Effect of Simvastatin on PTHrP-induced osteoclastic bone
resorption and mouse calvaria bone metabolism in
Author: Huang Lu Yu, Yun-Yu Hu, TAO people, Bilong
【Key Words】 Simvastatin
Abstract: [Objective] To investigate the simvastatin on the in vitro parathyroid
hormone-related peptide (PTHrP)-induced osteoclastic bone resorption in mice the
function of bone metabolism in mice and its effects. [Methods] PTHrP-induced
osteoclast cell culture of mouse bone marrow cells and mouse calvarial culture system
to detect the role of simvastatin after 8 d osteoclastic bone resorption, and calcium changes in the supernatant; test in mice calvaria bone alkaline phosphatase and calcium content of the supernatant, histological observation of morphological changes
in mice calvaria bone. [Results] simvastatin could significantly inhibit the in vitro PTHrP induced osteoclast formation and bone resorption of calcium release supernatant, simvastatin can enhance mouse calvaria in vitro culture supernatant of
bone alkaline phosphatase activity, histological observation of mice calvaria simvastatin increased bone mineralization. [Conclusion] simvastatin in vitro not only in mice calvaria can promote the osteogenic activity of bone, and can inhibit PTHrP-
induced mice, osteoclastic bone resorption function, bone resorption disease prevention and treatment has an important role.
Keywords: simvastatin; broken bone; bone resorption; parathyroid hormone-
related peptide (PTHrP)
Abstract: [Objective] To study the effect of simvastatin in the osteoclastic resorption stimulated by PTHrP and murine bone anabolism in vitro [Method] The bone resorption activities of the osteoclast stimulated by PTHrP were evaluated after treatment with simvastatin for 8 days in vitro; the concentration of Ca2 in the supernatant was also detected by atomic absorption spectrometerThe concentration of ALP and Ca2 of the supematant in murine calvarial organ culture were detectedThe histology of calvaria was observed [Result] Simvastatin greatly inhibited the
osteoclastic bone resorption stimulated by PTHrP in vitro and reduced the release of Ca2 Simvastatin increased the ALP activities and bone mineralization of murines calvarial organ culture in vitro [Conclusion] Simvastatin may inhibit the osteoclasric
resorption stimulated by PTHrP and promote osteoblast differentiation and bone mineralization in vitro, thus play an important role in the prevention and treatment of osteoporosis
Key words: Simvastatin; Osteoclast; Bone resorption; PTHrP
Statins by inhibiting the metabolism of cholesterol mevalonate pathway HMGCoA (3Hydroxy3MethylglutarylCoenzyme A) reductase activity, with a significant lipid-
lowering effect is now a widely used in clinical lipid-lowering drugs. 1999, Mundy et al
〔1〕 in the selection of more than 30,000 kinds of compounds and found that statin drugs are the only BMP2 can promote osteoblast fluorescent reporter gene activity of substances. Further studies have shown that statins can promote osteoblast cell lines
and rat bone marrow cells MC3T3E1 alkaline phosphatase activity and bone mineralization 〔2,3〕, and can promote new bone in osteoporotic rats formation 〔4〕. Under normal physiological conditions, bone resorption and bone formation in a
dynamic equilibrium state, once this balance is broken could lead to the occurrence of osteoporosis. Osteoclastic bone resorption function of cells are cells that absorb the functions of its hyper-active in osteoporosis and has an important significance.
Although previous studies have shown that statins may be through the promotion of osteoblast gene expression in BMP2 to promote bone formation, but its normal mature osteoclast function in bone resorption has been reported yet to see. In this study,
PTHrP-induced bone marrow cells in vitro formation of osteoclasts in culture system, as well as mouse calvarial bone culture, explore the nature of simvastatin on osteoclastic bone resorption and bone metabolism in mice, in order to clarify the statin drugs effects on osteoporosis prevention and treatment to provide a new theoretical basis.
1 Materials and methods
11 Main reagents and animals
API simvastatin (simvastatin), provided by the Shandong Lunan Pharmaceutical Group (production batch number: 050502). Parathyroid hormone-related peptide
(PTHrP): United States Cytolab companies; αMEM medium, the United States Gibco Company; fetal calf serum (FCS), Hycolon company; anti-liquor carbolic acid staining
kit, Japan Hokudo Corporation. BalB / C mice (clean grade): from the Fourth Military Medical University animal experiment center.
121 the production of thin bone
Fresh Niugu the backbone of cortical bone 25% glutaraldehyde fixation, to low-
speed saw-type cutting machine cross-cutting, hand saw Xiu 1 cm × 1 cm × 1 cm size,
leica hard tissue microtome cut into 30 μm thick slices, 100 mesh sandpaper on the polished wood, gradient alcohol dehydration, dilute ammonia ultrasonic cleaning 10 min × 3 times, cobalt-60 irradiation sterilization.
122 Isolation and culture of osteoclasts
Born 4 ~ 6 weeks of male Balb / C mice were sacrificed after the removal, 750 ml / L ethanol, soaked 5 min. To take bilateral femur and tibia, removing soft tissue and
epiphyses at both ends, using serum-free culture medium wash αMEM marrow, bone
marrow cells collected and washed twice with culture medium, and then re-suspended
in a cell containing 100 ml / L fetal calf serum The αMEM culture medium, cell count, the dubbed in 15 × 109 L-1 of cell suspension, 24-hole cell culture plate, each well 2 ml,
at 50 ml / L CO2 incubator at 37 ? for box train. Every 2 d to replace a sub-culture
medium, each fluid change, the removal of the old medium 1ml supplement equivalent new medium. Train 6 ~ 8 d.
123 Add simvastatin
Is divided into four groups: positive control group, in the changing culture medium every hole, when added to 45 ng / ml PTHrP: Hole in the experimental group for each culture when the culture medium by adding 45 ng / ml PTHrP and were added to 10-
5,10 -- 6,10-7 mol / L simvastatin, each two holes, respectively, a hole placed a piece of bone grinding.
124 Anti-tartaric acid staining
Staining, the culture plate adherent cells stained for tartrate resistant (TRAP
staining), culture 6 d after the careful removal of culture medium with phosphate buffer solution (PBS), after washing, by manual operation, the first ethanol - C ketone
(1:1) mixture of a fixed 5 min after the deionized water washing three times, then add
the substrate and the mixture of chromatin, 37 ? incubation box incubated 20 ~ 60
min, see the stain well, the removal of reaction solution, deionized water wash to terminate the reaction, dried at room temperature overnight.
Osteoclast cell count 125
Inverted microscope, all three, or more than 3 nuclei staining positive for tartrate-
resistant giant cells are osteoclasts. Other cells such as macrophages, osteoblasts, fibroblasts tartrate-resistant stain were negative. The experiment was repeated five
times and the results expressed as mean ? standard deviation.
126 osteoclasts absorption determination of
Cultured 6 d later, remove the bones grinding, de-ionized water, washed three times,
formaldehyde fixative fixed, toluidine blue staining and scanning electron microscopy, bone grinding absorption lacuna.
127 atomic spectrophotometer Ca
7 d in the culture medium supernatant obtained using PE 3060 atomic absorption spectrometer measurement of calcium content; conditions: wavelength of 3224 nm,
lamp current 75 mA, spectral width of 26 nm, the air pressure of 16 × 105 Pa, acetylene gas pressure 03 × 105 Pa.
13 Effect of simvastatin on mouse calvarial bone metabolism in
131 BalB / C mice calvaria bone isolation and culture
Born in 1 week Ba1B / C mice were 700 ml / L ethanol, soaked 5 min, PBS wash liquid ethanol, sterile cut the scalp, skull taken to the net internal and external surface of the skull periosteum and soft tissue, along the coronal suture and human characters
seam cut, complete separation of left parietal bone, placed containing 100 ml / L fetal bovine serum αMEM culture medium 24 h after pre-cultured calvaria bone into 24-
well plate hole; each hole by adding 2 ml re-suspended Yu containing 100 ml / L fetal
bovine serum αMEM culture medium, cell count, the dubbed in 15 × 109 L-1 of bone
marrow cell suspension; divided into four groups: positive control group. In the changing culture medium every hole, when added to 45 ng / ml PTHrP, Hole in the
experimental group for each culture when the culture medium by adding 45 ng / ml PTHrP, and were added to 10-5,10-6,10-7 mol / L Sim Simvastatin.
132 atomic spectrophotometer supernatant Ca
Mouse calvarial 8 d in vitro culture supernatants obtained measured calcium content of the supernatant, methods, ibid.
133 Determination of alkaline phosphatase activity in culture supernatant
Mouse calvarial 8 d in vitro culture supernatant obtained using alkaline
phosphatase detection kit Hitachi 7170 automatic biochemical analyzer was detected on alkaline phosphatase activity.
134 Preparation of paraffin sections and histological observation
Taken 8 d culture of calvaria bone, stationary fixed, paraffin-embedded, sliced thick
5 μm, HE staining, in the Leico LA (Germany) microscope and Pixaev automatic collection system consisting of digital image analyzer system, the image under the Acquisition , analysis of bone area (Figure 1).
14 Statistical analysis
All data using statistical package SPSS 100 treatment, t-test. Reposted elsewhere in
the paper for free download http://
21 Effect of Simvastatin on PTHrP-induced osteoclast formation and function of
211 Effect of Simvastatin on PTHrP-induced osteoclast formation of the role of
The results showed that five tests, 01 μmol / L simvastatin significantly inhibited the formation of osteoclasts, 1 μmol / L concentration, almost completely inhibited PTHrP-
induced osteoclast formation (Table 1, Figure 1). Table 1 Effect of Simvastatin on
PTHrP-induced osteoclast formation of bone grinding in the control group and simvastatin 10-5,10-6 mol / L group had no significant absorption lacuna formation, only in the symplectic cutting Statins 0,10-7mol / L groups had to absorb the formation
of lacunae, and the absorption of non-simvastatin group to form larger pits (Figure 2 ~
213 culture supernatant measured results of calcium
PTHrP-induced osteoclast 6 d, supernatants calcium tips, in the simvastatin concentration 10-5,10-6,10-7 mol / L, the treatment group significantly less than the control group, the difference statistically significance, with the increase of drug concentration lower calcium content (Table 2). Table 2 Effect of Simvastatin on PTHrP-induced osteoclast in vitro cell culture supernatant of calcium content in
221 supernatant determination of alkaline phosphatase and Ca2 Results
Determination of mouse calvarial the first 8 d in vitro activity of ALP in the supernatant, suggesting that with the simvastatin group in the concentration 10-5,10-6
mol / L when the supernatant was significantly higher ALP activity compared with the control group with statistical significance, simvastatin at the 10-7mol / L when the ALP
activity compared with the control group had no statistically significant difference; of the original content of the word Ca2 spectrophotometer, using simvastatin and control groups group had no statistically significant difference (Table 3). Table 3 Effect of simvastatin on mouse calvaria in vitro bone culture supernatant of Ca and ALP
Mouse calvarial culture 8 d, the control group shows significant absorption of bone tissue, with Simvastatin treatment group, Simvastatin concentrations 10-5,10-6,10-7
mol / L, the visible absorption significantly reduced bone mass, bone more dense (Figure 5,6). Figure 1 PTHrP stimulated, cultured bone marrow cells 6 d, a large number of osteoclastogenesis (TRAP × 200) Figure 2 in the PTHrP stimulated, cultured bone marrow cells, 8 d, the absorption of the formation of periosteum pieces lacunae (toluidine blue staining × 200), the absorption of the blue for the formation of lacunae Figure 3 PTHrP stimulated by adding 10-7mol / L of simvastatin, scanning electron
micrographs of absorption pits, see Figure 4 in the PTHrP significantly reduced the stimulation, Add 10-6mol / L of simvastatin, scanning electron micrographs of non-
absorption lacuna in vitro Figure 5 the first 8 d, the control group mice calvarial bone resorption significantly (HE × 100) Figure 6 in vitro No. 8 d, simvastatin 10-7 mol / L
group, mice calvarial bone as compared with group photos marked thickening (HE × 200)
Bone metastases due to hypercalcemia (humoral hypercalcemia of malignancy, HHM) and hyperparathyroidism due to hypercalcemia in the clinical symptoms are similar 〔4〕. In different cancer organizations have purified the parathyroid hormone-related protein (parathyroid hormonerelated protein, PTHrP), is considered the main factor mediated HHM 〔5〕, PTHrP can induce osteoclast formation, thereby
leading to resorption damage, resulting in HHM. TAO Hui-jung and many other
〔6,7〕 has successfully created PTHrP induced by whole spinal cord cells to form osteoclasts in culture system. To form osteoclasts in vitro function and regulation of research possible. It excludes the interference of complex factors in vivo to study the
single factors on the role of osteoclasts in vitro will be of osteoclasts and bone-chip co-
culture can reflect the ability of osteoclast bone resorption, in particular, the study of drugs on osteoclastic bone resorption in impact, but the shortcomings of this approach
is that they do not reflect the complex network of bone resorption in vivo regulation of the relationship between calvarial bone cells to maintain the original structure and the relationship between the comparative integrity of the benefit observed in the cell
culture system normal relations between the influence on each other as well as the role of local environmental regulation is to study the ideal model of bone tissue metabolism.
In osteoclasts to absorb in the process of osteoclasts and bone chip in close connection with the first, and then the formation of bone resorption organ cells polarized by intracellular carbonic anhydrase type ? (CAll) to CO2 and H2O
generated H2CO3, H2CO3 decomposed into H, CO3-, fold margin of the vacuole-type
proton pump (VATPase) to H secreted into the resorption of bone micro-environment
gives decalcified; osteoclasts to absorb bone as a migratory, and in a region after the end absorption can walk to the other regions of bone resorption, resulting in the
formation of a bone-chip absorption lacuna round and round. Therefore, bone
resorption, and free calcium in the supernatant reflects the ability of osteoclastic bone resorption a reliable indicator. The results of this study indicate that the concentration
of simvastatin in 10-5,10-6,10-7 mol / L will each make osteoclastic bone resorption
significantly reduced the number of simvastatin in the concentration of 10-5,10 -6,10-7
mol / L when you are so that the number of osteoclast bone formation, significantly
reduced calcium content in the supernatant also significantly reduced, and the dose-
effect relationship, suggesting that simvastatin can directly inhibit in vitro osteoclast The formation and resorption.
Alkaline phosphatase (ALP) is a specific marker of osteoblasts, ALP levels were correlated with differentiation of osteoblasts is closely related to. Cultured mouse calvarial section 8 d, the concentration of 10-5,10-6 mol / L simvastatin in the culture
supernatant ALP activity was significantly increased, histological observation is clearly prompted the concentration of simvastatin in 10 -5,10-6,10-7 mol / L when the mice
were significantly higher than the control group the skull to reduce bone resorption, bone more dense, mineralization increased. This indicates that simvastatin can inhibit in vitro osteoclastic bone resorption and contribute to osteoblast differentiation and function of simvastatin groups in this experiment using cultured mouse calvaria bone
calcium content and the supernatant There was no significant difference between the control group; may be due to calvarial culture system reflects an overall effect, not simply reflect the functional status of osteoclasts, we might as well just mature osteoclast cell culture system, due to less sensitive. However, experimental results can be seen with simvastatin groups in the concentration 10-5,10-6,10-7 mol / L culture
supernatant when the calcium content of a downward trend, suggesting that in the calvarial culture system Simvastatin can inhibit the activity of osteoclastic bone resorption.
Treatment of osteoporosis bisphosphonate drugs, by inhibiting mevalonate pathway downstream of the generation of products is the GPT protein, ras, rho family, the
delayed activation of a clear inhibition of the function of osteoclasts 〔8〕. In the
Mevalonate metabolic pathway of statins as an upstream pathway, HMGCoA
reductase inhibitor, is bound to affect its downstream product generation. Therefore, statins in the promotion of osteoblast activity at the same time, may also inhibit osteoclasts. Grasser WA 〔9〕 other studies have shown that lovastatin on cultured
neonatal rat osteoclast formation and differentiation have a significantly inhibited, by a two hydroxy acid metabolism in osteoclasts, the role of developmental studies confirm Long geranyl geraniol in Long osteoclast formation and differentiation plays an important role in lovastatin on the inhibition of osteoclasts through the key Prenylation protein in two bovine Long geranyl acetone completed, and the statin drugs that affect local bone effects of the number of osteoclasts. Von knoch F et al 〔10〕 were reported
in 2005, oral administration of 120 mgkg-1d-1 lovastatin can inhibit the ultra-high
molecular weight polyethylene (UHMWPE) particle-induced rat skull bone resorption,
and to local decrease in the number of osteoclasts. Huang Luyu 〔11〕 such reports in
the subcutaneous injection of 10 mg simvastatin