Cryptotanshinone Suppressed Postmenopausal Osteoporosis by Preventing RANKL-Mediated Osteoclastogenesis against Kidney Injury

Abstract

Background: Cryptotanshinone (CPT), an active component extracted from the root of Salvia miltiorrhiza Bunge, exhibits extensive favorable bioactive properties including anti-inflammatory, antioxidative, antibacterial, and antitumor effects. This study aims to investigate the effects of CPT on osteogenesis and explore related mechanisms both in vivo and in vitro.

Methods: In the in vivo experiment, ovariectomized (OVX) female rats were intragastrically administered with CPT at doses of 10 mg/kg and 20 mg/kg for 13 consecutive weeks. Dual-energy X-ray absorptiometry was employed to detect bone mineral density (BMD). ELISA assay was leveraged to detect the biochemical parameters such as BUN and creatinine in the kidney samples. Bone and kidney sections were dyed by H&E and Masson staining kits. In the in vitro experiment, the RAW 264.7 cells were stimulated through the receptor activation of the nuclear factor kappa B ligand (RANKL) to establish an osteoclast differentiation model, and CPT's protective effect against bone loss was evaluated. Differentiated osteoclasts were determined by TRAP staining. While, osteoclast-marker proteins such as NFATc1, c-Fos, and cathepsin K were identified by Western blot.

Results: The results from in vivo experiments revealed that CPT could elevate bone mass and increase bone formation markers in OVX rats. Intriguingly, CPT administration noticeably ameliorated the kidney injury in OVX rats by suppressing BUN and restoring creatinine levels. Furthermore, the results from in vitro experiments suggested that CPT downregulated the protein expression of osteoclast-associated genes such as cathepsin K, c-Fos, and NFATc1 which hinted the related potential mechanisms.

Conclusion: The evidence from in vivo and in vitro experiments suggested that CPT exerted antiosteoclastogenic effects by inhibiting the activation of osteoclast differentiation followed by suppressing the protein expressions of cathepsin K, c-Fos, and NFATc1 in osteoclast precursors, and it exhibited protective effects against kidney damage, which highlighted its advantage in clinical application.

Figure 1

Effect of CPT on OVX-induced bone microarchitecture. (a) Data from network pharmacology analysis. The rats were randomly divided into four groups (n = 10) and treated as described in the section of Materials and Methods: Sham group, OVX group, and OVX groups intragastrically receiving CPT at 10 and 20 mg/kg/day, respectively. (b) Representative micro-CT images. Femur surfaces were color coded to indicate the local thickness (0–0.800 mm). Characteristics of bone microarchitecture in femur including (c) trabecular separation (Tb.Sp,mm), (d) trabecular thickness (Tb.Th, mm), (e) trabecular number (Tb.N, mm−1), (f) bone mineral density (BMD, g/m3), (g) bone mineral content (BMC, mg), and (h) bone volume fraction(BV/TV%) were detected.

Figure 2

Effect of CPT on OVX-induced bone loss. (a) Morphological changes of bone loss in femur per group detected by H&E staining(magnification ×40). (b) Masson staining for femur (magnification ×40). Remarkable changes are marked in the black box.

Figure 3

Effect of CPT on OVX-induced kidney failure. The kidneys were isolated from rats of the Sham, OVX, and OVX rats with CPT (10 and 20 mg/kg/day) groups. (a) Morphological change in glomerulus via H&E staining. The releasing of (b) BUN and (c) creatinine detected by ELISA kits. The black arrow points to atrophic glomeruli.

Figure 4

(a) CPT exhibiting no toxicity to RAW 264.7 and reversed differentiation induced by RANKL. First, RAW 264.7 cells were cultured for toxicology trial with different concentrations of CPT (0, 0.1, 1, 10, and 100 µM). (b) Multinucleated cells with more than five nuclei, which were considered mature osteoclasts, observed under a light microscope after RANKL treatment, and they were suppressed by CPT cotreatment.

Figure 5

Role of osteoclast differentiation pathway in the process of CPT treatment-attenuated osteoclast differentiation. RAW 264.7 cells were pretreated with CPT (1, 10 μM) for 1 h in response to RANKL (50 ng/mL) for 5 days, and the expression of NFATc1, c-Fos, and cathepsin K was detected by Western blotting.