Effect of LPZ on bone density, serum biochemical indexes and femoral biomechanics property in mice
After continuous gavage administration for 6 months, there was no statistical difference in mouse weight among groups. Compared with the CMC-Na group, the low dose group and high dose group mice showed a decreasing trend in the maximum bone load (maximum breaking force) and bone structure strength (maximum crushing force) in femoral samples of material mechanical parameters in a dose-dependent manner (Fig. 1A, B). Furthermore, the low dose group (250 mg/kg) and high dose group (1000 mg/kg) mice showed a decreasing trend in femoral BMD, and there was a significant difference between the control group and high dose group (Fig. 1C). Bone serum biochemical results showed that serum-ALP (S-ALP) decreased slightly, serum-calcium (S-Ca) and serum-phosphorus (S-IP) decreased significantly (Fig. 1D, E, F). Similarly, compared with the CMC-Na group, trabecular bone micro-architecture was gradually thin and loose (Fig. 2).
Effect of LPZ on MC3T3-E1 cells viability
After discovering that LPZ had a damaging effect on mouse bone tissue, MC3T3-E1 cells, as a common osteoblast cell line, we used it to explore the mechanism of LPZ causing bone damage. First, we used an EDU kit to determine whether the LPZ promoted/inhibited MC3T3-E1 cells proliferation. The proportion of proliferating cells decreased significantly (P < 0.05) when cells were treated with LPZ at concentrations of 5, 10, 20, 50 µΜ (Fig. 3A, C). Next, we further explored whether the elevation of [Ca2+]i by LPZ could injure MC3T3-E1 cells through the apoptotic mechanism, MC3T3-E1 cells were incubated in α-MEM/10% FBS containing different concentrations (0, 5, 10, 20, and 50 μM) of LPZ and or none pre-incubated 10 µM BAPTA/AM for 30 min respectively. The apoptosis rate in the LPZ treatment group was increased in a dose-manner (Fig. 3B, D). Furthermore, LPZ could inhibit the viability of MC3T3-E1 cells in a dose-dependent manner (Fig. 3E). Above all, these results suggested that LPZ inhibited the viability of MC3T3-E1 cells
Calcium responses after LPZ treatment in MC3T3-E1 cells
1. LPZ induced calcium increase through ER Ca2+ release
We used confocal laser scanning microscopy technology to investigate calcium transient after LPZ treatment in MC3T3-E1 cells in real-time. We found that calcium fluorescence significantly increased in MC3T3-E1 cells treated with 50 µM LPZ (Fig. 4A). Moreover, the elevation of [Ca2+]i was slightly higher in calcium-contained solution than in calcium-free solution (Fig. 4B), but there was no significant difference between the two groups (Fig. 4C), which indicated that [Ca2+]i originated from both intracellular calcium release and extracellular calcium influx, and intracellular calcium release was mainly required for LPZ-induced [Ca2+]i response in MC3T3-E1 cells. Thapsigargin (TG) is an inhibitor of SERCA with high specificity. MC3T3-E1 cells were pre-incubated with TG in calcium-free solution to empty calcium storage in ER, and then treated with 50 µM LPZ, but there was almost no longer calcium transient in MC3T3-E1 cells (Fig. 4D). These findings suggested that LPZ-mediated calcium signaling in MC3T3-E1 cells was mainly from ER calcium release. Futhermore, we pre-incubated BAPTA/AM to chelate intracellular free Ca2+, and found MC3T3-E1 cells survival viability was significantly up-regulated (P < 0.05). LPZ inhibited the viability of MC3T3-E1 cells, however, the viability of MC3T3-E1 cells preincubated with BAPTA/AM was significantly higher than those without BAPTA/AM preincubation, which indicated that [Ca2+]i increase was an important factor for LPZ-induced cells impairment, and BAPTA/AM could effectively protect cell viability by blocking this process (Fig. 4E).
2. LPZ-induced calcium increases through IP3R and SOCE pathway
Cells were pre-incubated with 2-APB (25 µM) or Ryanodine (20 µM) in calcium-free medium, blocking the IP3R Ca2+ release channel and SOC Ca2+ entry pathway, or Ryr Ca2+ release channel respectively. Then cells were treated with 10 µM LPZ (clinically relevant concentrations). It turned out that Ryanodine had little effect on [Ca2+]i activated by LPZ (Fig. 5B), but 2-APB almost made the calcium fluorescence intensity consistent with the vehicle group (Fig. 5A). In addition, in order to confirm which calcium channel is responsible for the calcium influx, MC3T3-E1 cells pre-incubated with verapamil (10µM), a L-type calcium channel blocker(Jung et al. 2015), or BTP-2 (20µM), store-operated calcium (SOC) channel in calcium-contained medium, then cells treated with 10 µM LPZ. It was found that BTP-2 could partly restrain calcium fluorescence enhancement (Fig. 5D), however, verapamil had little influence on [Ca2+]i (Fig. 5C). Taken together, these results above revealed LPZ-mediated calcium transient mainly from ER released by IP3R channel and could promote the activation of SOCE.
BTP-2 in Ca2+-contained medium (n = 3).
3. LPZ induced a decrease in [Ca2+]ER and an increase [Ca2+]mito
Furthermore, to investigate whether LPZ had an effect between ER and mitochondria, MC3T3-E1 cells were loaded with Mag-fluo4/AM and Rhod-2/AM probe to identify the calcium levels in ER and mitochondria respectively. As we expected, ER Ca2+ fluorescence decreased after LPZ (10 µM) was added, meanwhile Ca2+ in mitochondria slightly increased. (Fig. 6)
Assessment of calcium in flow cytometry
1. Elevated calcium in long term LPZ exposed MC3T3-E1 cells
Then we used flow cytometry to investigate the intracellular calcium changes after long-term treatment (24 h) of LPZ in MC3T3-E1 cells. MC3T3-E1 cells were pre-incubated with TG (2 μM) in calcium-free medium, or pre-incubated with BTP-2 (20 μM) in calcium-contained medium. Then MC3T3-E1 cells were added with 50 μM LPZ for another 1 h. It was found that [Ca2+]i was significantly higher either in calcium-contained or free medium than the vehicle group (0.1% DMSO). Furthermore, the level of calcium elevation in MC3T3-E1 cells pre-incubated with BTP-2 under calcium-contained conditions was not statistically different from that cells treated with LPZ only under calcium-free conditions. However, taking vehicle as reference, the elevation of [Ca2+]i was significantly lower when MC3T3-E1 cells were pre-incubated with TG under calcium-free conditions (Fig. 7).
2. [Ca2+]i remained at a high level after LPZ exposed in MC3T3-E1 cells
We selected TG (2 μM) and LPZ (50 µM) in calcium-contained medium for 24 h. It was found that the intracellular calcium level remained high in the LPZ group, but the intracellular calcium level was low in the TG group, and there was no significant difference between the TG and the vehicle group. These reminded us that LPZ may have an inhibitory effect on SERCA. When MC3T3-E1 cells were treated with TG, TG could not make [Ca2+]i remain at a high level for a long time, and Ca2+ would be expelled by PMCA and NCX on the plasma membrane in time. However, LPZ could keep [Ca2+]i at a high level for a long time, LPZ might inhibit calcium efflux transporters. Ca2+ stress seems to be more associated with long-term stress conditions, thus persistent Ca2+ elevation triggered cell apoptosis. We hypothesized that LPZ also had an inhibitory effect on PMCA and/or NCX, so that LPZ-treated cells remained calcium overloading after 24 h (Fig. 8). (n = 3)
NCX current changes
In osteoblasts, PMCA and NCX are responsible for calcium balance. We examined the effect of LPZ on NCX transporter using the patch clamp technique. It was shown that the 10 µM LPZ increased the reverse current density from -3.29 ± 1.00 pA/PF (0 µM) to -5.62 ± 1.46 pA/pF by 70.91%, the current density was 18.9 ± 5.96 pA/pF by 475.10% at 50 µM, and the current density 100 µM was -5.21 ± 2.33 pA/pF by 58.52% at 100 µM (Fig. 9). Taken together, patch clamp results showed that 10, 50, and 100 µM lansoprazole could significantly strengthen the reverse current (forward mode) of Na+-Ca2+ exchange current in MC3T3-E1 cells (P < 0.05). It indicated that at the concentration of 10, 50, 100 µM LPZ, Ca2+ could be transported out through Na+ - Ca2+ exchangers. We hypothesized that lansoprazole might stimulate Ca2+ overload to prompt an increase in sodium-calcium exchanger activity in order to uphold a low intracellular calcium balance.
Effects of LPZ on MC3T3-E1 mRNA expression levels
MC3T3-E1 cells were treated with 0.1% DMSO (the vehicle group), 10 µM LPZ, 25 µM 2-APB, and 2 µM TG for 24 h, and then we detected the effects of LPZ on MC3T3-E1 cells genes related to differentiating and ER stress apoptosis pathway. We found that LPZ could reduce the mRNA expression levels of differentiating and maturate in MC3T3-E1 cells (ALP, OCN, Runx2, CoLIα) and increase the mRNA expression levels of ER stress markers (Caspase12, ATF4). (Fig. 10).
Expression of ERS and osteoblast functional protein
Previous studies had reported that Caspase-12, GRP78, and CHOP are the particular mediators of ERS, and Bcl-2 is an anti-apoptotic protein, while Bax is a pro-apoptotic protein(Chiu et al. 2018). TG could induce a rapid calcium release from ER and promote ER stress. We treated the TG group as positive control(Chen NX 2000 May). In this study, compared with the vehicle group, LPZ increased ERS-mediated caspase-12, Grp78, ATF4, and CHOP protein levels, and promoted the expression of cleave-caspase-3, a terminal cleavage enzyme during the process of apoptosis. However, pre-incubation of 2-APB lessened ER stress protein expression and reduced the elevation of Bax/BCL-2 ratio induced by LPZ. It is well documented that Bcl-2 also regulates endoplasmic reticulum calcium homeostasis(Chiu et al. 2018). Beyond these, the expression of calpain-2, a calcium-dependent ER stress protein, was up-regulated in LPZ and TG group, 2-APB could alleviate the expression of calpain-2, which means that LPZ caused a rise in intracellular calcium at the protein level and ER stress. Moreover, the expression of MC3T3-E1 osteoblast functional protein ALP, OPG/Rankl, OCN were reduced in the LPZ group and the TG group compared with the vehicle group, which illustrated that LPZ impaired the function of MC3T3-E1 osteoblasts, and affected the differentiation, secretion, and mineralization process, and 2-APB could protect the viability of MC3T3-E1 cells after the treatment of LPZ for 24h. LPZ could cause ERS in MC3T3-E1 cells in a dose-dependent manner, and BAPTA could mitigate the damage caused by LPZ to OBs (Fig. 11).
Ca-ATPase activity assay
At the same time, the activity of Ca-ATP enzyme in MC3T3-E1 cells was determined by phosphorus assay, and the results showed that the activity of Ca-ATP enzyme decreased in LPZ group (Fig. 12). After treatment with 10 μM LPZ, the expression of Ca2+-ATP enzyme (ATP2B1) gene in plasma membrane decreased in a time-dependent manner. Although the kit measured the activity of all Ca-ATP enzymes, it also provided some reference for us. It is suggested that LPZ had a negative effect on calcium pump activity of MC3T3-E1 cells.
HE staining and immunohistochemical staining in bone
In vivo, HE staining showed that the control group, the trabecular bone microstructure was dense, with full cancellous bone and few vacuoles; In the low dose group, trabecular bone (cancellous bone) was loosely arranged and partially broken, with a decrease in thickness and some vacuoles in the bone marrow; In the high dose group, trabecular bone (cancellous bone) was fractured, with thinner thickness and with worsened structural integrity, increased separation and more vacuoles in the medulla (Fig. 13A). To test whether ER stress (ERS) occurred in mice, we used immunohistochemical staining to detect the expression of CHOP, an ER stress marker, in distal femoral of different groups. Immunohistochemical results showed that after long-term intragastric administration, the expression of CHOP was gradually increasing with the increase of LPZ dose. It was suggested that long-term LPZ administration may have a negative effect on bone tissue and caused ER stress in the distal femur (metaphysis), the osteoblasts on the trabecular bone surface also decreased, influencing bone remodeling. It indicated that endoplasmic reticulum stress occurred in the bone tissues of mice (Fig. 13B, C). In conclusion, in vivo and in vitro experiments show that LPZ could lead to increased apoptosis of bone cells and thus bone injury by endoplasmic reticulum stress.