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Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway

  • Authors:
    • Yiwei Zhao
    • Zechao Qu
    • Lin Liu
    • Yong Zhang
    • Xiaohao Wang
    • Bo Zhang
    • Yining Gong
    • Liang Yan
  • View Affiliations / Copyright

    Affiliations: Department of Spine Surgery, Honghui Hospital, Xi'an Jiao Tong University, Xi'an, Shaanxi 710054, P.R. China, Department of Critical Care Medicine, Honghui Hospital, School of Medicine, Xi'an Jiao Tong University, Xi'an, Shaanxi 710054, P.R. China
    Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 187
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    Published online on: May 13, 2026
       https://doi.org/10.3892/ijmm.2026.5858
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Abstract

Osteoporosis (OP) is a systemic disease characterized by a reduction in the number of trabecular bone structures and damage to the bone microstructure. It is commonly found in people who are aging or have estrogen deficiency. Oxidative stress and chronic inflammation caused by pathological factors such as aging and estrogen deficiency are key pathogenic factors. Betulinic acid (BA), a natural pentacyclic triterpenoid compound, exhibits anti‑inflammatory and antioxidant biological effects. However, its role and potential mechanisms in the inflammatory injury of osteoblasts in OP remain unclear. In the present study, in vivo experiments were conducted using an ovariectomized (OVX) rat model of OP, with bone microstructure analyzed by micro‑CT, protein expression detected by immunohistochemistry, and serum inflammatory factors measured by ELISA. BA was revealed to alleviate bone loss in OVX rats and inhibit the expression of NOD‑like receptor pyrin domain‑containing 3 (NLRP3), Asc and caspase‑1 in the femur of OVX rats, as well as suppress the release of inflammatory factors such as interleukin‑1 β, interleukin‑6, and tumor necrosis factor‑αin the serum of rats. The inflammatory injury osteoblast model of BA intervention was also studied with hydrogen peroxide (H2O2) in vitro, with reactive oxygen species (ROS) levels assessed by fluorescence assay, osteogenic differentiation evaluated by ALP staining and alizarin red staining, and autophagy‑related proteins detected by western blotting. BA pretreatment reduced production of ROS, inhibited expression of NLRP3 and downstream pathway activation, improved alkaline phosphatase activity, mineralization ability, and osteogenic differentiation ability of MC3T3‑E1 cells. Administration of BA increased the autophagy of MC3T3‑E1 cells treated with H2O2, which was confirmed by the increased expression levels of LC3b II and Beclin‑1 and the decreased expression levels of P62. In addition, BA could enhance the phosphorylation of AMPK in MC3T3‑E1 cells treated with H2O2 and reduce the phosphorylation of mTOR, but this effect could be rescued by Compound C (an AMPK blocker). BA can protect osteoblasts from inflammatory injury by reducing the production of ROS and inhibiting the activation of NLRP3 through autophagy mediated by the AMPK/mTOR pathway.
View Figures

Figure 1

Protective effect of BA on
OVX-induced bone loss in rats. (A) Flow chart representing the
study design protocol to evaluate the therapeutic effect of BA on
OVX rats. (B) Representative images of femurs in each group of rats
detected by micro-CT. (C-F) Quantitative analyses of bone
structural parameters: BMD, BV/TV, Tb.Sp and Tb.N. (G) Masson's
staining and H&E staining of metaphyseal tissue sections of
femurs. Scale bar, 200 μm. Data are expressed as the mean ±
SEM. **P<0.01 vs. sham group; #P<0.05
and ##P<0.01 vs. corresponding OVX group. BA,
betulinic acid; OVX, ovariectomized; BMD, bone mineral density;
BV/TV, bone volume fraction; Tb.N., trabecular number; Tb.Sp,
trabecular separation; IHC, immunohistochemical.

Figure 2

Protective effect of BA on
OVX-induced inflammation in rats. (A) Immunohistochemical analysis
was performed to detect the expression of NLRP3, Asc and Caspase 1
in rat femoral epiphyseal tissue. Scale bar, 100 μm. (B)
Positive areas of NLRP3, Asc and Caspase 1 in rat femoral
epiphyseal tissue. (C) The expression levels of IL-1β, IL-6 and
TNF-α in rat serum. Data are expressed as the mean ± SEM.
*P<0.05 and **P<0.01 vs. sham group;
##P<0.01 vs. corresponding OVX group. BA, betulinic
acid; OVX, ovariectomized; NLRP3, NOD-like receptor pyrin
domain-containing 3.

Figure 3

H2O2-induced
osteogenic differentiation decline in MC3T3-E1 cells related to
ROS/NLRP3. (A) MC3T3-E1 was cultured with ROS inhibitor NAC (20
μM) or NLRP3 inhibitor MCC950 (100 μM) in a medium
containing H2O2 (200 μM) to produce
ROS, and then intracellular ROS was detected by DCFH-DA. Scale bar,
100 μm. (B) The average relative DCF fluorescence intensity
on each pore cell was evaluated and quantified. (C) The protein
levels of NLRP3 were determined by western blotting. (D)
Quantification of the results shown in C. (E and F) Quantitative
analysis of the expression of osteoblast marker mRNA in osteoblasts
treated with NAC or MCC950 with or without
H2O2 by reverse transcription-quantitative
PCR. (G) The protein levels of related markers in the process of
osteoblast induction were determined by western blotting. (H and I)
Quantification of the results shown in G. Data are expressed as the
mean ± SEM. **P<0.01 vs. control group;
#P<0.05 and ##P<0.01 vs. corresponding
H2O2 group; &P<0.01 vs.
corresponding H2O2 group. ROS, reactive
oxygen species; NAC, N-acetyl-L-cysteine; NLRP3, NOD-like receptor
pyrin domain-containing 3; RUNX2, runt-related transcription factor
2.

Figure 4

BA attenuates NLRP3-induced
inflammatory injury and osteogenic differentiation decline in
H2O2-exposed MC3T3-E1 cells. (A) The chemical
structure of BA. (B) MC3T3-E1 cells were treated with different
concentrations of BA (0, 5, 10, 20, 30 and 40 μM) for 24 and
48 h. Cell viability was measured by Cell Counting Kit-8 assay. (C)
MC3T3-E1 cells were incubated in the presence of
H2O2 (200 μM) medium with the BA (10
μM) or BA (20 μM), western blot analysis was
performed against NLRP3, Asc, caspase-1, cleaved caspase-1 and
IL-1β. (D-H) Quantification of the results shown in C. (I) ALP and
ARS staining were performed in MC3T3-E1 cells. (J and K)
Quantitative analysis of osteoblast marker gene expression after
treatment with different concentrations of BA using reverse
transcription-quantitative PCR. Data are expressed as the mean ±
SEM. **P<0.01 vs. control group;
#P<0.05 and ##P<0.01 vs. corresponding
H2O2 group. BA, betulinic acid; NLRP3,
NOD-like receptor pyrin domain-containing 3; ALP, alkaline
phosphatase; ARS, alizarin red staining; RUNX2, runt-related
transcription factor 2; N.S., not significant.

Figure 5

BA enhances autophagy in
H2O2-exposed MC3T3-E1 cells. (A) MC3T3-E1
cells were incubated in the presence of H2O2
(200 μM) medium with the BA (20 μM) or BA (20
μM) + 3-MA, western blot analysis was performed against
LC3b, beclin-1 and p62. (B-D) Quantification of the results shown
in A. (E) The expression of lc3b protein was detected by
immunofluorescence. (F) Quantification of the results shown in E.
(G) MDC detected the production of autophagosomes. Intracellular
reactive oxygen species were detected by DCFH-DA. (H and I)
Quantification of the results shown in G. Data are expressed as the
mean ± SEM. *P<0.05 and **P<0.01 vs.
control group; #P<0.05 and ##P<0.01 vs.
corresponding H2O2 group;
&P<0.05 and &&P<0.01 vs.
corresponding H2O2 + BA group. BA, betulinic
acid; MDC, monodansylcadaverine; 3-MA, 3-methyladenin; N.S., not
significant.

Figure 6

BA activates the AMPK/mTOR signaling
pathway in H2O2-exposed MC3T3-E1 cells. (A)
MC3T3-E1 cells were incubated in the presence of
H2O2 (200 μM) medium with the BA (20
μM) or BA (20 μM) + CC (AMPK inhibitor), western blot
analysis was performed against p-AMPK and AMPK, p-mTOR and mTOR. (B
and C) Quantification of the results shown in A. (D) Western blot
analysis was performed against LC3b, beclin-1 and p62. (E-G)
Quantification of the results shown in D. (H) The expression of
lc3b protein was detected by immunofluorescence. (I) Quantification
of the results shown in H. (J) MDC detected the production of
autophagosomes. (K) Quantification of the results shown in J. Data
are expressed as the mean ± SEM. #P<0.05 and
##P<0.01 vs. corresponding H2O2
group; &P<0.05 and &&P<0.01
vs. corresponding H2O2 + BA group. BA,
betulinic acid; CC, compound c; p-, phosphorylated; MDC,
monodansylcadaverine; N.S., not significant.

Figure 7

Schematic illustration of the working
model demonstrating that betulinic acid activates autophagy via the
AMPK/mTOR pathway to inhibit ROS production, thereby suppressing
NLRP3 inflammasome activation and alleviating inflammatory damage
to osteoblasts. ROS, reactive oxygen species; NLRP3, NOD-like
receptor pyrin domain-containing 3.
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Copy and paste a formatted citation
Spandidos Publications style
Zhao Y, Qu Z, Liu L, Zhang Y, Wang X, Zhang B, Gong Y and Yan L: Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway. Int J Mol Med 58: 187, 2026.
APA
Zhao, Y., Qu, Z., Liu, L., Zhang, Y., Wang, X., Zhang, B. ... Yan, L. (2026). Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway. International Journal of Molecular Medicine, 58, 187. https://doi.org/10.3892/ijmm.2026.5858
MLA
Zhao, Y., Qu, Z., Liu, L., Zhang, Y., Wang, X., Zhang, B., Gong, Y., Yan, L."Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway". International Journal of Molecular Medicine 58.1 (2026): 187.
Chicago
Zhao, Y., Qu, Z., Liu, L., Zhang, Y., Wang, X., Zhang, B., Gong, Y., Yan, L."Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway". International Journal of Molecular Medicine 58, no. 1 (2026): 187. https://doi.org/10.3892/ijmm.2026.5858
Copy and paste a formatted citation
x
Spandidos Publications style
Zhao Y, Qu Z, Liu L, Zhang Y, Wang X, Zhang B, Gong Y and Yan L: Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway. Int J Mol Med 58: 187, 2026.
APA
Zhao, Y., Qu, Z., Liu, L., Zhang, Y., Wang, X., Zhang, B. ... Yan, L. (2026). Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway. International Journal of Molecular Medicine, 58, 187. https://doi.org/10.3892/ijmm.2026.5858
MLA
Zhao, Y., Qu, Z., Liu, L., Zhang, Y., Wang, X., Zhang, B., Gong, Y., Yan, L."Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway". International Journal of Molecular Medicine 58.1 (2026): 187.
Chicago
Zhao, Y., Qu, Z., Liu, L., Zhang, Y., Wang, X., Zhang, B., Gong, Y., Yan, L."Betulinic acid alleviates the inflammatory injury of osteoblasts in osteoporosis by augmenting autophagy via the AMPK-mTOR signaling pathway". International Journal of Molecular Medicine 58, no. 1 (2026): 187. https://doi.org/10.3892/ijmm.2026.5858
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