Effect of the lipoxygenase-inhibitors baicalein and zileuton on the vertebra in ovariectomized rats

Bone 101 (2017) 134–144

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Effect of the lipoxygenase-inhibitors baicalein and zileuton on the vertebra in ovariectomized rats D. Saul a,⁎, S. Gleitz a, H.H. Nguyen a, R.L. Kosinsky b, S. Sehmisch a, D.B. Hoffmann a, M. Wassmann c, B. Menger a, M. Komrakova a a b c

Department of Trauma, Orthopaedics and Reconstructive Surgery, Georg-August-University of Goettingen, Goettingen, Germany Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, 37075 Goettingen, Germany Medical Institute of General Hygiene and Environmental Health, University of Goettingen, 37075 Goettingen, Germany

a r t i c l e

i n f o

Article history: Received 16 June 2016 Revised 13 April 2017 Accepted 19 April 2017 Available online 26 April 2017 Keywords: Baicalein Zileuton Lipoxygenase-inhibitors Osteoporosis Spine

a b s t r a c t Osteoporosis is one of the most common diseases worldwide. In osteoporosis, vertebral fractures represent a major burden. Lipoxygenase (LOX) inhibitors such as baicalein and zileuton may represent a promising therapeutic option owing to their antioxidative effects and suppression of various inflammatory processes in muscle and bone. The effect of these LOX inhibitors on the spine was studied in osteopenic rats. Female Sprague-Dawley rats were divided two times into five groups: four groups each were ovariectomized (OVX) and one control group was non-ovariectomized (NON-OVX). Eight weeks after ovariectomy, three concentrations of baicalein (1 mg/kg body weight [BW], 10 mg/kg BW, and 100 mg/kg BW) were administered subcutaneously daily in three OVX groups for 4 weeks. Similarly, zileuton was administered in three concentrations via food for 5 weeks. In vivo computed tomography (pQCT) of the spine was performed before the treatments and at the end of the experiment. Lumbar vertebrae were subjected to a compression test, micro-CT, and ashing analyses. After baicalein treatment, cortical bone mineral density (BMD) was improved; trabecular connectivity and trabecular BMD were diminished at high dose. After zileuton treatment, the total BMD, anorganic weight, trabecular nodes, and trabecular area were improved. The in vivo stress-strain index was increased and alkaline phosphatase activity in serum was enhanced after both treatments. A dose-dependent effect was not clearly observed after both treatments. The treatments using baicalein for 4 and zileuton for 5 weeks were not sufficient to change the biomechanical properties and bone volume fraction (BV/TV). Overall, baicalein improved the cortical bone parameters whereas zileuton had a favorable effect on the trabecular structure. Moreover, both treatments increased the bone formation rate. Longer trials, a combination of both LOX inhibitors, and their effect at the cellular and molecular levels should be investigated in further studies. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Osteoporosis is a common disease in humans, with a prevalence of ~ 10.3% in Germany [1]. Osteoporosis increases mortality and reduces the quality of life [2]. Vertebral fractures indicative of injury are a major burden of this disease [3]. One therapy that is currently used is calcium and vitamin D supplements [4]; however, this is not sufficient for the treatment of severe osteoporosis. Other therapeutic options include a limited range of medications such as bisphosphonates, human parathyroid hormone (hPTH), selective estrogen receptor modulators (SERMs), strontium ranelate, anti-receptor activator of NF-κB ligand

⁎ Corresponding author. E-mail address: [email protected] (D. Saul).

http://dx.doi.org/10.1016/j.bone.2017.04.011 8756-3282/© 2017 Elsevier Inc. All rights reserved.

(RANKL) antibodies, and newly investigated drugs that are based on the inhibition of cathepsin K or of sclerostin [5–8]. However, all recently prescribed anti-osteoporosis drugs have serious negative side-effects ([12–15], and therefore, new therapeutic alternatives need to be developed. For example, lipoxygenase (LOX) inhibitors were found to improve bone density and strength [9]. In particular, 5-lipoxygenase (5-LOX) inhibitors suppressed osteoclast formation [10], and they can potentially enhance bone formation [11] and help to treat bone resorption diseases [16]. Baicalein is a phytochemical agent extracted from the plant Scutellaria baicalensis Georgi. It acts as a LOX (especially cyclooxygenase [COX-I]) inhibitor, and it is a potent inhibitor of 12-LOX and 15-LOX, thereby producing an antioxidative effect. Baicalein also inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and this, in turn, suppresses the function of tumor necrosis factor

D. Saul et al. / Bone 101 (2017) 134–144

alpha 8 (TNFα) and interleukin 6 (IL-6), all of which are mediators of inflammation cascades. Baicalein affects degenerative bone diseases such as rheumatoid arthritis [17,18]. It also activates alkaline phosphatase by the mTORC1-pathway, thereby inducing osteoblast-differentiation markers in osteoblasts and even increasing bone parameters in distal femurs [19]. Baicalein also inhibited the bone resorptive activity of osteoclasts by inhibiting osteoclast differentiation and promoting osteoclast apoptosis [20,20]. Mechanistically, baicalein inhibits 5-LOX translocation to the nucleus and p38 phosphorylation, whereas zileuton does not [21]. Zileuton (Zyflo®) is a therapeutic drug approved for treating asthma since 1997 in the USA [22,23]. As 5-LOX downregulates fracture healing [24], zileuton, in a manner similar to 5-LOX inhibitors, is expected to have a positive effect on bone. Zileuton has been found to lead to reduced bone resorption in the maxilla [25]. However, it enhances fracture repair in femoral fractures with increased callous size and earlier bone formation [26]. The effects of the LOX inhibitors baicalein and zileuton on osteoporotic vertebrae have not yet been examined, although LOX inhibitors could be potential candidates for treating bone resorptive diseases [10, 25,27]. Thus, this study aims to examine their effects on both the cortical and trabecular bones of lumbar vertebrae in ovariectomy-induced osteoporotic rats.

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100 mg/kg BW were administered [34,35]. The effective dose calculated based on the daily food intake was, on average, 0.71, 7.72, and 64.44 mg/kg BW for C1, C2, and C3, respectively. The OVX and NONOVX groups received a soy-free diet without zileuton, as mentioned above. At completion, the rats were anesthetized using CO2 and decapitated, and the weight of the rats and their uteri were recorded. The lumbar vertebral bodies were isolated and stored at −20 °C until further analysis. The second vertebral body (L2) was used in an ashing analysis, L3 was subjected to a compression test, and L4 was analyzed by in vivo peripheral quantitative computed tomography (pQCT) and microcomputed tomography (micro-CT). 2.2. In vivo quantitative computed tomography (pQCT) pQCT was performed in isoflurane-anesthetized rats (n = 5 per group) using the pQCT device (XCT Research SA, Stratec Medizintechnik GmbH, Pforzheim, Germany), as described in a previous study [36]. L4 was scanned before the treatment (8 weeks after OVX) and at the end of the experiment. The total bone mineral density (BMD, mg/cm3) and stress-strain index (SSI, mm3) were evaluated using XCT-6.20C software (Stratec Medizintechnik GmbH, Pforzheim, Germany). 2.3. Compression test

2. Materials and methods 2.1. Animals and treatment For this study, 129 three-month-old virgin female Sprague-Dawley rats (Winkelmann Company, Borken, Germany) were housed at 20 °C and relative humidity of 55% in Makrolon IV® cages. Two experiments were performed in conformity with the ethical standards of animal care and with the approval of the local district government. The baicalein experiment involved 60 female rats, with 12 rats per group. The zileuton experiment involved 69 rats with 13 rats each in the non-ovariectomized (NON-OVX) group and ovariectomized (OVX) groups, 14 rats each in the C1 (1 mg/kg body weight [BW]) and C2 (10 mg/kg BW) groups, and 15 rats in the C3 (100 mg/kg BW) group. After acclimatization for 1 week, bilateral OVX was performed or the ovaries were left intact, as described by Iwasa et al. [28]. Surgery was performed under ketamine/xylazine (ketamine: 62.5 mg/kg BW; Hostaket®, Hoechst, Bad Soden, Germany; xylazine: 7.5 mg/kg BW; Rompun®, Bayer, Leverkusen, Germany) anesthesia (0.1 ml/100 g BW, intraperitoneal [i.p.]). After shaving, anesthesia, and disinfection, the skin was incised on both sides of the lower abdomen, and the adnexa were dissected, clamped, and removed before the wound was closed. After 8 weeks, an osteotomy of tibia metaphysis with plate osteosynthesis [29] was performed as a part of other studies. Based on previous studies [29–31], we assumed that at around this time point, that is, eight weeks after OVX, the rats would have developed osteoporosis. One day after the osteotomy, baicalein and zileuton treatments were started. For injections, baicalein (98%; Sigma-Aldrich Chemie Gmbh, Munich, Germany) was dissolved in 100% dimethyl sulfoxide (DMSO). Both control groups (NON-OVX and OVX, each n = 10) received only DMSO. Baicalein was injected subcutaneously, as described previously [32, 33], at different concentrations (C1: 1 mg/kg BW, C2: 10 mg/kg BW, and C3: 100 mg/kg BW, respectively) in three groups with 10 animals in each group. The injections were administered every 24 h for 4 weeks. Zileuton was administered with food for 5 weeks after osteotomy. A soy-free diet (ssniff® special diet; GmbH, Soest, Germany, ingredients in Table 2) was supplemented with zileuton (Zyflo ®; Cornerstone Therapeutics Inc., Cary, NC, USA) at three different concentrations. The rats received demineralized water throughout the experiments. Body weight and food intake were recorded weekly (Fig. 1). The average daily food intake was calculated. The zileuton dosages of 1, 10, and

The biomechanical properties of the vertebra were tested according to Sehmisch et al.'s protocol [37]. A Zwick machine (145 660 Z020/TND; Ulm, Germany) was used to measure the mechanical resistance of lumbar vertebrae. The dissected vertebrae, located on the base of the Zwick machine, were fixed. Next, a slender stamper was dipped onto the vertebra at a rate of 50 mm/min and with initial force of 1 N to fix the vertebra on the plate. Subsequently, measurements were obtained with accuracy of 0.2–0.4% over 2–500 N, as described previously by our group [38]. Live testing showed a linear increasing curve, and testing was stopped when the curve declined by N10 N. The test was recorded using testXpert software (Zwick GmbH & Co. KG, Ulm, Germany). The stability was measured in increments of 0.1 mm. We quantified the maximum load (Fmax), yield load (yL), and stiffness (S) as described by Sehmisch et al. [39] and Stuermer et al. [40]. Fmax is the highest force that the ground plate can withstand. yL is the bending point from elastic to plastic deformation. Stiffness measures the bone's elasticity [38]. 2.4. Micro-CT analysis The vertebral bodies were scanned using Quantum FX micro-CT (Caliper Sciences, Hopkinton, MA, USA). The scan protocol was as follows: 70 kVp, 200 μA, 2-min exposure time, 360° rotation, 3600 projections, 20 × 20 mm2 field of view, 512-pixel matrix, and 40-μm resolution. A phantom block with five hydroxyapatite elements of several mineral densities was scanned with each vertebra to interpret the gray scale in terms of density (g/cm3). Fig. 2A shows the lumbar vertebrae scanned with the phantom three dimensionally (3D). 3DOsteoAnalyze developed in our laboratory was used to assess the bone parameters according to the American Society for Bone and Mineral Research (ASBMR) criteria [41,42]. Trabecular, cortical, and total bone densities (g/cm3); tissue and total volume (mm3); and bone volume fraction (BV/TV) were assessed. The cortical area (mm2) was measured at the dorsal and ventral segments of the vertebral body cut on the sagittal plane of the 3D images (Fig. 2B). Structural analyses were performed using 2D images (transformation with sectional plane is shown in Fig. 2C). Four images of sagittal cut vertebral bodies were analyzed using MetaMorph Basic Acquisition Software (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany, Fig. 2 D–J). The trabecular nodes (N.Nd), trabecular connectivity (N.Nd/mm2),

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D. Saul et al. / Bone 101 (2017) 134–144

Fig. 1. Body weight of rats after baicalein treatment (A) and zileuton treatment (C). Average daily food intake in baicalein (B) and zileuton (D) treatment. + different vs. others, # different vs. OVX, † different vs. C1, ‡ different vs. C2, \ different vs. C3 (P b 0.05, Tukey's test).

cortical and trabecular density/thickness (Fig. 2 I, J), trabecular bone area (Fig. 2 I), and trabecular thickness (Tb.Wi) were recorded, as described previously by our group [38,43].

3. Results

2.5. Ashing

Initially, the BW of the rats did not differ significantly among the groups (Fig. 1); however, at the end of the experiment, the NON-OVX group was the lightest (Fig. 1). Neither baicalein nor zileuton treatment affected BW. After ovariectomy, BW increased significantly in all OVX groups compared to the NON-OVX group after week two. Food intake was significantly higher in OVX groups during the first three weeks. Thereafter, it remained the same across all groups (Fig. 1). The uterine weight in the NON-OVX group was significantly higher than in all OVX groups, indicating successful OVX (Table 1).

To analyze the organic and anorganic contents of the vertebral body, L2 was ashed at 750 °C for 1 h. The calcium content was quantified using an atomic absorption spectrometer (4100; PerkinElmer, Baesweiler, Germany). The orthophosphate content was scaled using the colorimetric method (Spectral Photometer DM4; Zeiss, Jena, Germany) [43]. 2.6. Serum analyses Serum analyses were conducted at the Department of Clinical Chemistry, University of Goettingen, using an Architect c16000 analyzer (Abbott, Wiesbaden, Germany) to detect the alkaline phosphatase (ALP) activity by the para-nitro-phenyl phosphate method (Abbott) at 404 nm. 2.7. Statistics Statistical analysis was performed using the one-way analysis of variance (ANOVA) and Tukey Post-hoc test (α = 0.05) with the aid of GraphPad Prism (GraphPad Software 5.04, San Diego, CA, USA). The figures and tables show the mean values and standard error of mean (SEM).

3.1. Rat characteristics

3.2. In vivo pQCT During the experiment, we performed pQCT to evaluate the BMD and SSI. BMD was significantly reduced in all OVX groups compared to the NON-OVX group before the treatment (Fig. 3A, E). In the end, the results were comparable (Fig. 3B, F). Similarly, in the beginning, SSI was significantly reduced in all OVX groups compared to the NON-OVX group in both experiments (Fig. 3C, G). However, in the end, baicalein C1 and zileuton C1 led to significantly higher values compared to OVX (Fig. 3D, H). 3.3. Compression test Biomechanical testing revealed significantly reduced stiffness in all baicalein-treated groups compared to the NON-OVX group (Table 1)

D. Saul et al. / Bone 101 (2017) 134–144

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Fig. 2. 3D and 2D scans of lumbar vertebrae. 3D (A, B, D, E, G, H) and 2D (C, F, I, J) analyses of lumbar vertebral bodies (a sample from NON-OVX group). A scan of three vertebral bodies with phantom of five different densities (A), measurements of dorsal (Ct. d) and ventral (Ct. v) cortical areas (B), standard thresholds indicated in green in 2D images (D, E, G, H): D) total tissue, E) soft tissue, G) cortical bone, H) trabecular bone. Creation of 10 slices of 0.001-mm thickness with 0.1-mm spacing (C). 2D images for analyses (F). Area (arrow) for analysis of trabecular bone (Tb.Ar) (I). Measurements of Ct. v and Ct. d. cortical densities (J).

and lower yield load and maximum load in the OVX and baicalein C1/ C3-treated groups (Table 1) compared to the NON-OVX group. In OVX and zileuton-treated groups, slight but nonsignificant effects of reduced stiffness and yield load were detected compared to the NON-OVX group (Table 1); at the maximum load, the difference between the NON-OVX and the OVX groups was significant (Table 1). Furthermore, at the lowest zileuton concentration, nonsignificantly higher stiffness, yield load, and maximum load were observed compared to the OVX group. As with the baicalein-treated groups, the zileuton-treated groups did not differ significantly among each other.

3.4. Micro-CT

3.4.1. Baicalein 3D The 3D scans showed that the total BMD and BV/TV quotient were significantly lower in the OVX and baicalein-treated groups compared to the NON-OVX group (Fig. 4A, B). The cortical BMD was significantly greater in the baicalein-treated groups compared to the OVX group (Fig. 4C). The ventral cortical area was smaller in the baicalein-treated groups C1-C3 (Fig. 4D), and the dorsal cortical area was larger, especially in C3 compared to NON-OVX (Fig. 4E). The OVX and C1/C3 groups showed higher tissue volume than the NON-OVX group (Fig. 4F). The trabecular BMD was lower in the OVX and all baicalein-treated groups compared with the NON-OVX group, with the C3 group showing the lowest BMD (Fig. 4G). No significant differences were observed in the total volume (Fig. 4H).

To assess the bone volume and density, 3D analysis was performed using micro-CT scans of the vertebrae. 2D images were used for analyzing the trabecular structure. In the untreated OVX groups, a rare trabecular network and thin cortical bone can be seen (Fig. 2).

3.4.2. Zileuton 3D 3D analysis revealed that the BV/TV quotient and total BMD were significantly lower in the OVX and zileuton-treated groups compared

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Table 1 Uterus weight, biomechanics, and ashing analyses of lumbar vertebrae. Parameters

OVX

C1

C2

C3

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Uterus weight Baicalein [g] Zileuton [g]

0.64 0.56

0.05 0.03

0.11e 0.14e

0.01 0.01

0.13e 0.15e

0.03 0.01

0.14e 0.16e

0.01 0.01

0.15e 0.14e

0.01 0.01

AP in serum Baicalein [U/l] Zileuton [U/l]

96.50d 91.09c

11.51 5.49

104.90 129.80

5.51 9.57

84.20d 127.70

4.66 13.74

117.40 140.30

21.18 12.37

151.80 122.80

17.59 6.59

133.5

4.7

113.5

4.3

102.0e

5.6

109.5e

7.2

100.7e

5.9

317.5 151.4

33.1 12.6

246.8 116.8e

26.3 8.8

264.1 119.1e

23.6 5.0

254.1 134.0

23.9 5.8

229.4 107.2e

22.8 4.3

231.3 152.7 246.3

16.9 12.7 14.2

185.2 118.0e 187.8e

12.0 8.8 12.0

207.3 120.6e 211.6

15.6 5.6 16.4

193.2 134.8 196.6

15.6 5.8 15.9

203.1 108.3e 209.9

9.9 4.2 8.8

0.90

0.01

0.91

0.02

0.90

0.01

0.90

0.02

0.92

0.02

1.29 58

0.05 0.7

1.33 66e

0.01 0.6

1.32 65e

0.01 0.6

1.35 65e

0.04 0.7

1.33 66e

0.01 0.7

65 42

0.4 0.7

70e 34e

0.8 0.6

69e 36e

0.5 0.6

70e 35

0.6 0.7

68e,a 34

0.4 0.7

35

0.4

30e

0.8

31e

0.5

30e

0.6

32e,a

0.4

Biomechanics Stiffness [N/mm] Baicalein Zileuton Yield load [N] Baicalein Zileuton Maximum load [N] Baicalein Zileuton Ashing Ca2+/ PO34 Baicalein Zileuton Organic weight [%] Baicalein Zileuton Anorganic weight [%] Baicalein Zileuton a

NON-OVX Mean

Differs from OVX. b Differs from C1. c Differs from C2. d Differs from C3. e Differs from NON-OVX.

to the NON-OVX group; in particular, the C1 group showed significantly greater BV/TV than the C3 and C2 groups and significantly higher total BMD than the OVX group (Fig. 5A, B). The C1 group showed significantly lower cortical BMD compared to the NON-OVX and C2/C3 groups (Fig. 5C). The C1 group showed the highest ventral cortical area (Fig. 5D) and dorsal cortical area (Fig. 5E) compared to the NON-OVX group, and the NON-OVX and OVX groups showed differences. The tissue volume (Fig. 5F) was the highest in the OVX group, and all groups differed significantly compared to the NON-OVX-group, with the OVX group showing a significantly greater value than the C2 group. All groups showed lower trabecular BMD than the NON-OVX group (Fig. 5G). The OVX and C1 groups showed significantly higher total volume compared to the NON-OVX group (Fig. 5H). The 3D analysis of the micro-CT data revealed a different effect of zileuton on the trabecular and cortical bone; therefore, the bone structure was analyzed in detail using 2D images. 3.4.3. Baicalein 2D The 2D analysis showed that the number of trabecular nodes (Fig. 6A), trabecular bone area (Fig. 6C), trabecular thickness (Fig. 6D), and trabecular density (Fig. 6E) were reduced significantly in all OVX treated and untreated rats. The intermediate concentration of baicalein had a slight, but nonsignificant, protective effect on trabecular bone. The trabecular connectivity (Fig. 6B) showed similar results; however, the highest baicalein concentration showed a significantly lower number of nodes per mm2 compared to the OVX and C2 groups. The cortical density was significantly higher in the C3 group than in the OVX and C1 groups; the C2 group also showed significantly higher density than the C1 group (Fig. 6F). Furthermore, the trabecular and cortical parameters were significantly impaired in the OVX group compared to the NON-OVX group. 3.4.4. Zileuton 2D The 2D analysis of the trabecular bone showed a lower number of trabecular nodes (Fig. 7A) and trabecular bone area (Fig. 7C) in the

OVX and zileuton groups compared to the NON-OVX group. However, these parameters were improved in the C3 group compared to the OVX and C1 groups (p b 0.05). The trabecular connectivity, calculated as the number of nodes per mm2 (Fig. 7B), was reduced in the C1 group. The trabecular thickness (Fig. 7D) and density (Fig. 7E) were highest in the C3 group compared to the C2 and C1 groups, but without nullifying the OVX effect. The cortical density was reduced in the C1 group (Fig. 7F). 3.5. Ashing analysis After micro-CT analysis, L2 was subjected to ashing to assess the organic and anorganic contents and mineral components. No differences in calcium and phosphate or in their ratio (Table 1) were found. In the baicalein experiment, the organic content was significantly lower in the NON-OVX group compared to all other groups; conversely, the anorganic content was significantly higher in the NON-OVX group compared to all other groups (Table 1). In the zileuton experiment, the organic and anorganic content in the C3 group was significantly different from that in the OVX group (Table 1). 3.6. Serum analysis In the baicalein-treated groups, alkaline phosphatase was significantly higher in the C3 group compared to the NON-OVX group, whereas in the zileuton-treated group, it was increased in the C2 group (Table 1). 4. Discussion Bone is chiefly affected through osteoporosis in the trabecular and cortical components. Analyzing the lumbar vertebrae enables both the trabecular and cortical bone to be evaluated; both work as functional units, and they may be influenced by osteoprotective treatment [49,50].

D. Saul et al. / Bone 101 (2017) 134–144 Table 2 Composition of rat diets (Ssniff special diet, GmbH, Soest, Germany) used at the breeding facility (Janvier Labs, Saint-Berthevin Cedex, France) or during experiment (University of Goettingen, Goettingen, Germany). Ingredients

Breeding facilitya

Experimental diet (soy-free)b

Fat (kJ%) Protein (kJ%) Carbohydrate (kJ%) Crude protein (%) Crude fat (%) Crude fiber (%) Crude ash (%) Sugar (%) Calcium (%) Phosphorus (%) Sodium (%) Magnesium (%) Potassium (%) Iron (mg/kg) Manganese (mg/kg) Zinc (mg/kg) Cooper (mg/kg) Iodine (mg/kg) Selenium (mg/kg) Vitamin A (IU/kg) Vitamin D3 (IU/kg) Vitamin E (mg/kg) Vitamin K (mg/kg) Thiamine (B1; mg/kg) Riboflavin (B2; mg/kg) Pyridoxine (B6; mg/kg) Cobalamin (B12; μg/kg) Nicotinic acid (mg/kg) Pantothenic acid (mg/kg) Folic acid mg/kg) Biotin (μg/kg) Choline (mg/kg)

17 34 49 21 7 4 6.2 4.6 1.10 0.80 0.25 0.20 0.94 176 64 89 16 2.2 0.3 25,000 1500 135 80 85 32 31 150 140 59 10 690 3370

10 23 67 19.1 3.4 4.4 6 3.3 1 0.7 0.21 0.22 0.73 184 75 98 14 2.1 0.4 25,000 1500 135 20 86 31 30 150 149 57 9 660 1370

a Diet composition: grain and grain products, oil seed products, minerals, vegetable oils, brewer's yeast, amino acids, vitamins, trace elements. b Diet composition: wheat and wheat products, barley, corn gluten, potato protein, minerals, oat hulls, amino acids, vitamins, trace elements, vegetable oil.

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We investigated the impact of LOX inhibitors, baicalein, and zileuton on the cortical and trabecular properties in lumbar vertebrae using an ovariectomized rat model. After ovariectomy, the properties of the vertebra were diminished in rats [31,44,45]; this mimics observations in humans, where vertebra fractures are indicative of osteoporosis [46– 48]. Ovariectomy was confirmed by the lower weight of the atrophied uteri in OVX rats. Biomechanical analyses revealed reduced stiffness, yield load, and maximum load at the lowest and highest baicalein concentrations compared to the NON-OVX group, whereas these parameters did not differ for the OVX group. Similarly, zileuton did not improve the biomechanical parameters. To the best of our knowledge, ours is the first study to conduct biomechanical analysis of osteopenic bone after treatments with these substances. For baicalein, cell culture experiments next to the distal femur breaking tests showed promising results for bone structures [19,51, 20]. Some molecular mechanisms underlying baicalein's impact have yet to be clarified. It seems to act as a 12/15-LOX inhibitor by decreasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, protein ubiquitination, and ubiquitin-proteasome-mediated proteolytic degradation [52] and by stimulating osteoblast differentiation via mitogen-activated protein (MAP) kinases, NF-κB, activator protein 1 (AP-1), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) [53]. To further determine the impact of these LOX inhibitors on vertebrae, we analyzed the cortical and trabecular bone properties. pQCT performed in vivo showed that SSI, which was negatively affected through OVX and serves as an in vivo parameter of bone strength [54,36], could be significantly improved at the lowest baicalein and zileuton concentrations, even reversing the OVX effect. BMD was not changed by either baicalein or zileuton treatment. The human pQCT in vivo approaches [55–57] make it feasible to assess shifts after baicalein or zileuton treatment in patients, which should be considered in future studies. Micro-CT analyses revealed an improvement in cortical BMD after baicalein treatment, whereas trabecular BMD was diminished. The 2D analyses confirmed these findings; they showed reduced trabecular

Fig. 3. pQCT of vertebrae L4 in baicalein (A–D) and zileuton (E–H) experiments. BMD was determined before the treatments (A, E) and at the end of the experiments (B, F). SSI was measured before the treatments (C, G) and at the end of the experiments (D, H).

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Fig. 4. 3D analysis of micro-CT data of L4 in the baicalein experiment. The assessed parameters were bone volume related to total volume (A), total BMD (B), cortical BMD (C), ventral (D) and dorsal cortical area (E), tissue volume (F), trabecular BMD (G), and total volume (H).

connectivity and improved cortical density in the baicalein C3 group compared to OVX rats. The fact that baicalein reduces cortical bone loss significantly makes it especially interesting because 70% of all bone loss is cortical [58]. Osteoporotic bone loss is pronounced near the endocortical surface [59], and osteoporosis does not affect

trabecular bone alone. Indeed, the deficiency of cortical bone leads to the loss of trabecular bone [60], and baicalein can strengthen cortical BMD. On the other hand, the improvement of cortical bone could be due to the adaptive changes that attenuate the effect of trabecular loss in vertebra [61].

Fig. 5. 3D analysis of micro-CT data of L4 in the zileuton experiment. The assessed parameters were volume related to total volume (A), total BMD (B), cortical BMD (C), ventral (D) and dorsal cortical area (E), tissue volume (F), trabecular BMD (G), and total volume (H).

D. Saul et al. / Bone 101 (2017) 134–144

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Fig. 6. 2D analysis of micro-CT data of L4 in the baicalein experiment. We investigated the number of trabecular nodes (N.Nd, A), trabecular connectivity (N.Nd, B), trabecular bone area (C), trabecular thickness (Tb.Wi, D), trabecular density (E), and cortical density (%, F).

While baicalein had a more favorable effect on cortical bone, zileuton was more effective in improving not only total BMD but also the trabecular nodes and trabecular bone area. The effect on cortical bone was less or even unfavorable at low dose. Increased cortical BMD through the inhibition of 5-LOX was reported by Bonewald et al. [62] and Traianedes et al. [63], who described 5-LOX metabolites as negative regulators for bone formation. Other researchers did not find any differences in BV/TV and total BMD in rats after 5-LOX inhibition [11]; however, 5-LOX knockout showed no change in cortical bone but a decrease in trabecular bone portions [64]. Because bone defects are currently left to heal without medicinal support, our findings are highly relevant. Small molecules like alendronate increase primary cortical bone formation in a manner similar to how baicalein is thought to do so, namely, via inhibition of osteoclast development [65]. Zileuton treatment, which improved the trabecular structure and enhanced the anorganic mass (ashing analysis) in OVX rats, may also be a promising agent in osteoporosis treatment. Inhibition of 5-LOX suppressed bone loss in in vivo mouse experiments through the suppression of RANKL-induced osteoclast formation [16]; however, the exact trabecular/cortical effects remain unknown. Though glycoprotein 130 (gp130)/interleukin-6 (IL-6) signaling maintains trabecular bone formation [66] and zileuton inhibits IL-6 [67,68], one possible mechanism could lead through this axis; however, further studies through molecular methods are needed to reveal the underlying pathway, in which the two different LOXinhibitors act. The fact that the organic weight of bone increased and the anorganic weight decreased after OVX is an typical effect of estrogen depletion on bone [69–71]. Baicalein treatment did not change this, whereas highdose zileuton treatment increased the anorganic weight. To better understand bone mineralization and formation, serum alkaline phosphatase (AP) was measured. It was found to be increased in the baicalein

C3 group and the zileuton C2 group. This indicates increased bone formation rate [72] after treatment with both LOX inhibitors. In conclusion, treatments with baicalein for 4 weeks and zileuton for 5 weeks had an impact on the spine. Baicalein increased the cortical BMD and SSI with little effect on trabecular parameters, whereas highdose zileuton treatment primarily had a positive effect on cancellous bone and improved SSI. Both substances enhanced AP activity and, in turn, the bone formation rate. The dose-dependent effect was not clearly observed after both treatments. Some findings, such as the low-dose effect on cortical bone, indicate the need to take precautions when using zileuton. Both treatments were not sufficient to change biomechanical parameters. Longer trials, a combination of both LOX inhibitors, and their effect at the cellular and molecular levels will be investigated in further studies. Moreover, the effects of LOX inhibitors on fracture healing will be analyzed to see whether they could be administered to improve bone formation processes. Population-based studies on humans treated with zileuton because of asthmatic disease, starting, for example, with pQCT-analysis in this population, could show whether the bone microarchitecture is modified over time or whether bone fractures occur more often after this treatment. 5. Limitations Baicalein was injected subcutaneously in this experiment. Unfortunately, this route of administration led to necrotic lesions at the injection sites; these were increasingly observed in baicalein-treated rats compared to rats treated with DMSO alone. This is not previously described in the literature. These lesions did not impinge on overall health, as indicated by body weight, food intake, and general conditions (clean coat, absence of porphyrin around eyes, etc.). However, in the future, it is advisable to use an alternative route of administration, such as oral [73,74] or intravenous [75].

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Fig. 7. 2D analysis of micro-CT data of L4 in the zileuton experiment. We investigated the number of trabecular nodes (N.Nd, A), trabecular connectivity (N.Nd, B), trabecular bone area (C), trabecular thickness (Tb.Wi, D), trabecular density (E), and cortical density (%, F).

The administration of zileuton with food was well tolerated by the rats; therefore, in the future, oral administration of baicalein should be more preferable. To elucidate the effect of baicalein in the presence of estrogen, that is, in healthy NON-OVX rats, an extra group should be included. This could also help to distinguish whether baicalein or OVX had a superior effect. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors would like to thank Elsbeth Bonhoff Stiftung for financially supporting the present study (Grant N114). The authors are also grateful to R. Castro-Machguth and A. Witt for their technical support. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2017.04.011. References [1] C. Lange, Daten und Fakten: Ergebnisse der Studie “Gesundheit in Deutschland aktuell 2012”, Robert-Koch-Inst, Berlin, 2014. [2] R. Doyle, D. Rajacich, The Roy adaptation model. Health teaching about osteoporosis, AAOHN. J. 39 (1991) 508–512.

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