The Relationship Between Oxidative Stress And Age-related Osteopenia In Rats

BackgroundsOsteoporosis(OP) is the most common metabolic bone disorder, which was estimated that the people with osteoporosis had been more than 200 millions worldwide according to International Osteoporosis Foundation Translation(IOF). There had been more than 70 million patients in our country in the investigation of Editorial Board of Osteoporosis Prevention and Treatment. According to WHO in 2001, OP is characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. The most important risk of osteoporosis is osteoporotic fracture, and hip fractures, spinal fractures and distal radius fractures are common, and the largest risk of which is hip fracture, because of its high morbidity and high mortality. With increasing age, the incidence of osteoporosis increased significantly, the ethiopathogenesis of osteoporosis is related to aging, hypogonadism, genetic, endocrine, nutrition and other factors. Loss of bone mass and decrease of bone turnover are the main pathological features in senile osteoporosis. The 20th century, it was distinct that estrogen deficiency is a reason of in osteoporosis, but the pathophysiology is unclear. In the study of the ethiopathogenesis of aging, reactive oxygen species (ROS) is the main reason of aging in free radical theory. It is indicate that oxidative stress caused by imbalance between oxidation and anti-oxidation may be another reason of senile osteoporosis.Free oxygen radicals or reactive oxygen species (ROS) include hydroxyl radicals (OH-), superoxide anion radical (O2-), hydrogen peroxide (H2O2) and lead to the specific oxidation of some enzymes, protein oxidation and degradation. Their effects are eliminated by enzymatic antioxidant mechanisms such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase. Oxidative stress is an imbalance between the free radicals and antioxidant mechanism in biological systems. Abnormally high levels of oxidants and/or low levels or activity of antioxidants can specifically impair the balance. i.e., Lipid peroxidation caused by attacking on polyunsaturated fatty acid can impair the biofilm's structure and function and cell surface receptors; to damage amina and thiol of proteins induce protein denatuation,crosslinking and loss of enzyme activity;damaging DNA strand breaks and mutations.Free radicals can cause protein aggregation and cross-linked peptide strand breaks, the formation of protein and lipid-bound polymers induce the protein the loss of function. Free radicals attack on amino acid side-chain protein molecules, such as lysine, arginine, proline, threonine, can produce the corresponding protein carbonyl derivatives induced decrease of protein sulfhydryl levels in vivo.protein oxidized by hypochlorous acid (HOC1) in macrophage and oxidized glycosylated protein can produce advanced oxidation protein products (AOPP) and advanced glycation end products (AGE) which can be used as protein marker of oxidative damage. Unsaturated fatty acids attacked by free redicals on biofilm which induces cell deformability decreased and brittleness increased that affecting the ion channel function, increasing membrane permeability. Lipid peroxidation can also add new free radicals and lead to advanced lipid oxidation products such as malondialdehyde (malondialchehyche MDA),4-hydroxy-nonnenal, oxidized low-density lipoprotein (oxidized low density lipoprotein ox-LDL) and iso-prostaglandin. Free radicals can also be related to the base which impairs the DNA strand. i.e.,8-oxo-guanine oxidized by guanine, under the action of the repair enzymes to 8 hydroxy-deoxyguanosine form of excision. Oxygen free radicals can make the collagen fibers of collagen cross-linked, hyaluronic degradated and extracellular matrix demolish. Oxidative stress (OS) caused by increased ROS status in vivo will lead to great damage. The negative role of free radicals is mainly due to its unpaired electrons in structure and it is highly reactive and able to quickly react with the surrounding, then its half-life is very short and difficult to measure. Therefore, oxidation products caused by cross-linked ROS and molecules (lipids, proteins, DNA) can be the markers of oxidative stress.Advanced oxidation protein products (AOPP) are proposed by Witko Sarsat in 1996, which are the dityrosinecontaining and cross-linking protein products. They are formed during oxidative stress by the reaction between proteins and chlorinated oxidants such as chloramines or hypochlorous acid (HOC1). Segmented neutrophils and monocytes can produce the respiratory burst in the appropriate stimulation, which can produce a large number of high activity reactive oxygen species (reactive oxygenspecies, ROS). Phagocytes generates chlorine dioxide under the effect of myeloperoxidase which can make hydrogen peroxide generated hypochlorite (one of the most lively chemical properties and toxic substances produced by phagocytic) in duration of ROS formation. Recently, increased levels of AOPP have been found in the patients with diabetes, uremia, coronary artery disease, chronic inflammatory bowel diseases, as well as obesity, implying that accumulation of AOPP may be relevant in a number of pathophysiologicconditions. AOPP as a new uremic toxins and inflammatory mediators can be used as a novel marker of oxidant-mediated protein damage.Malondialdehyde (MDA) is the principal and most studied product of polyunsaturated fatty acid peroxidation. The main source of MDA in biological samples is the peroxidation of polyunsaturated fatty acids with two or more methylene-interrupted double bonds. Excessive MDA with proteins, nucleic acids and other molecules to form lipofuscin deposition in the cell, and this is an important reason for the body cell senescence. At the same time, it can also react with phospholipid protein, changing cell membrane permeability, causing tissue damage. MDA is the products of lipid peroxidation, its content directly reflects the rate of lipid peroxidation. Therefore, it used as markers to determine the damage of free radicals, indirectly reflects the level of cell damage by free radicals.In 1938, Mann and Keilin were the first one who discovered superoxide dismutase (SOD) isolated from bovine red blood cells. In 1969, Mccord and Fridovich found this protein had function of catalyticing superoxide anion and disproportionation. Superoxide anion radical is a substrate of SOD with not only a negative charge but also one unpaired electronic which can disproportionate free radicals to be non-toxic H2O and O2 in catalase. Superoxide anion radical is necessary to sustain normal life, but it will damage the health when high concentrations. SOD as a specific scavenger, it can disproportionat superoxide anion and keep balance with free radicals avoiding adverse reaction due to excessive concentration of superoxide anion. Decrease in the SOD has a profound effect on the cellular resistance to oxidant induced damage and cell killing. Therefore, SOD are considered as the markers of antioxidant defense mechanismObjectives:There is increasing evidence suggesting the role of free radicals in bone resorption and bone loss. In this study, we investigated 60 Wistar rats at 2,9 and 18 months old (n=20 in each group).To explore the relationship between oxidative stress and age-related osteopenia of rats, and to provide the theoretic basis for the intervention of age-related osteoporosis.Methods:Sixty Wistar rats were divided into 3 groups according to their age:young(2 months n=20),adult(9 months n=20)and old(18 months n=20).The colorimetry was adopted to measure the serum levels of advanced oxidation protein products (AOPP),malonaldehyde (MDA)and superoxide dismutase (SOD)for the rats. Then rats were killed and the right femurs were removed, the tissue homogenates were made and used for the estimation of AOPP, MDA and SOD. The left femur was used for measurement of bone mineral density (BMD). Histomorphometry was done on the left tibia of another 18 animals aged 2,9 and 18 months(n=6). Results:(1)The BMD in femur decreased significantly in 18 months old rats compared with 2 and 9 months old groups (P<0.01),but it was not significant different between 2 and 6 months old groups(P>0.05)(2)From 2 months of age and there-after, trabecular thickness(Tb.Th) decreased and trabecular separation(Tb.SP) increased progressively(P<0.05);In the tibia, mineral apposition rate (MAR) and bone formation rate (BFR/BS) decreased with aging (P<0.05 or P<0.01).(3) The serum levels of AOPP,MDA increased significantly in 18 months old rats compared with 2 and 9 months old ones (P<0.01), but they were not significant different between 2 and 9 months old groups (P>0.05). The levels of SOD showed a decrease with aging (P<0.05). There was a negative correlation between AOPP and femur BMD levels (r=-0.640; p<0.01). The same trend was observed between MDA and femur BMD (r=-0.421; p<0.01). A positive correlation was observed between SOD and femur BMD levels (r=0.470; p<0.01).(4) The supernatant levels in femur of AOPP increased with aging (P< 0.01);Compared with 2 and 9 months old rats, the levels of MDA increased in 18 months old ones (P<0.05); A negative correlation was observed between AOPP and femur BMD levels (r=-0.595; p<0.01).There was a negative correlation between SOD and femur BMD levels (r=0.353;p<0.05).ConclusionsIncrease level of oxidative stress is involved into age-related osteopenia in rats. Oxidative stress might play an important role in the pathophysiology of age-related osteoporosis.