Tag Archives: animal

NAC Stimulates Osteoblastogenesis in Rats

Abstract

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration.

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

Yamada M, Tsukimura N, Ikeda T, Sugita Y…
Biomaterials Aug 2013
PMID: 23711675

NAC Prevents Skeletal Aging in Transgenic Mice

Abstract

Swedish mutant APP suppresses osteoblast differentiation and causes osteoporotic deficit, which are ameliorated by N-acetyl-L-cysteine.

Reduced bone mineral density and hip fracture are frequently observed in patients with Alzheimer’s disease (AD). However, mechanisms underlying their association remain poorly understood. Amyloid precursor protein (APP) is a transmembrane protein that is ubiquitously expressed in bone marrow stromal cells (BMSCs), osteoblasts (OBs), macrophages (BMMs), and osteoclasts (OCs). Mutations in the APP gene identified in early-onset AD patients are believed to cause AD. But little is known about APP’s role in bone remodeling. Here, we present evidence for Swedish mutant APP (APPswe) in suppression of OB differentiation and function in culture and in mouse. APP expression in BMSCs increases during aging. Ubiquitous expression of APPswe in young adult Tg2576 transgenic mice (under the control of a prion promoter) recaptured skeletal “aging-like” deficits, including decreased OB genesis and bone formation, increased adipogenesis and bone marrow fat, and enhanced OC genesis and bone resorption. Remarkably, selective expression of APPswe in mature OB-lineage cells in TgAPPswe-Ocn mice (under the control of osteocalcin [Ocn] promoter-driven Cre) also decreased OB genesis and increased OC formation, resulting in a trabecular bone loss. These results thus suggest a cell-autonomous role for APPswe in suppressing OB formation and function, but a nonautonomous effect on OC genesis. Notably, increased adipogenesis and elevated bone marrow fat were detected in young adult Tg2576 mice, but not in TgAPPswe-Ocn mice, implying that APPswe in BMSCs and/or multicell types in bone marrow promotes bone marrow adipogenesis. Intriguingly, the skeletal aging-like deficits in young adult Tg2576 mice were prevented by treatment with N-acetyl-L-cysteine (NAC), an antioxidant, suggesting that reactive oxygen species (ROS) may underlie APPswe-induced osteoporotic deficits. Taken together, these results demonstrate a role for APPswe in suppressing OB differentiation and bone formation, implicate APPswe as a detrimental factor for AD-associated osteoporotic deficit, and reveal a potential clinical value of NAC in the treatment of osteoporotic deficits.

Xia WF, Jung JU, Shun C, Xiong S…
J. Bone Miner. Res. Oct 2013
PMID: 23649480

NAC Prevents Bone Loss in Diabetic Rats

Abstract

N-acetylcysteine decreases alveolar bone loss on experimental periodontitis in streptozotocin-induced diabetic rats.

The purpose of this study was to evaluate the morphometric and histopathological changes associated with experimental periodontitis in diabetic rats in response to systemic administration of N-acetylcysteine (NAC), a sulfhydryl-containing thiol antioxidant.
Sixty Wistar rats were divided into six experimental groups: nonligated (NL) group; ligature-only (L) group; streptozotocin-only (STZ) group; STZ and ligature (STZ + L) group; and systemic administration of NAC and ligature (70 and 100 mg/kg body weight per day, respectively) (NAC70 and NAC100 groups). Diabetes mellitus was induced by 60 mg/kg of streptozotocin. Silk ligatures were placed at the gingival margin of the lower first molars of the mandibular quadrant. The study duration was 30 d and the animals were killed at the end of this period. Changes in alveolar bone levels were clinically measured and tissues were histopathologically examined to assess the differences among the study groups.
At the end of the 30-d study period, alveolar bone loss was significantly higher in the STZ + L group compared with the other groups (p < 0.05). Also, alveolar bone loss in all the NAC groups was significantly lower than in the STZ + L and L groups (p < 0.05). The osteoblastic activity in the NAC100 group was significantly higher than in the other groups (p < 0.05).
Within the limits of this study, it can be suggested that NAC, when administered systemically, prevents alveolar bone loss in the diabetic rat model.

Toker H, Ozdemir H, Balcı H, Ozer H
J. Periodont. Res. Dec 2012
PMID: 22712627

Homocysteine Decreases Bone Quality and is Antagonized by NAC

Abstract

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1.

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1β, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality.

Vijayan V, Khandelwal M, Manglani K, Singh RR…
Free Radic. Biol. Med. Mar 2013
PMID: 23500899

MMPs a Potential Target

Abstract

Hydrogen sulfide and sodium nitroprusside compete to activate/deactivate MMPs in bone tissue homogenates.

Bone microvascular remodeling is the primary predictor of bone structure and function. Remodeling by its very nature implies synthesis and degradation of the extracellular matrix. Normally, 50% of total protein in the vessel wall is elastin. During remodeling, elastin is degraded by specialized matrix metalloproteinases (MMPs). Because the turnover of elastin is 1000-fold slower than that of collagen, most of the elastin is replaced by stiffer collagen. Stiffer vessels impose pressure on the aortic valve, causing regurgitation and increased pulse pressure. On the other hand, high MMP activity will cause vascular dilatation, leading to aneurysm. Therefore, balanced constitutive remodeling is necessary for adequate bone structure and function. Interestingly, collagen-degrading MMPs are involved in various pathological conditions, including osteoporosis, osteoarthritis, and cardiovascular disease.

Sodium nitroprusside is a nitric oxide donor that could potentially alter MMP activity via vasodilation in vivo, but can also produce peroxynitrite, which activates MMPs by combining with superoxide. Moreover, hydrogen sulfide is a known antioxidant as well as a vasodilator, and is also speculated to contribute directly to MMP activity. We hypothesized that hydrogen sulfide reduced activity of MMP in ex vivo bone tissue homogenates and that sodium nitroprusside would increase MMP activity in vitro.
We surgically removed the tibia and femur from anesthetized mice, and prepared bone tissue homogenates using a mortar and pestle, measured the protein concentration with a spectrophotometer, and detected MMP activity using gelatin gel zymography.
Our data showed increased MMP activity at a sodium nitroprusside concentration of 1 μM, and MMP activity increased exponentially. There was a decrease in MMP activity with increasing hydrogen sulfide, beginning at 16 μM (P < 0.01) and continuing to 40 μM. Moreover, sodium nitroprusside 3 μM was able to overcome the decrease in MMP activity that occurred with hydrogen sulfide 40 μM; this resulted in a more pronounced exponential increase in MMP activity.
There are several substances that can potentially be used to decrease MMP activity and to alleviate pathological remodeling by MMPs.

Vacek TP, Qipshidze N, Tyagi SC
Vasc Health Risk Manag 2013
PMID: 23658491 | Free Full Text

Folate Prevents Bone Loss in Cyclosporine-Treated Rats

Abstract

Protective effect of folic acid on cyclosporine-induced bone loss in rats.

Hyperhomocysteinemia is seen in patients with decreased bone mineral density. Cyclosporine can cause alveolar bone loss and osteopenia. It is also associated with elevated serum homocysteine levels. We aimed to investigate the effect of cyclosporine on serum homocysteine level, bone volume, and bone density, and determine whether folic acid had a protective effect against bone loss. In an experimental study, 40 male Sprague-Dawley rats were randomly assigned to five groups and received dietary supplementation for 6 weeks with olive oil (Group A), cyclosporine (Group B), folic acid (Group C), and cyclosporine plus folic acid (Group D), or no supplementation (Group F, control). Serum homocysteine, calcium, alkaline phosphatase, total bone volume, periodontal ligament volume, and volume density of bone were compared between groups. Mean serum homocysteine level (10.84 ± 0.93 μmol/l) was significantly higher in group B (cyclosporine supplementation) compared with the other groups (P = 0.001). Mean total mandibular volume was 46.3 ± 13.6 mm(3) in rats treated with cyclosporine, 80.4 ± 15.70 mm(3) in rats treated with folic acid (P = 0.004), and 73.9 ± 21.3 mm(3) in rats treated with cyclosporine plus folic acid (P = 0.028). In our experimental model, cyclosporine increased serum homocysteine levels and decreased bone volume and density. Folic acid may have a preventive role against bone loss in rats treated with cyclosporine.

Mohammadi A, Omrani L, Omrani LR, Kiani F…
Transpl. Int. Jan 2012
PMID: 22039919

AKG Prevents Bone Loss in Dexamethasone-Treated Pigs

Abstract

Postnatal administration of 2-oxoglutaric acid improves articular and growth plate cartilages and bone tissue morphology in pigs prenatally treated with dexamethasone.

The potential effects of prenatal administration of dexamethasone (DEX) and postnatal treatment with 2-oxoglutaric acid (2-Ox) on postnatal development of connective tissue of farm animals were not examined experimentally. The aim of this study was to establish changes in morphological parameters of bone and articular and growth plate cartilages damaged by the prenatal action of DEX in piglets supplemented with 2-Ox. The 3 mg of DEX was administered by intramuscular route every second day from day 70 of pregnancy to parturition and then piglets were supplemented with 2-Ox during 35 days of postnatal life (0.4 g/kg body weight). The mechanical properties, BMD and BMC of bones, and histomorphometry of articular and growth plate cartilages were determined. Maternal treatment with DEX decreased the weight by 48%, BMD by 50% and BMC by 61% of the tibia in male piglets while such action of DEX in female piglets was not observed. DEX led to thinning of articular and growth plate cartilages and trabeculae thickness and reduced the serum GH concentration in male piglets. The administration of 2-Ox prevented the reduction of trabeculae thickness, the width of articular and growth plate cartilages in male piglets connected with higher growth hormone concentration compared with non-supplemented male piglets. The result showed that the presence of 2-Ox in the diet had a positive effect on the development of connective tissue in pigs during suckling and induced a complete recovery from bone and cartilage damage caused by prenatal DEX action.

Tomaszewska E, Dobrowolski P, Wydrych J
J. Physiol. Pharmacol. Oct 2012
PMID: 23211309 | Free Full Text

AKG shows Only a Slight Influence on Bone in Rats

Abstract

The effect of dietary administration of 2-oxoglutaric acid on the cartilage and bone of growing rats.

2-Oxoglutaric acid (2-Ox), a precursor to hydroxyproline – the most abundant amino acid in bone collagen, exerts protective effects on bone development during different stages of organism development; however, little is known about the action of 2-Ox on cartilage. The aim of the present study was to elucidate the influence of dietary 2-Ox supplementation on the growth plate, articular cartilage and bone of growing rats. A total of twelve male Sprague-Dawley rats were used in the study. Half of the rats received 2-oxoglutarate at a dose of 0·75 g/kg body weight per d in their drinking-water. Body and organ weights were measured. Histomorphometric analyses of the cartilage and bone tissue of the femora and tibiae were conducted, as well as bone densitometry and peripheral quantitative computed tomography (pQCT). Rats receiving 2-Ox had an increased body mass (P<0·001) and absolute liver weight (P=0·031). Femoral length (P=0·045) and bone mineral density (P=0·014), overall thickness of growth plate (femur P=0·036 and tibia P=0·026) and the thickness of femoral articular cartilage (P<0·001) were also increased. 2-Ox administration had no effect on the mechanical properties or on any of the measured pQCT parameters for both bones analysed. There were also no significant differences in histomorphometric parameters of tibial articular cartilage and autofluorescence of femoral and tibial growth plate cartilage. Dietary supplementation with 2-Ox to growing rats exerts its effects mainly on cartilage tissue, having only a slight influence on bone. The effect of 2-Ox administration was selective, depending on the particular bone and type of cartilage analysed.

Dobrowolski P, Tomaszewska E, Bienko M, Radzki RP…
Br. J. Nutr. Aug 2013
PMID: 23308390

AKG Unimpressive in Omeprazole-Induced Bone Loss in Rats

Abstract

Can 2-oxoglutarate prevent changes in bone evoked by omeprazole?

Proton-pump inhibitors, such as omeprazole, are widely used in the prevention and treatment of gastroesophageal diseases. However, an association between proton-pump inhibitors and the increased risk of bone fractures has been observed, especially in patients treated for extended periods. Conversely, 2-oxoglutarate, a precursor of hydroxyproline, the most abundant amino acid in bone collagen, counteracts the bone loss. The aim of the present study was to elucidate the influence of omeprazole on bone and investigate whether dietary 2-oxoglutarate supplementation could prevent the effects of omeprazole.
Eighteen male Sprague-Dawley rats were used. Rats received omeprazole in the diet and 2-oxoglutarate in the drinking water. Body and organ weights and serum concentrations of cholecystokinin and gastrin were measured. The femurs, tibias, and calvarias were collected. Histomorphometric analysis of bone and cartilage tissues was conducted. Bone densitometric and peripheral quantitative computed tomographic analyses of the femur and tibia were performed.
Omeprazole decreased the femur and tibia weights, the mechanical properties of the femur, the volumetric bone density and content, the trabecular and cortical bone mineral content, the total, trabecular, and cortical bone areas, the mean cortical thickness, and the periosteal circumference of the femur. Omeprazole had a minor effect on the examined bone morphology and exerted negligible effects on the cartilage. 2-Oxoglutarate lowered the gastrin concentration.
Omeprazole treatment exerts its effects mostly on bone mineralization and cancellous bone, adversely affecting bone properties. This adverse effect of omeprazole was not markedly abolished by 2-oxoglutaric acid, which acted as an anti-hypergastrinemic agent.

Dobrowolski P, Tomaszewska E, Radzki RP, Bienko M…
Nutrition Mar 2013
PMID: 23218481

AKG Helps Prevent Induced Osteopenia in Pigs

Abstract

2-Oxoglutaric acid administration diminishes fundectomy-induced osteopenia in pigs.

Partial surgical removal of the stomach (fundectomy, FX) leads to osteopenia in animals and humans. FX adversely affects the bone. 2-oxoglutaric acid is a precursor of glutamine and hydroxyproline–the most abundant amino acid of collagen. The aim of the study was to investigate the effects of 2-oxoglutaric acid on FX-evoked osteopenia in pigs.

Eighteen castrated male pigs of the Puławska breed were used. Twelve pigs were subjected to FX and divided into two groups: FX + AKG (the AKG group; AKG at the daily dosage of 0.4 g/kg of body weight) and FX + Placebo (the FXC group; received CaCO(3) as placebo). Remaining six pigs were sham-operated (the SHO group). The pigs were euthanized at the age of 8 months and long bones were collected. Area bone mineral density (aBMD) and bone mineral content (BMC) were measured; morphology, geometry and biomechanical properties were determined. Moreover, the serum concentrations of selected hormones and one marker of bone metabolism were determined. FX caused osteopenia in the pigs and treatment with AKG greatly reduced these effects of FX in pigs. Negative effect of fundectomy on the skeletal system leading to decreased bone mass in pigs is associated with lowered body gain and activity of the gastric-hypothalamic-pituitary axis. Better definitions of each of the local and systemic hormonal and structural components associated with fundectomy-induced decreased bone mass that separately and together determine the whole bone properties may lead to identify opportunities for prevention.

Śliwa E
J Anim Physiol Anim Nutr (Berl) Oct 2010
PMID: 20487101