Lansoprazole-induced osteoporosis via the IP3R- and SOCE-mediated calcium signaling pathways

Abstract

Background: Many clinical studies have shown a correlation between proton pump inhibitors (PPIs) and osteoporosis or fractures. The purpose of this study was to establish a murine model of chronic oral PPI administration to verify whether PPIs caused bone metabolic impairment and investigate the relevant molecular mechanism underlying the effects of PPIs on MC3T3-E1 murine osteoblasts.

Methods: A lansoprazole-induced bone loss model was used to investigate the damaging effects of PPIs. In vivo, immunohistochemistry, Hematoxylin-Eosin (HE) staining, micro-CT analysis, and blood biochemical analyses were used to evaluate the effect of lansoprazole on bone injury in mice. In vitro, the effects of lansoprazole and related signaling pathways in MC3T3-E1 cells were investigated by CCK-8 assays, EdU assays, flow cytometry, laser confocal microscopy, patch clamping, reverse transcription-quantitative polymerase chain reaction and Western blotting.

Results: After 6 months of lansoprazole gavage in ICR mice, the micro-CT results showed that compared with that in the vehicle group, the bone mineral density (BMD) in the high-dose group was significantly decreased (P < 0.05), and the bone microarchitecture gradually degraded. Biochemical analysis of bone serum showed that blood calcium and phosphorus were both decreased (P < 0.01). We found that long-term administration of lansoprazole impaired skeletal function in mice. In vitro, we found that lansoprazole (LPZ) could cause calcium overload in MC3T3-E1 cells leading to apoptosis, and 2-APB, an inhibitor of IP3R calcium release channel and SOCE pathway, effectively blocked increase in calcium caused by LPZ, thus protecting cell viability.

Conclusions: Longterm administration of LPZ induced osteoporotic symptoms in mice, and LPZ triggered calcium increases in osteoblasts in a concentration-dependent manner. Intracellular calcium ([Ca²⁺]_i) persisted at a high concentration, thereby causing endoplasmic reticulum stress (ERS) and inducing osteoblast apoptosis.

Keywords: Calcium overload; ER stress; IP3R; Lansoprazole; Osteoporosis; SOCE.

Fig. 1

After 6 months of LPZ gavage, the right femurs of male and female mice were scanned by micro-CT to calculate BMD (n = 10); the remaining mice were randomly sacrificed, and blood was collected to examine bone and serum biochemical indicators (n = 10). A, B Femoral biomechanical strength properties. C BMD of the right femur. D–F Bone serum biochemical parameters

Fig. 2

Representative microcomputed tomography (micro-CT) images of the distal femurs. A Micro-CT images of femur specimens showed a reduction in trabecular bone microarchitecture after treatment with increasing doses of LPZ (red box). B Micro-CT images of cancellous bone structures near the growth plate (n = 7)

Fig. 3

Cell proliferation, viability and apoptosis were examined by CCK-8 assays, EdU assays and flow cytometry. A MC3T3-E1 cells were treated with increasing concentrations of LPZ, and the EdU assay showed a reduction in proliferation. B Apoptosis in osteoblasts pretreated with LPZ (0, 5, 10, 20, 50 µM) for 24 h. C Number of cells, as determined by the EdU assays. D Quantitative analysis of apoptotic rates. E The viability of MC3T3-E1 cells after LPZ treatment. *P < 0.05, **P < 0.01 (n = 3)

Fig. 4

Calcium regulation in MC3T3-E1 cells was observed by confocal microscopy. A Representative confocal images (40 ×): After LPZ administration, intracellular calcium fluorescence increased significantly. B Quantification of Fluo-3 fluorescence: Ca²⁺ fluorescence was slightly higher with calcium than without calcium. C Area under the curve of Fluo-3 fluorescence: There was no significant difference in the area under the curve of calcium fluorescence with or without calcium. D Fluo-3 fluorescence after preincubation with TG (n = 3): After preincubation with TG to deplete calcium stores, LPZ did not cause a significant increase in cellular calcium fluorescence. E The viability of MC3T3-E1 cells after preincubation with BAPTA/AM and LPZ treatment with CCK-8: BAPTA prevented the increase in Ca²⁺ and could effectively protect cell viability

Fig. 5

Relative calcium fluorescence change curves. A Relative [Ca²⁺]_i response in MC3T3-E1 cells that were preincubated with 2-APB in Ca²⁺-free medium. B Relative [Ca²⁺]_i response in MC3T3-E1 cells that were preincubated with ryanodine in Ca²⁺-free medium. C Relative [Ca²⁺]_i response in MC3T3-E1 cells that were preincubated with verapamil in Ca²⁺-containing medium. D Relative [Ca²⁺]_i response in MC3T3-E1 cells that were preincubated with BTP-2 in Ca²⁺-containing medium (n = 3)

Fig. 6

Representative confocal images of MC3T3-E1 cells. A MC3T3-E1 cells were double-loaded with ER tracker (red) and Mag-fluo4/AM (green) to determine ER Ca²⁺ levels. B MC3T3-E1 cells were double-loaded with MitoTracker Green and Rhod-2AM (red) to determine mitochondrial Ca²⁺ levels. (20×)

Fig. 7

[Ca²⁺]_i after longterm treatment with LPZ. A [Ca²⁺]_i levels in MC3T3-E1 cells that were preincubated with BTP-2 in calcium-containing medium, as detected by flow cytometry. B [Ca²⁺]_i levels in MC3T3-E1 cells that were preincubated with TG in calcium-free medium, detected by flow cytometry. C Quantitative analysis of [Ca²⁺]_i levels. (n = 3)

Fig. 8

[Ca²⁺]_i after longterm treatment with LPZ and TG. A [Ca²⁺]_i levels in MC3T3-E1 cells that were preincubated with TG in calcium-containing medium, as detected by flow cytometry. B Quantitative analysis of [Ca²⁺]_i after treatment with LPZ or TG for 24 h (n = 3)

Fig. 9

Patch clamp recording of currents. A The recorded current was determined to be NCX. B Initial induced current of NCX and the effect of different lansoprazole concentrations and Ni²⁺ on I_NCX; gray: 5 mM NiCl₂; brown: 0 µM lansoprazole; blue: 10 µM lansoprazole; black: 50 µM lansoprazole; red: 100 µM lansoprazole. C NCX current peak statistics of MC3T3-E1 cells in response to different lansoprazole concentrations; forward current density peak statistics, ^#P < 0.05 vs. the normal group; reverse current density peak statistics, *P < 0.05 vs. the normal group, **P < 0.01 vs. normal group. (x + s, n = 6)

Fig. 10

The mRNA expression of OB functional genes and ER stress and apoptosis pathway-related genes (ALP, OCN, Runx2, COLΙα, Caspase-12, ATF4). TG was used as a positive control (n = 3)

Fig. 11

The expression of OB functional genes and ER stress- and apoptosis pathway-related proteins. A Western blot analysis after LPZ, 2-APB, and TG treatment. B Semiquantitative analysis of protein levels in MC3T3-E1 cells

Fig. 12

Effects of LPZ on Ca²⁺-ATPase in MC3T3-E1 cells. A LPZ reduced Ca²⁺-ATPase activity. B LPZ inhibited plasma membrane Ca²⁺-ATPase gene expression (n = 3)

Fig. 13

HE staining of femurs and the expression of CHOP after intragastric administration of LPZ for 6 months (n = 3, Scale = 2 mm). A Mice treated with increasing doses of LPZ showed bone damage, as determined by HE staining. B Mouse femur immunohistochemical analysis of CHOP (n = 3, Scale = 200 µm). C Semiquantitative calculation of CHOP was performed with ImageJ

Fig. 14

Possible mechanisms underlying the changes in [Ca²⁺]_i