the three-dimensional structures of biomacromolecules (proteins, nucleic acids, sugars). The 3D chemical structure of GA-A was obtained from the ZINC database (http://zinc.docking.org/). Before molecular docking, the monomeric protein structure PIK3CA was picked out from the original PDB file and carried out energy minimization and water removal, and hydrogenation operations. Molecular docking of proteins to GA-A was performed using Autodock software (Version 4.2.1), and the free binding energy and binding status were determined. The docking pocket refers to the small ligand molecules already presented in the PDB file.

2.4 Detection of Serum Indicators

The levels of the serum indicators including malondialdehyde (MDA), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) were measured by detection kits based on the manufacturer's instructions (Nanjing Jiancheng Bioengineering Ltd., Nanjing, China).

2.5 Cell Culture

In this study, primary osteoblasts (POB) and MC3T3-E1 (MC) cells were used. POB were isolated from the cranium of 3–7-day-old mice and digested with type-II collagenase and then transferred to cell flasks for culture. After 1–3 days, the cells were reseeded, and the medium was replaced every other day. MC cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Both cell lines were cultured in a DMEM medium containing 10% FBS and 100 U/mL penicillin–streptomycin. Cell culture flasks were incubated in incubators at 37°C with 5% CO₂.

2.6 Animal Experiments

Eight-week-old C57BL/6J female mice (purchased from Liaoning Changsheng Biotechnology Co., LTD.) were selected. After 1 week of adaptive feeding, they were randomly divided into 5 groups, with 10 mice in each group, which were the control group, model group, low (5 mg/kg), medium (10 mg/kg), and high (20 mg/kg) doses of GA-A groups. The doses for the mice experiments were determined by referring to previous studies based on mice models (Lu etal.2021; Abudumijiti etal.2021). The animals were reared in a house with constant temperature and were provided with food and water adlibitum during the study. Ovariectomy (OVX) was used to establish the postmenopausal osteoporosis model, and the control group underwent sham surgery, from which only part of the fat tissue around the ovaries was removed. One week after the operation, the mice in the low, medium, and high doses of GA-A groups were treated with different doses of GA-A by intraperitoneal injection. Eight weeks later, the mice were sacrificed under an over-dose of anesthesia (Urethane 1.2 g/kg), and the serum and bone tissues were collected and stored in a refrigerator at −80°C for further analysis. All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Research Ethics Committee of Dalian Medical University (Dalian, China; ethical permit No. CSE202303003). All efforts were made to minimize the number and suffering of the animals used.

2.7 Cell Proliferation Assay

Both kinds of cells were seeded in 96-well plates. When the cell density was about 80% confluence (8 × 10^3 cells per well), different doses of Ganoderic acid A (0, 5, 10, 20, 40, 80, 160 μM, dissolved in DMSO) were given for 24 h, and then Cell Counting Kit-8 (cck8) reagent (purchased from Beyotime Biotechnology, Shanghai, China) was given for treatment. After 4 h, the drug toxicity of Ganoderic acid A was detected by measuring the absorbance (a) at 450 nm using a thermo354 microplate reader (Thermo Fisher Scientific, China). Hydrogen peroxide (H₂O₂) at a certain concentration can cause oxidative stress damage to cells; thus, it was used for the invitro model. Different concentrations of hydrogen peroxide (0, 100, 150, 200, 250, 300 μM, dissolved in DMEM) were added for 4 h, and the cck8 reagent was added after 4–6 h to detect the effect of hydrogen peroxide on cell survival rate.

2.8 Western Blot

The cell or tissue proteins were mixed with 2 × loading buffer and then separated by electrophoresis. The corresponding gel blocks were cut off according to the different molecular weights labeled by the markers, and the proteins were transferred to the PVDF membranes by membrane transfer apparatus. After completion of the transfer, the membranes were blocked with 5% skim milk powder for 2 h, then cleaned with TTBS and incubated with the primary antibodies against the specific targets in a refrigerator at 4°C overnight. The following day, the secondary antibodies conjugated with horseradish peroxidase were incubated for 1.5 h in a water bath at 37°C. After the incubation was completed, the membranes were rinsed with TTBS and, using a gel imager (BioSpevtrum, USA), the protein expressions on PVDF membranes were observed.

2.9 Immunofluorescence

The 12-well plates of pretreated cells were removed, the culture medium was discarded, rinsed with PBS, and the cells were fixed with 4% paraformaldehyde for 30 min. Then the cells were treated with 0.1% TritonX-100 for 20 min and blocked with 1% BSA for 30 min. After washing, the cells were incubated with the primary antibodies overnight. On the following day, the primary antibody was washed off and incubated with a fluorescent secondary antibody in the dark for 1 h at 37°C in a water bath, followed by 10 min with DAPI in the dark. The images were finally captured by using a fluorescence inverted microscope (Olympus, Japan).

2.10 Osteogenic Induction

MC cells were used for osteogenic induction, and the osteogenic induction solution consisted of dexamethasone sodium phosphate injection (5 μL, 1 mL:5 mg), β-glycerophosphate sodium (1.5 g), and vitamin C (4.3 mg), which were added to DMEM blank medium for later use. Cells were seeded in 12-well plates and treated with ganoderic acid A for another 24 h after the addition of hydrogen peroxide for 4–6 h, and then replaced with osteogenic induction solution diluted 100-fold as described above and replaced every other day. To detect osteogenic differentiation activity, an alkaline phosphatase staining was performed after 14 days of induction, and an alizarin red staining was performed after 21 days of induction to detect the mineralized precipitation of the cells; the specific experimental operations were referred to the kit instructions.

2.11 Gene Silencing

MC cells were seeded in six-well plates at a density of 1 × 10⁶ per well, and the designed si-RNA sequence (sense 5′-3′:GGUACAUCGACUUCCUGUATT and antisense 5′-3′:UACAGGAAGUCGAUGUACCTT for Twist1 silencing, sense 5′-3′:GCAGAGCAAUGUAUGUCUATT and antisense 5′-3′:UAGACAUACAUUGCUCUGCTT for PIK3CA silencing; purchased from Jima Gene Co., LTD., Suzhou, China) was transfected into the cells via lipo2000 according to the manufacturer's recommended conditions. After 4–6 h of transfection, the transfection medium was removed and the cells were re-cultured in a complete medium for24 h. Then the proteins were extracted for Western blot assay to detect the silencing of Twist1 and the expression changes of related proteins.

2.12 Dose–Response Cellular Thermal Shift Assay (CETSA)

Cellular Thermal shift assays (CETSA) can be used to understand the thermal stability of proteins upon binding to ligands. Briefly, the cells were heated to 65°C for 5 min after being treated with different concentrations of GA-A or solvent control, then the thermal stability of target proteins was detected via WB assay.

2.13 Statistical Analysis

GraphPad Prism 8.0 software was used for the statistical analysis of experimental data, and the results obtained by the analysis were expressed as mean ± standard deviation (SD). The student t-test was used for comparisons between any two different groups, one-way analysis of variance was used for more than two groups, and the resulting P value < 0.05 was considered statistically significant.

3.1 GA-A Dose-Dependently Reverses the Bone Loss and Changes in Bone Microstructure in the OVX Mice

To investigate the effects of Ganoderic acid A on osteoporosis, the femurs of the mice were selected for H&E staining, and the results showed that the bone tissue in the OVX group showed increased fat cavities, discontinuous bone trabeculae, and fracture of bone microstructure compared with the sham-operated group, indicating the bone loss and decreased osteogenesis in the OVX mice, and the model of osteoporosis was successfully established. In contrast, the internal cavities of the femur and the fracture of the bone microstructure were obviously improved in the Ganoderic acid A treatment groups (Figure1A). The results of the micro-CT examination of the femur showed that the bone mineral density of the femur in the OVX mice was significantly reduced and the bone microstructure was severely damaged (Figure1B). Among the specific bone microstructural measurements, bone mineral density (BMD), bone volume fraction (BV/TV), bone surface area and tissue volume ratio (BS/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were all found to be markedly decreased in the OVX group, which could be reversed by GA-A treatment (Figure1C). In contrast, parameters including the trabecular surface area to volume ratio (BS/BV), trabecular separation (Tb.Sp), and trabecular pattern factor (Tb.Pf) were increased obviously in the OVX group compared with the sham group and were decreased significantly after GA-A treatment (Figure1D).

The activity of ALP detecting results showed that compared with the sham group, the activity of ALP in the OVX group was significantly increased, indicating the increased bone loss (Llorente-Pelayo etal.2020). However, the activity of ALP was gradually decreased after GA-A intervention (Figure1E). In addition, in order to verify the oxidative stress damage caused by OVX and the antagonizing effects of GA-A, the oxidative stress-related kits were adopted to examine the oxidative stress-related markers of LDH, MDA, SOD levels. The results showed that the activities of LDH and MDA in the OVX group were significantly higher, while the activity of SOD was markedly lower than those in the sham group. However, after the administration of GA-A, the above oxidative stress-related markers were significantly reversed dose dependently, indicating the anti-oxidative effects of GA-A in preventing against OVX-induced osteoporosis (Figure1F).

Western blotting analysis of the protein expression of the osteogenic-related markers RUNX2, OPN, and β-catenin in the mice femur showed that the levels of all the osteogenic markers were decreased in the OVX group, but they were significantly increased after GA-A administration (Figure1G). Collectively, the above results indicated that GA-A could combat bone loss and changes in the bone microstructure in the OVX mice.

3.2 Network Pharmacology and Molecular Docking Analysis

Then the mechanisms of GA-A suppressing bone loss were investigated. Firstly, bioinformatics was used to analyze the targets of GA-A protecting against osteoporosis. Through Batman, SuperPred, and Swiss websites, the acting targets of Ganoderic acid A were obtained respectively, and the intersection was selected to obtain the Venn diagram (Figure2A). The targets of osteoporosis were obtained through Disgenet, OMIM, and Gene card websites, respectively, and the intersection was selected to obtain the Venn diagram (Figure2B). The Venn intersection results of GA-A and osteoporosis were performed to obtain the common targets between Ganoderic acid A and osteoporosis (Figure2C). The String database was used to construct a protein–protein interaction (PPI) of the common targets and 3 central targets were identified. According to the relevant literature, the effects of AR and CTNNB1 have already been studied well in the related studies (Huang etal.2023; Chen etal.2003; Liu etal.2019), while the effects of PIK3CA still remain unclear in osteoporosis (Figure2D,E). Moreover, the results of molecular docking showed that the binding energy of PIK3CA to GA-A was −7.12 kcal/mol, foreshadowing that this protein is likely to be the target of Ganoderic acid A against OP (Figure2F). Subsequently, we performed the Dose–Response CETSA experiment on the PIK3CA protein, and the results showed that GA-A could make the protein more stable, which proved that PIK3CA could bind with GA-A (Figure2G). Therefore, PIK3CA was selected for the subsequent mechanistic study for GA-A against osteoporosis.

3.3 Ganoderic Acid A Modulates the PIK3CA/p-Akt/TWIST1 Signaling Pathway in the OVX Mice

The feasibility of the above screening targets was first verified by Western blot invivo. The results showed that the protein expression of PIK3CA was significantly downregulated in the OVX group. However, it was up-regulated significantly after the administration of gradient Ganoderic acid A (Figure3A,B), which suggested that PIK3CA was possibly the acting target of GA-A and PIK3CA was positively related to osteoblastic differentiation. Next, the Western blot results verified the decreased ratio of p-Akt/Akt and the increased protein expression of TWIST1 in the OVX mice, which could be reversed by GA-A treatment (Figure3C–E). Then the immunofluorescence experiments were used to confirm the result. The results demonstrated consistent results with the protein expressions in Western blot analysis (Figure3F,G). These results further supported the feasibility of GA-A acting on the PIK3CA/p-Akt/TWIST1 signaling pathway to prevent bone loss induced by OVX.

3.4 GA-A Promotes Osteogenic Differentiation in the MC3T3-E1 Cells and Primary Osteoblasts

MC cell line and primary osteoblasts were used to verify the effects of GA-A promoting osteogenic formation invitro, and H₂O₂ was used to induce oxidative stress injury in the cells (Sies2017). According to the results of the cck8 assay, the concentration of H₂O₂ used in the MC cells was 1 mM, and in POB was 250 μM. Also, 5, 20, and 80 μM of GA-A were used for the following study (Figure4A,B).

In MC cells, low, medium, and high doses of GA-A were given for 24 h, followed by 4 h of H₂O₂ exposure. Then the cells were induced for 14 days to detect alkaline phosphatase (ALP) activity by ALP staining and an ALP detecting kit. Alizarin red (ARS) staining was performed 21 days after induction. The results of the ALP assay showed that compared with the control group, the number of dark staining nodules of ALP was significantly reduced and the ALP activity was significantly decreased in the H₂O₂- induced group; however, GA-A could increase the number of dark staining nodules and ALP activity in a dose-dependent manner. Alizarin red results also showed that the number of mineralized nodules in the model group was significantly reduced, and the color became lighter. Remarkably, in the GA-A treatment group, the number of mineralized nodules was obviously increased and the color was deepened in a dose-dependent manner (Figure4C–E). Consistent with the invivo results, the protein expressions of osteogenic related markers RUNX2, OPN, and β-catenin in both cell lines were decreased after exposure to H₂O₂ detected by Western blot analysis, whereas GA-A acted in an opposite way (Figure4 F, G). What is more, when compared with estrogen (E2), an approved medicine used for the treatment of postmenopausal OP, a high dose of GA-A (80 μM) exhibited almost the same effects on osteogenic related markers (Figure4H). These results implied that GA-A could promote osteogenic formation in H₂O₂-induced MC3T3-E1 cells and primary osteoblasts.

3.5 Effects of GA-A on the PIK3CA/ Akt/TWIST1 Signaling Pathway InVitro

The expression of the target proteins in MC cells and POB was also detected by Western blotting. The results showed that the changes in the target proteins were consistent in both cell lines. Compared with the control group, PIK3CA and p-Akt protein expressions in the model group decreased, and they were increased in a dose-dependent manner after GA-A treatment; However, the protein expression of TWIST1 was contrary to that of PIK3CA (Figure5A,B). In order to further verify the expression changes of the above signal proteins, the fluorescence intensity of PIK3CA and TWIST1 was then identified by immunofluorescence staining. The results showed that the fluorescence intensity of both proteins was consistent with the invivo immunofluorescence results, with a decrease in the fluorescence intensity of PIK3CA and an increase in the fluorescence intensity of TWIST1 in the H₂O₂-induced group, which was reversed by GA-A administration (Figure5C,D).

3.6 PIK3CA Promotes Osteogenic Differentiation via Increasing Akt Phosphorylation to Down-Regulate TWIST1 Expression in MC3T3-E1 Cells

To investigate the specific mechanism of GA-A activating PIK3CA signaling protein on bone formation, the si-PIK3CA silencing sequence was transfected into the MC cells. The silencing efficiency of PIK3CA in the cells was detected by Western blot and qPCR, and the result showed that the silencing efficiency was about 50% (Figure6A). After the knockdown of PIK3CA, the expression of p-Akt was also significantly decreased, indicating that PIK3CA could activate the PI3K/Akt signaling pathway to upregulate the protein expression of p-Akt (Figure6B,H).

Next, to verify the regulating effect of p-Akt on TWIST1, p-Akt expression was inhibited with the PI3K/Akt inhibitor (LY294002) (Figure6C). According to existing research, phosphorylation of TWIST1 by Akt is required for β-TrCP-mediated TWIST1 ubiquitination and degradation (Li etal.2016). Therefore, after the p-Akt protein was inhibited, we examined p-TWIST1 protein and TWIST1 protein and found that p-TWIST1 level was significantly decreased, while TWIST1 level was significantly increased, suggesting that p-Akt regulates TWIST1 by phosphorylating it for degradation (Figure6D).

Finally, to verify the effects of TWIST1 on bone formation, the si-TWIST1 silencing sequence was transfected into the cells by transfection technology, and the silencing efficiency of TWIST1 in the cells was detected by Western blot and qPCR. The result showed that the silencing efficiency was about 80% (Figure6E). Expectedly, after the knockdown of TWIST1, the expression of OPN and β-catenin in the si-TWIST1 group was significantly higher than that in the control group (Figure6F,R), confirming that TWIST1 negatively regulates osteogenic differentiation.

Some studies have found that the expression of the transcription factor TWIST1 is correlated with p-Akt (Yuan etal.2014; Inoue etal.2017). However, the specific relationship between the two factors has not been proven. To verify the relationship between p-Akt and TWIST, the Co-IP experiment was used to verify the interaction of the two factors. As shown in the results, p-Akt could act on TWIST1 to affect its expression, which was consistent with the experimental results of the input group (Figure6G).

Therefore, these results suggested that Ganoderic acid A could promote osteoblastic bone formation and protect against osteoporosis through activating PIK3CA and subsequently activating the PI3K/Akt signaling pathway to down-regulate TWIST1 expression.