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Antiosteoporotic effects of L-Arginine in ovariectomied rats
Biomedicine & Aging Pathology 4 (2014) 117–122
Available online at
ScienceDirect www.sciencedirect.com
Original article
Antiosteoporotic effects of L-Arginine in ovariectomied rats Hong Guo a , Yu Gao b , Bin Gu a , Jing Wang c , Hongchen Liu a,∗ a Institute of Dental Research, Chinese PLA General Hospital and Postgraduate Military Medical School, No.28, Fu Xing Road, Hai Dian District, Beijing 100853, PR China b Department of Stomatology, Peking University People’s Hospital, No.11, Xizhimen South Street, Xicheng District, Beijing 100044, PR China c Department of Pathology, Peking University School and Hospital of Stomatology, Beijing 100081, PR China
a r t i c l e
i n f o
Article history: Received 13 January 2014 Accepted 18 February 2014 Available online 24 April 2014 Keywords: L-Arginine Osteoporosis Rats
a b s t r a c t Objective: To investigate the antiosteoporotic effects of L-Arginine on ovariectomied rats. Methods: Forty twelve-week-old female Sprague-Dawley rats were randomly divided into four groups of bilaterally ovariectomy and one group of Sham animals, with 8 animals in each group. Twelve additional weeks elapsed before initiation of the treatment with L-Arginine in order to induce significant bone loss in the ovariectomied animals. A 4-week daily treatment (5 days a week, Monday–Friday) at doses of 5 mg/kg/d, 10 mg/kg/d, 20 mg/kg/d of L-Arginine or vehicle only was administered by s.c. injection to the ovariectomied groups respectively. At the end of the treatment period, blood samples from all animals were collected for biochemical analysis. Micro-computerized tomography analysis, histopathological study and biomechanical test were performed on the femur of each animal. Results: Serum alkaline phosphatase and osteocalcin were reduced in ovariectomied rats after the administration of different dosage of L-Arginine. L-Arginine of 10 mg/kg showed the most significant effect. Micro-computerized tomography 3-D images revealed that the increase of bone mass in 5 mg/kg and 10 mg/kg groups were significant greater than that in Sham group. The biomechanical parameters of femur were improved significantly in L-Arginine 10 mg/kg group when compared to untreated ovariectomied rats. Conclusions: L-Arginine (10 mg/kg) contributes significantly to the treatment of the bone loss induced by ovariectomy on rats, demonstrated by increased bone mass, improved bone structure and recovery of bone biomechanical activity. © 2014 Published by Elsevier Masson SAS.
The prevalence of osteoporosis is increasing rapidly worldwide while the aging population rises. However, a limited number of medications are currently available for the treatment of osteoporosis, including calcium and vitamin D, hormone replacement therapy (HRT), selective estrogen receptor modulator (SERM), calcitonin (CT), diphosphonate, parathyroid hormone (PTH), fluoride, statin, as well as herbal estragon [1]. Diphosphonates have been reported as one of the most efficient and widely used antiosteoporotic drugs. However, its side effects including abrupt symptoms to the digest
Abbreviations: NO, nitric oxide; L-Arg, L-Arginine; SD, Sprague-Dawley; OVX, Ovariectomy; ALP, Alkaline phosphatase; OC, Osteocalcin; microCT, Microcomputerized tomography; BV/TV, Bone volume/tissue volume; Tb.Ar/T.Ar, Tabecular area/tissue area; Tb.Th, Tabecular thickness; Tb.N, Tabecular number; Tb.Sp, Tabecular separation; BMD, Bone mineral density; BMC, Bone mineral content. ∗ Corresponding author. Fax: +86 10 66936254. E-mail addresses: [email protected] (H. Guo), liuhc [email protected] (H. Liu). http://dx.doi.org/10.1016/j.biomag.2014.02.002 2210-5220/© 2014 Published by Elsevier Masson SAS.
system, risk of a bone turnover when used for long period, hampered its clinic application [2–4]. A medication with high efficacy and low side effects is needed. Established researches have shown that nitric oxide (NO) suppresses osteoclasts while having little oppressive effect on the growth of osteoblasts. It has thus been suggested exogenous NO may function as a promising candidate for treatment of osteoporosis. Meanwhile, substance that can regulate the metabolism of NO have also attracted attention of researchers worldwide [5–8]. Currently, there are two different types of NO associated medications in use: the nitric oxide synthase (NOS) inhibitor or NO Scavenger group, and the NO generator group. L-Arginine (L-Arg) is one of the NO generators [9–11]. It has been recently discovered that NO has some important effect on bone turnover and has some effects on the treatment of osteoporosis [12–14]. The purpose of this study is to investigate the therapeutical potential of L-Arg using ovariectomized (OVX) rat models at different dosages. The effects were evaluated by means of biochemical analysis, such as determination of serum levels of Ca, P, alkaline phosphatase (ALP) and osteocalcin
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(OC), micro-computerized tomography (MicroCT) study, pathological observation as well as 3 point bending biomechanical test [15–17].
biomechanical apparatus (MTS858Mini Bionix, USA). A supporter with two loading points, 25 mm apart from each other, was used on the stage of the testing machine. A rounded press head compressed the middle of the femur shaft until fracture occurred [18,19].
1. Material and methods 1.6. Statistical studies 1.1. Animals Twelve-week-old female Sprague-Dawley (SD) rats were purchased from Vitalriver company, Beijing, China, and maintained at the animal research facility of the Chinese PLA General Hospital. The animals were housed in an air-conditioned room with 12 h/12 h light-dark illumination cycles at constant temperature (24 ± 0.5 ◦ C) and humidity (45–50%). Animals were fed diet containing 1.33% calcium, 0.89% phosphorus and water ad libitum throughout the experiment. 1.2. Experimental protocol The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. The rats were randomly divided into four groups of bilaterally OVX and one group of Sham animals (only fat tissue was removed), with 8 animals in each group. Twelve additional weeks elapsed before initiation of the treatment with L-Arg allowing significant bone loss to occur in the OVX animals. A 4-week daily treatment (5 days a week, Monday–Friday) at the doses of 5 mg/kg/d, 10 mg/kg/d, 20 mg/kg/d of L-Arg (Sigma) or vehicle (NS, Sigma) only was then performed by s.c. injection to the OVX groups respectively. At the end of the treatment period, blood samples from all animals were collected from tail vein for biochemical analysis. All rats were then euthanized using overdose anesthetic. The femora and tibia were separated and cleaned of soft tissues. The tibia were fixed in 10% phosphate buffered formalin (pH 7.2) for 48 h, and then kept in 70% ethanol until further use. The femora were stored in a freezer at –80 ◦ C until examination for biomechanical testing. 1.3. Micro-computed tomography studies For a detailed qualitative and quantitative 3-D evaluation, tibial bones were examined by a desktop micro-computed tomography system (eXplore Locus MicroCT, the General Electric Company, New York, USA), voltage 80V, current 450 A, number of views 400, exposure time 2000 ms, bin mode 1 × 127 m. To scan the entire tibial head, a total of 500 microtomographic slices were acquired at a 27 m slice increment. Morphometrical parameters were determined using a direct 3-D approach in three different pre-selected analysis regions: 1.8 × 1.5 × 1.4 mm3 area in the proximal tibial metaphysis [13,14].
Data were analyzed using one-way ANOVA followed by Student’s t-test using SPSS software (version 13.0). Results are reported as mean ± standard deviation (SD). Statistical significance was established for two-tailed values of P < 0.05. 2. Results 2.1. Serum biochemical studies Results of serum parameters in rats of different groups are in Table 1. The results indicated that L-Arg treatment reduced serum ALP and OC levels significantly (P < 0.05) in OVX model group compared to Sham group. L-Arg 10 mg/kg showed the most significant effect on reduction of the serum ALP level among three different dosages. As result, there was no significant difference of the ALP level between L-Arg 10 mg/kg group and sham group. 2.2. Micro-computed tomography studies A significant difference of tibial parameters determined by micro-CT between different groups was observed. Trabecular BV/TV, Tb.Th, Tb.N, BMC and BMD in L-Arg (5 mg/kg) group and LArg (10 mg/kg) groups were greater than those of Sham group, but trabecular Tb.Sp were lower (P < 0.05) in both L-Arg groups than that in Sham group (Fig. 1). Trabecular Tb.Th of L-Arg 20 mg/kg group was greater than that of OVX group, but Tb.Sp and other parameters became lower than those of OVX group (Table 2). 2.3. Histopathological studies Histopathological studies of the L-Arg 10 mg/kg group exhibited the most significant effect on OVX rat’s tibia while bone trabecular re-connected and turn thicker, the number of bone trabecular increased and the separation degree decreased. The bone trabecular re-connected but there were a number of fissure in the tibia section of L-Arg 5 mg/kg group’s rats (Fig. 2). L-Arg 20 mg/kg had negative effect on the OVX rats’ tibia while most of bone parameters turned worse. 2.4. Biomechanical studies
Following mCT image acquisition, the specimens were fixed in 10% phosphate buffered formalin for 24 h, then dehydrated in progressive concentrations of ethanol, defatted in xylene and embedded undecalcified in methyl methacrylate. The frontal sections were cut at 10-micrometer thicknesses with a microtome (Leica RM2155, Germany) followed by HE staining. The sections were mounted and then observed under microscope for histopathological changes.
The biomechanical properties of femur decreased significantly in OVX group compared to those of Sham group. Following administration of L-Arg (10 and 20 mg/kg) for four weeks, the maximal stress and elastical stress increased significantly, compared to those in untreated OVX group. There was no significant effect including maximal load, elastical load, maximal stress, or elastical stress or interaction in L-Arg 5 mg/kg group. Especially, a major effect, maximal load and elastical load, was observed to be increased significantly in L-Arg 10 mg/kg group (Figs. 3 and 4). Thus, the treatment of L-Arg 10 mg/kg had beneficial effects on femoral breaking strength as assessed by three-point bending.
1.5. Biomechanical studies
3. Discussion
The femur specimen stored at –80 ◦ C were transferred into a freezer for twenty-four hours before they were assessed for their biomechanical strength by three-point bending test using the
In this study, different dosages (5, 10, 20 mg/kg) of L-Arg were examined on the treatment of OVX rats [8,10,14]. OVX rat is one of the commonly used animal model for the evaluation of drug effect
1.4. Histopathological studies
–4 ◦ C
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Table 1 Effect of L-Arginine (L-Arg) on ovariectomized (OVX) rats after 4 weeks as determined by serum biochemical examination. Parameter
Ca (mmol/L)
Sham OVX L-Arg 5 mg/kg L-Arg 10 mg/kg L-Arg 20 mg/kg
2.50 2.48 2.33 2.20 2.41
± ± ± ± ±
0.11 0.08 0.08a,b,d 0.08a,b,c,e 0.08d
P (mmol/L) 1.57 1.55 1.71 1.71 1.29
± ± ± ± ±
0.13 0.11 0.07a,b,e 0.05a,b,e 0.07a,b,c,d
ALP (U/L) 89.66 161.46 149.66 57.84 128.70
± ± ± ± ±
OC (ng/mL) 14.06 8.86a 3.07a,d,e 1.13b,c,e 7.1a,b,c,d
14.56 18.41 23.36 17.33 20.29
± ± ± ± ±
1.52 1.94a 1.27a,b,d,e 0.81a,c,e 2.33a,b,c,d
All groups were n = 8 SD rats; data are presented as mean ± standard error. Groups were compared by one-way ANOVA method. F test was used when compared the difference between groups, and T test was used when compared two out of the groups. ALP: alkaline phosphatase; OC: osteocalcin. a P < 0.05, when compared with Sham group. b P < 0.05, when compared with OVX group. c P < 0.05, when compared with OVX group treated with L-Arg 5 mg/kg. d P < 0.05, when compared with OVX group treated with L-Arg 10 mg/kg. e P < 0.05, when compared with OVX group treated with L-Arg 20 mg/kg.
on osteoporosis. The measurement of the activity of serum ALP can be used as a parameter of bone formation and it may be used in clinic. Total serum ALP activity is the most commonly used marker of bone formation. It is a sensitive marker of the increased bone turnover in postmenopausal women and is an accurate index of the effects of antiresorptive drugs on bone turnover. The concentration
of serum OC was not only used for the evaluation of rat osteoporosis model, it was also used as an important parameter on evaluation of drug effects on the treatment of osteoporosis [1]. Our results showed that ALP was reduced in OVX rats after the administration of different dosage of L-Arg. L-Arg of 10 mg/kg showed the most significant effect, and ALP reduced to a level which was not significant
Fig. 1. 3-D mCT images of the proximal tibial cancellous bone after 4 weeks L-Arginine (L-Arg) treatment. A. L-Arg 5mg/kg. B. L-Arg 10mg/kg. C. L-Arg 20mg/kg. D. OVX. E. Sham.
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Table 2 Effects of L-Arginine (L-Arg) on rats femurs after 4 weeks as determined by micro-CT. Parameter
BV/TV (%)
Sham OVX L-Arg 5 mg/kg L-Arg 10 mg/kg L-Arg 20 mg/kg
0.25 0.19 0.50 0.60 0.11
± ± ± ± ±
0.02 0.01a 0.04a,b,d,e 0.06a,b,c,e 0.04a,b,c,d
Tb.Th (mm) 0.07 0.06 0.10 0.10 0.08
± ± ± ± ±
0.01 0.01a 0.02a,b,e 0.02a,b,e 0.02a,c,d
Tb.N (1/mm) 3.31 2.88 4.19 6.61 1.33
± ± ± ± ±
0.11 0.08a 0.10a,b,d,e 0.27a,b,c,e 0.05a,b,c,d
Tb.Sp (mm) 0.20 0.27 0.13 0.08 0.68
± ± ± ± ±
0.01 0.01a 0.03b,e 0.03a,b,e 0.04a,b,c,d
BMC (mg) 0.73 0.57 1.00 1.32 0.37
± ± ± ± ±
0.04 0.04a 0.13b,d,e 0.07a,b,c,e 0.05a,b,c,d
BMD (mg/cc) 218.69 168.44 303.80 389.41 107.95
± ± ± ± ±
10.82 11.18a 13.07a,b,d,e 5.93a,b,c,e 5.06a,b,c,d
All groups were n = 8 SD rats; data are presented as mean ± standard error. Groups were compared by one-way ANOVA method. F test was used when compared the difference between groups, and T test was used when compared two out of the groups. BV/TV: bone volume/tissue volume; Tb.Th: tabecular thickness; Tb.N: tabecular number; Tb.Sp: tabecular separation; BMC: bone mineral content; BMD: bone mineral density; OVX: ovariectomized. a P < 0.05, when compared with Sham group. b P < 0.05, when compared with OVX group. c P < 0.05, when compared with OVX group treated with L-Arg 5 mg/kg. d P < 0.05, when compared with OVX group treated with L-Arg 10 mg/kg. e P < 0.05, when compared with OVX group treated with L-Arg 20 mg/kg.
different from that in the Sham group; OC in the 10 mg/kg group was also reduced while OC in the other two OVX groups increased. Low calcium and high phosphorus in periphery blood were seen in all L-Arg treatment groups. It is then suggested that treatment of osteoporosis should accompanied with the administration of calcium and monitoring of serum calcium and phosphorus levels.
The proximal tibial metaphysis and the distal femural metaphysis consist of abundant cancellous bone and could be easily induced bone loss by ovariectomy on rats, then they are the ideal area for the verification of the effects of anti-osteoporosis drug on OVX rats in a relatively short period of time. It has to be emphasized that the histopathological observation should be carried out
Fig. 2. Photomicrographs of the proximal tibial metaphyseal trabecular bone after 4 weeks L-Arginine (L-Arg) treatment. A. L-Arg 5mg/kg. B. L-Arg 10mg/kg. C. L-Arg 20mg/kg. D. OVX. E. Sham.
H. Guo et al. / Biomedicine & Aging Pathology 4 (2014) 117–122
Fig. 3. Effect of L-Arginine (L-Arg) on biomechanical parameter-Load.
in the secondary cancellous bone area as bone grows constantly. Early morphological changes are very important on the evaluation of animal model and the treatment effect. Although the test of bone density is a widely used parameter on evaluating animal model, in the diagnostic of osteoporosis in clinic as well as the parameter of drug evaluation, it only shows its effect when a significant loss of bone can be observed, thus its sensitivity and repeatability are poor. In the meantime, the biochemical parameters of blood and urine can only partly reflect the changes in animal model and therefore, it’s more often used as co-parameters. Compared with bone density measurement and biochemical parameters, morphometric parameters can not only describe the changes of quantity of bone and morphology, but also reflects the dynamic process of bone metabolism. Change of morphology is an early indicator of pathophysiological process and an indicator of pharmacological function. The quantitive determination of bone bio-function is an important parameter relating to bone function, such as possibility of bone fracture. It’s better to combine the morphometric and biochemical parameters to evaluate the physiological, pathological and function changes of bone. It will also help the access the efficacy of new drugs. In this study, we found that bone trabecular re-connected and turn thicker in L-Arg 10 mg/kg group, the number of trabecular
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increased and the tabecular separation decreased. In this group, the bone mass increased and the bone micro-architecture improved. MicroCT has attracted attention among medical professionals for its widely application on cancer screening, disease monitoring and drug evaluation. A 3-D image of scanned microstructure can be displayed on MicroCT, and the sub-cellular structure can be observed on MicroCT at highest resolution. The description parameters obtained from Micro-CT, which related to 3-D image, is more accurate than the 2-D data. To scan the representative target and reconstruct the 3-D image are very important. MicroCT has been widely used in drug evaluation [16,17]. MicroCT 3-D image revealed that the increase of bone mass in 5 mg/kg and 10 mg/kg groups were significant greater than that in Sham group. A number of parameters, including BV/TV, Tb.Th, Tb.N, BMD and BMC in 5 mg/kg and 10 mg/kg groups were significantly improved than those in Sham group, while the 5 mg/kg group showed less effect although the difference between 5 mg/kg and 10 mg/kg group was not significant. However, in L-Arg 20 mg/kg group, Tb.Sp increased significantly. It implied that 5 mg/kg and 10 mg/kg L-Arg could be used to treat the osteoporosis induced by ovariectomy in rats. The loss of bone mass and changes of bone structure will eventually affect the bio-function and reduce the strength of bone. Therefore, changes of biomechanics are used as a unique parameter for evaluation of the outcome of antiosteoporotic treatment. Three-point bending test is one of the commonly used methods for this purpose [18,19]. The fundamental principal of treatment is to increase the quantity of bone mass and improve the bone structure, as results, the mechanic properties of bone can be improved. In our study, the biomechanical parameters of femur were improved significantly in L-Arg 10 mg/kg group when compared to untreated OVX group. The maximal load together with elastic load of L-Arg 10 mg/kg group were not significant different from those in Sham group. It has been found in this study that not only the bone mass was increased and the bone structure was improved after L-Arg (10 mg/kg) treatment but also the bone biomechanical activity was recovered. L-Arg 5 mg/kg treatment had no effect on the OVX bone mechanical parameters. 4. Conclusions L-Arg, one of the generators of NO in vivo, is effective on the treatment of osteoporosis in OVXrats. L-Arg (10 mg/kg) contributes significantly to the treatment of the bone loss induced by ovariectomy of rats. Not only the bone mass was increased and the bone structure was improved after L-Arg (10 mg/kg) treatment but also the bone biomechanical activity was recovered. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments We are grateful to Professor Xiaojun Xu, Bojiang Shen and Dr. Dajun Wang for the language amendment and their helpful advices. This work was supported by grants from the Chinese National Natural Sciences Foundation Projects (30772450) and Beijing Natural Sciences Foundation Projects (7082086). References
Fig. 4. Effect of L-Arginine (L-Arg) on biomechanical parameter-Stress.
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Available online at
ScienceDirect www.sciencedirect.com
Original article
Antiosteoporotic effects of L-Arginine in ovariectomied rats Hong Guo a , Yu Gao b , Bin Gu a , Jing Wang c , Hongchen Liu a,∗ a Institute of Dental Research, Chinese PLA General Hospital and Postgraduate Military Medical School, No.28, Fu Xing Road, Hai Dian District, Beijing 100853, PR China b Department of Stomatology, Peking University People’s Hospital, No.11, Xizhimen South Street, Xicheng District, Beijing 100044, PR China c Department of Pathology, Peking University School and Hospital of Stomatology, Beijing 100081, PR China
a r t i c l e
i n f o
Article history: Received 13 January 2014 Accepted 18 February 2014 Available online 24 April 2014 Keywords: L-Arginine Osteoporosis Rats
a b s t r a c t Objective: To investigate the antiosteoporotic effects of L-Arginine on ovariectomied rats. Methods: Forty twelve-week-old female Sprague-Dawley rats were randomly divided into four groups of bilaterally ovariectomy and one group of Sham animals, with 8 animals in each group. Twelve additional weeks elapsed before initiation of the treatment with L-Arginine in order to induce significant bone loss in the ovariectomied animals. A 4-week daily treatment (5 days a week, Monday–Friday) at doses of 5 mg/kg/d, 10 mg/kg/d, 20 mg/kg/d of L-Arginine or vehicle only was administered by s.c. injection to the ovariectomied groups respectively. At the end of the treatment period, blood samples from all animals were collected for biochemical analysis. Micro-computerized tomography analysis, histopathological study and biomechanical test were performed on the femur of each animal. Results: Serum alkaline phosphatase and osteocalcin were reduced in ovariectomied rats after the administration of different dosage of L-Arginine. L-Arginine of 10 mg/kg showed the most significant effect. Micro-computerized tomography 3-D images revealed that the increase of bone mass in 5 mg/kg and 10 mg/kg groups were significant greater than that in Sham group. The biomechanical parameters of femur were improved significantly in L-Arginine 10 mg/kg group when compared to untreated ovariectomied rats. Conclusions: L-Arginine (10 mg/kg) contributes significantly to the treatment of the bone loss induced by ovariectomy on rats, demonstrated by increased bone mass, improved bone structure and recovery of bone biomechanical activity. © 2014 Published by Elsevier Masson SAS.
The prevalence of osteoporosis is increasing rapidly worldwide while the aging population rises. However, a limited number of medications are currently available for the treatment of osteoporosis, including calcium and vitamin D, hormone replacement therapy (HRT), selective estrogen receptor modulator (SERM), calcitonin (CT), diphosphonate, parathyroid hormone (PTH), fluoride, statin, as well as herbal estragon [1]. Diphosphonates have been reported as one of the most efficient and widely used antiosteoporotic drugs. However, its side effects including abrupt symptoms to the digest
Abbreviations: NO, nitric oxide; L-Arg, L-Arginine; SD, Sprague-Dawley; OVX, Ovariectomy; ALP, Alkaline phosphatase; OC, Osteocalcin; microCT, Microcomputerized tomography; BV/TV, Bone volume/tissue volume; Tb.Ar/T.Ar, Tabecular area/tissue area; Tb.Th, Tabecular thickness; Tb.N, Tabecular number; Tb.Sp, Tabecular separation; BMD, Bone mineral density; BMC, Bone mineral content. ∗ Corresponding author. Fax: +86 10 66936254. E-mail addresses: [email protected] (H. Guo), liuhc [email protected] (H. Liu). http://dx.doi.org/10.1016/j.biomag.2014.02.002 2210-5220/© 2014 Published by Elsevier Masson SAS.
system, risk of a bone turnover when used for long period, hampered its clinic application [2–4]. A medication with high efficacy and low side effects is needed. Established researches have shown that nitric oxide (NO) suppresses osteoclasts while having little oppressive effect on the growth of osteoblasts. It has thus been suggested exogenous NO may function as a promising candidate for treatment of osteoporosis. Meanwhile, substance that can regulate the metabolism of NO have also attracted attention of researchers worldwide [5–8]. Currently, there are two different types of NO associated medications in use: the nitric oxide synthase (NOS) inhibitor or NO Scavenger group, and the NO generator group. L-Arginine (L-Arg) is one of the NO generators [9–11]. It has been recently discovered that NO has some important effect on bone turnover and has some effects on the treatment of osteoporosis [12–14]. The purpose of this study is to investigate the therapeutical potential of L-Arg using ovariectomized (OVX) rat models at different dosages. The effects were evaluated by means of biochemical analysis, such as determination of serum levels of Ca, P, alkaline phosphatase (ALP) and osteocalcin
118
H. Guo et al. / Biomedicine & Aging Pathology 4 (2014) 117–122
(OC), micro-computerized tomography (MicroCT) study, pathological observation as well as 3 point bending biomechanical test [15–17].
biomechanical apparatus (MTS858Mini Bionix, USA). A supporter with two loading points, 25 mm apart from each other, was used on the stage of the testing machine. A rounded press head compressed the middle of the femur shaft until fracture occurred [18,19].
1. Material and methods 1.6. Statistical studies 1.1. Animals Twelve-week-old female Sprague-Dawley (SD) rats were purchased from Vitalriver company, Beijing, China, and maintained at the animal research facility of the Chinese PLA General Hospital. The animals were housed in an air-conditioned room with 12 h/12 h light-dark illumination cycles at constant temperature (24 ± 0.5 ◦ C) and humidity (45–50%). Animals were fed diet containing 1.33% calcium, 0.89% phosphorus and water ad libitum throughout the experiment. 1.2. Experimental protocol The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. The rats were randomly divided into four groups of bilaterally OVX and one group of Sham animals (only fat tissue was removed), with 8 animals in each group. Twelve additional weeks elapsed before initiation of the treatment with L-Arg allowing significant bone loss to occur in the OVX animals. A 4-week daily treatment (5 days a week, Monday–Friday) at the doses of 5 mg/kg/d, 10 mg/kg/d, 20 mg/kg/d of L-Arg (Sigma) or vehicle (NS, Sigma) only was then performed by s.c. injection to the OVX groups respectively. At the end of the treatment period, blood samples from all animals were collected from tail vein for biochemical analysis. All rats were then euthanized using overdose anesthetic. The femora and tibia were separated and cleaned of soft tissues. The tibia were fixed in 10% phosphate buffered formalin (pH 7.2) for 48 h, and then kept in 70% ethanol until further use. The femora were stored in a freezer at –80 ◦ C until examination for biomechanical testing. 1.3. Micro-computed tomography studies For a detailed qualitative and quantitative 3-D evaluation, tibial bones were examined by a desktop micro-computed tomography system (eXplore Locus MicroCT, the General Electric Company, New York, USA), voltage 80V, current 450 A, number of views 400, exposure time 2000 ms, bin mode 1 × 127 m. To scan the entire tibial head, a total of 500 microtomographic slices were acquired at a 27 m slice increment. Morphometrical parameters were determined using a direct 3-D approach in three different pre-selected analysis regions: 1.8 × 1.5 × 1.4 mm3 area in the proximal tibial metaphysis [13,14].
Data were analyzed using one-way ANOVA followed by Student’s t-test using SPSS software (version 13.0). Results are reported as mean ± standard deviation (SD). Statistical significance was established for two-tailed values of P < 0.05. 2. Results 2.1. Serum biochemical studies Results of serum parameters in rats of different groups are in Table 1. The results indicated that L-Arg treatment reduced serum ALP and OC levels significantly (P < 0.05) in OVX model group compared to Sham group. L-Arg 10 mg/kg showed the most significant effect on reduction of the serum ALP level among three different dosages. As result, there was no significant difference of the ALP level between L-Arg 10 mg/kg group and sham group. 2.2. Micro-computed tomography studies A significant difference of tibial parameters determined by micro-CT between different groups was observed. Trabecular BV/TV, Tb.Th, Tb.N, BMC and BMD in L-Arg (5 mg/kg) group and LArg (10 mg/kg) groups were greater than those of Sham group, but trabecular Tb.Sp were lower (P < 0.05) in both L-Arg groups than that in Sham group (Fig. 1). Trabecular Tb.Th of L-Arg 20 mg/kg group was greater than that of OVX group, but Tb.Sp and other parameters became lower than those of OVX group (Table 2). 2.3. Histopathological studies Histopathological studies of the L-Arg 10 mg/kg group exhibited the most significant effect on OVX rat’s tibia while bone trabecular re-connected and turn thicker, the number of bone trabecular increased and the separation degree decreased. The bone trabecular re-connected but there were a number of fissure in the tibia section of L-Arg 5 mg/kg group’s rats (Fig. 2). L-Arg 20 mg/kg had negative effect on the OVX rats’ tibia while most of bone parameters turned worse. 2.4. Biomechanical studies
Following mCT image acquisition, the specimens were fixed in 10% phosphate buffered formalin for 24 h, then dehydrated in progressive concentrations of ethanol, defatted in xylene and embedded undecalcified in methyl methacrylate. The frontal sections were cut at 10-micrometer thicknesses with a microtome (Leica RM2155, Germany) followed by HE staining. The sections were mounted and then observed under microscope for histopathological changes.
The biomechanical properties of femur decreased significantly in OVX group compared to those of Sham group. Following administration of L-Arg (10 and 20 mg/kg) for four weeks, the maximal stress and elastical stress increased significantly, compared to those in untreated OVX group. There was no significant effect including maximal load, elastical load, maximal stress, or elastical stress or interaction in L-Arg 5 mg/kg group. Especially, a major effect, maximal load and elastical load, was observed to be increased significantly in L-Arg 10 mg/kg group (Figs. 3 and 4). Thus, the treatment of L-Arg 10 mg/kg had beneficial effects on femoral breaking strength as assessed by three-point bending.
1.5. Biomechanical studies
3. Discussion
The femur specimen stored at –80 ◦ C were transferred into a freezer for twenty-four hours before they were assessed for their biomechanical strength by three-point bending test using the
In this study, different dosages (5, 10, 20 mg/kg) of L-Arg were examined on the treatment of OVX rats [8,10,14]. OVX rat is one of the commonly used animal model for the evaluation of drug effect
1.4. Histopathological studies
–4 ◦ C
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Table 1 Effect of L-Arginine (L-Arg) on ovariectomized (OVX) rats after 4 weeks as determined by serum biochemical examination. Parameter
Ca (mmol/L)
Sham OVX L-Arg 5 mg/kg L-Arg 10 mg/kg L-Arg 20 mg/kg
2.50 2.48 2.33 2.20 2.41
± ± ± ± ±
0.11 0.08 0.08a,b,d 0.08a,b,c,e 0.08d
P (mmol/L) 1.57 1.55 1.71 1.71 1.29
± ± ± ± ±
0.13 0.11 0.07a,b,e 0.05a,b,e 0.07a,b,c,d
ALP (U/L) 89.66 161.46 149.66 57.84 128.70
± ± ± ± ±
OC (ng/mL) 14.06 8.86a 3.07a,d,e 1.13b,c,e 7.1a,b,c,d
14.56 18.41 23.36 17.33 20.29
± ± ± ± ±
1.52 1.94a 1.27a,b,d,e 0.81a,c,e 2.33a,b,c,d
All groups were n = 8 SD rats; data are presented as mean ± standard error. Groups were compared by one-way ANOVA method. F test was used when compared the difference between groups, and T test was used when compared two out of the groups. ALP: alkaline phosphatase; OC: osteocalcin. a P < 0.05, when compared with Sham group. b P < 0.05, when compared with OVX group. c P < 0.05, when compared with OVX group treated with L-Arg 5 mg/kg. d P < 0.05, when compared with OVX group treated with L-Arg 10 mg/kg. e P < 0.05, when compared with OVX group treated with L-Arg 20 mg/kg.
on osteoporosis. The measurement of the activity of serum ALP can be used as a parameter of bone formation and it may be used in clinic. Total serum ALP activity is the most commonly used marker of bone formation. It is a sensitive marker of the increased bone turnover in postmenopausal women and is an accurate index of the effects of antiresorptive drugs on bone turnover. The concentration
of serum OC was not only used for the evaluation of rat osteoporosis model, it was also used as an important parameter on evaluation of drug effects on the treatment of osteoporosis [1]. Our results showed that ALP was reduced in OVX rats after the administration of different dosage of L-Arg. L-Arg of 10 mg/kg showed the most significant effect, and ALP reduced to a level which was not significant
Fig. 1. 3-D mCT images of the proximal tibial cancellous bone after 4 weeks L-Arginine (L-Arg) treatment. A. L-Arg 5mg/kg. B. L-Arg 10mg/kg. C. L-Arg 20mg/kg. D. OVX. E. Sham.
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Table 2 Effects of L-Arginine (L-Arg) on rats femurs after 4 weeks as determined by micro-CT. Parameter
BV/TV (%)
Sham OVX L-Arg 5 mg/kg L-Arg 10 mg/kg L-Arg 20 mg/kg
0.25 0.19 0.50 0.60 0.11
± ± ± ± ±
0.02 0.01a 0.04a,b,d,e 0.06a,b,c,e 0.04a,b,c,d
Tb.Th (mm) 0.07 0.06 0.10 0.10 0.08
± ± ± ± ±
0.01 0.01a 0.02a,b,e 0.02a,b,e 0.02a,c,d
Tb.N (1/mm) 3.31 2.88 4.19 6.61 1.33
± ± ± ± ±
0.11 0.08a 0.10a,b,d,e 0.27a,b,c,e 0.05a,b,c,d
Tb.Sp (mm) 0.20 0.27 0.13 0.08 0.68
± ± ± ± ±
0.01 0.01a 0.03b,e 0.03a,b,e 0.04a,b,c,d
BMC (mg) 0.73 0.57 1.00 1.32 0.37
± ± ± ± ±
0.04 0.04a 0.13b,d,e 0.07a,b,c,e 0.05a,b,c,d
BMD (mg/cc) 218.69 168.44 303.80 389.41 107.95
± ± ± ± ±
10.82 11.18a 13.07a,b,d,e 5.93a,b,c,e 5.06a,b,c,d
All groups were n = 8 SD rats; data are presented as mean ± standard error. Groups were compared by one-way ANOVA method. F test was used when compared the difference between groups, and T test was used when compared two out of the groups. BV/TV: bone volume/tissue volume; Tb.Th: tabecular thickness; Tb.N: tabecular number; Tb.Sp: tabecular separation; BMC: bone mineral content; BMD: bone mineral density; OVX: ovariectomized. a P < 0.05, when compared with Sham group. b P < 0.05, when compared with OVX group. c P < 0.05, when compared with OVX group treated with L-Arg 5 mg/kg. d P < 0.05, when compared with OVX group treated with L-Arg 10 mg/kg. e P < 0.05, when compared with OVX group treated with L-Arg 20 mg/kg.
different from that in the Sham group; OC in the 10 mg/kg group was also reduced while OC in the other two OVX groups increased. Low calcium and high phosphorus in periphery blood were seen in all L-Arg treatment groups. It is then suggested that treatment of osteoporosis should accompanied with the administration of calcium and monitoring of serum calcium and phosphorus levels.
The proximal tibial metaphysis and the distal femural metaphysis consist of abundant cancellous bone and could be easily induced bone loss by ovariectomy on rats, then they are the ideal area for the verification of the effects of anti-osteoporosis drug on OVX rats in a relatively short period of time. It has to be emphasized that the histopathological observation should be carried out
Fig. 2. Photomicrographs of the proximal tibial metaphyseal trabecular bone after 4 weeks L-Arginine (L-Arg) treatment. A. L-Arg 5mg/kg. B. L-Arg 10mg/kg. C. L-Arg 20mg/kg. D. OVX. E. Sham.
H. Guo et al. / Biomedicine & Aging Pathology 4 (2014) 117–122
Fig. 3. Effect of L-Arginine (L-Arg) on biomechanical parameter-Load.
in the secondary cancellous bone area as bone grows constantly. Early morphological changes are very important on the evaluation of animal model and the treatment effect. Although the test of bone density is a widely used parameter on evaluating animal model, in the diagnostic of osteoporosis in clinic as well as the parameter of drug evaluation, it only shows its effect when a significant loss of bone can be observed, thus its sensitivity and repeatability are poor. In the meantime, the biochemical parameters of blood and urine can only partly reflect the changes in animal model and therefore, it’s more often used as co-parameters. Compared with bone density measurement and biochemical parameters, morphometric parameters can not only describe the changes of quantity of bone and morphology, but also reflects the dynamic process of bone metabolism. Change of morphology is an early indicator of pathophysiological process and an indicator of pharmacological function. The quantitive determination of bone bio-function is an important parameter relating to bone function, such as possibility of bone fracture. It’s better to combine the morphometric and biochemical parameters to evaluate the physiological, pathological and function changes of bone. It will also help the access the efficacy of new drugs. In this study, we found that bone trabecular re-connected and turn thicker in L-Arg 10 mg/kg group, the number of trabecular
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increased and the tabecular separation decreased. In this group, the bone mass increased and the bone micro-architecture improved. MicroCT has attracted attention among medical professionals for its widely application on cancer screening, disease monitoring and drug evaluation. A 3-D image of scanned microstructure can be displayed on MicroCT, and the sub-cellular structure can be observed on MicroCT at highest resolution. The description parameters obtained from Micro-CT, which related to 3-D image, is more accurate than the 2-D data. To scan the representative target and reconstruct the 3-D image are very important. MicroCT has been widely used in drug evaluation [16,17]. MicroCT 3-D image revealed that the increase of bone mass in 5 mg/kg and 10 mg/kg groups were significant greater than that in Sham group. A number of parameters, including BV/TV, Tb.Th, Tb.N, BMD and BMC in 5 mg/kg and 10 mg/kg groups were significantly improved than those in Sham group, while the 5 mg/kg group showed less effect although the difference between 5 mg/kg and 10 mg/kg group was not significant. However, in L-Arg 20 mg/kg group, Tb.Sp increased significantly. It implied that 5 mg/kg and 10 mg/kg L-Arg could be used to treat the osteoporosis induced by ovariectomy in rats. The loss of bone mass and changes of bone structure will eventually affect the bio-function and reduce the strength of bone. Therefore, changes of biomechanics are used as a unique parameter for evaluation of the outcome of antiosteoporotic treatment. Three-point bending test is one of the commonly used methods for this purpose [18,19]. The fundamental principal of treatment is to increase the quantity of bone mass and improve the bone structure, as results, the mechanic properties of bone can be improved. In our study, the biomechanical parameters of femur were improved significantly in L-Arg 10 mg/kg group when compared to untreated OVX group. The maximal load together with elastic load of L-Arg 10 mg/kg group were not significant different from those in Sham group. It has been found in this study that not only the bone mass was increased and the bone structure was improved after L-Arg (10 mg/kg) treatment but also the bone biomechanical activity was recovered. L-Arg 5 mg/kg treatment had no effect on the OVX bone mechanical parameters. 4. Conclusions L-Arg, one of the generators of NO in vivo, is effective on the treatment of osteoporosis in OVXrats. L-Arg (10 mg/kg) contributes significantly to the treatment of the bone loss induced by ovariectomy of rats. Not only the bone mass was increased and the bone structure was improved after L-Arg (10 mg/kg) treatment but also the bone biomechanical activity was recovered. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments We are grateful to Professor Xiaojun Xu, Bojiang Shen and Dr. Dajun Wang for the language amendment and their helpful advices. This work was supported by grants from the Chinese National Natural Sciences Foundation Projects (30772450) and Beijing Natural Sciences Foundation Projects (7082086). References
Fig. 4. Effect of L-Arginine (L-Arg) on biomechanical parameter-Stress.
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