Ganoderic Acid A Prevented Osteoporosis by Modulating the PIK3CA/p-Akt/TWIST1 Signaling Pathway

Abstract

Osteoporosis is a disorder of decreased bone mass, microarchitectural deterioration, and fragility fractures. Ganoderma lucidum has been reported to have a variety of pharmacological activities, including immune regulation, anti-inflammation, antioxidation, sedative hypnosis, blood sugar and lipid regulation, and so on. However, the effective ingredients and the underlying mechanism of Ganoderma lucidum against osteoporosis are rarely clarified. Ganoderic acid A (GA-A), a triterpenoid, is one of the main components of Ganoderma lucidum. Our previous preliminary bioinformatic study found that it may affect bone metabolism, and it has been reported that GA-A has anti-osteoporosis potential via regulating MC3T3-E1 cells' osteogenic differentiation activity. Therefore, the aim of this study is to investigate the effects of Ganoderic acid A in preventing osteoporosis and uncover the potential mechanisms. In vivo, the 8-week-old C57BL/6J female mice were used to establish the osteoporosis model by ovariectomy (OVX). Two cell lines, MC3T3-E1 cells and primary osteoblasts, were used and induced with hydrogen peroxide (H₂O₂) to the state of oxidative stress in osteoporosis in vitro. We showed that Ganoderic acid A could inhibit OVX-induced bone loss in a dose-dependent manner and promote H₂O₂-induced osteogenic differentiation of primary osteoblasts and MC3T3-E1 cells. The mechanism-related signaling pathways were identified by network pharmacology screening and verified by bioinformatics. Results predicted that the target of Ganoderic acid A might be PIK3CA. Mechanistically, we found that PIK3CA activated the Akt receptor, then inhibited the expression of TWIST1 in the osteoblasts to up-regulate the protein expression of the osteogenic-related markers. Our results suggested that Ganoderic acid A could prevent OVX-induced osteoporosis and promote H₂O₂-induced osteogenic differentiation of primary osteoblasts and MC3T3-E1 cells. Ganoderic acid A might play an important role in the prevention of osteoporosis by modulating the PIK3CA/p-Akt/TWIST1 signaling pathway.

Keywords: PIK3CA; TWIST1; ganoderic acid a; network pharmacology; osteoporosis; p‐Akt.

FIGURE 1

GA‐A inhibited bone loss in OVX mice. (A) Representative images of H&E staining. (B) Representative 3D micro‐CT analysis images of the femur were reconstructed. (C) The BMD, BV/TV, BS/TV, Tb. Th, Tb. N values of micro‐CT analysis were evaluated. (D) BS/BV, Tb. Sp, and Tb. Pf values of micro‐CT analysis were evaluated. (E) Effect of GA‐A on serum ALP activity of mice. (F) GA‐A inhibited OVX‐induced increase in LDH and SOD; and inhibited OVX‐induced decrease in MDA. (G) Protein expression of osteogenesis‐related markers RUNX2, OPN, and β‐catenin in mice. n = 6, #p < 0.05, ##p < 0.01 and ###p < 0.001 compared with the control group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the OVX group.

FIGURE 2

The workflow for screening target genes using the network pharmacology and computational bioinformatics analysis approach. (A) Identification of GA‐A‐related targets. (B) Identification of OP‐related targets. (C) Obtaining 20 GA‐A–OP‐related targets. (D) PPI network construction and analysis. (E) Analysis of the 3 center targets in the PPI subnetwork. (F) Molecular docking of the PIK3CA center target in the PPI subnetwork. (G) Dose–Response CETSA Experiment on the PIK3CA protein.

FIGURE 3

Validation of GA‐A's therapeutic target for OP. (A, B) Putative target PIK3CA validation by Western blot analysis. (A, C, D, E) Detection of p‐Akt, TWIST1, and p‐TWIST1 indicators by western blot. (F, G) Fluorescence intensity visualization of PIK3CA and TWIST1 production by fluorescent microscope n = 6, # p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the control group. *p < 0.05 and **p < 0.01 compared with the OVX group (scare bar = 100 μm).

FIGURE 4

GA‐A suppressed H₂O₂‐induced inhibition of osteogenesis differentiation and mineralization in MC3T3‐E1s and primary osteoblasts. (A, B) CCK‐8 analysis. (C) ALP staining and (D) ALP activity detection. (E) ARS staining of MC3T3‐E1s. (F, G) Protein expression of osteogenesis‐related markers RUNX2, OPN, and β‐catenin in MC3T3‐E1s and primary osteoblasts. n = 6, #p < 0.05, ##p < 0.01 and ###p < 0.001 compared with the control group. *p < 0.05 and **p < 0.01 compared with the H₂O₂ group. (H) qPCR assay of RUNX2, OPN, BMP2, and β‐catenin after treated with E2 or GA‐A in MC3T3‐E1 (*p < 0.05 and **p < 0.01 compared with the control group. #p < 0.05 and ##p < 0.01 compared with the H₂O₂ group).

FIGURE 5

Validation of GA‐A's therapeutic target for OP in MC3T3‐E1s and primary osteoblasts. (A) Putative target PIK3CA, p‐Akt, TWIST1, and p‐TWIST1 validation by Western blot in MC3T3‐E1s. (B) Putative target PIK3CA, p‐Akt, TWIST1, and P‐TWIST1 validation by Western blot in primary osteoblasts. (C, D) Fluorescence intensity visualization of PIK3CA and TWIST1 production by fluorescent microscope in MC3T3‐E1 cells. n = 6, # p < 0.05, ##p < 0.01 and ###p < 0.001 compared with the control group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the H₂O₂ group.

FIGURE 6

The mechanism of PIK3CA's inhibiting for OP. (A, B) Knockdown of PIK3CA by siRNA and detection of PIK3CA and p‐Akt indicators by Western blot. n = 6, ***p < 0.001 compared with the si‐Control group. (C, D) PI3K/Akt inhibitor (LY294002) inhibits p‐Akt and detection of p‐Akt, TWIST1, and p‐TWIST1 indicators by Western blot. n = 6, ***p < 0.001 compared with the control group. (E, F) Knockdown of TWIST1 by siRNA and detection of osteogenesis‐related indicators by Western blot. n = 6, ***p < 0.001 compared with the si‐Control group. (G) Detection of the protein interaction of p‐Akt with TWIST1 by Co‐IP western blot. (H) qPCR assay of PIK3CA and Akt after PIK3CA knocking down (n = 4, **p < 0.001 compared with the control group). (I) qPCR assay of β‐catenin and OPN after Twist1 knocking down (n = 4, **p < 0.001 compared with the control group).