The effect of Hyperhomocysteinemia on the Osteoclasts activity in Male New Zealand White Rabbits

 

Mohammed Majid AL-qanbar¹*, Wefak Jbori AL-Bazi¹, Hepa A. Abd-Alsalam²

¹Department of Physiology, College of Veterinary Medicine, University of Kerbala, Iraq.

²College of Education for Pure Science, University of Kerbala, Iraq.

 

ABSTRACT:

Methionine is a specific amino acid which contains sulfur, and can be used to make proteins, found in fish, meat, and dairy products, the excess intake of L-methionine lead to elevated homocysteine (Hcy) level that known as Hyperhomocysteinemia (HHcy). Increased Hcy plasma may represent an independent risk factor for osteoporotic fractures, and therefore may also negatively affect bone metabolism. This study was designed to examine the impact of Hcy on osteoclast activity in Male Rabbits, following methionine overload. To achieve this study's aims, we recruiting (20) males of New Zealand white rabbits that were divided into (10/group) control group and a group treated with methionine. Then after the intubation of methionine overload, we measured the "Receptor Activator of Nuclear factor Kappa-b" (RANK) and "Receptor Activator of Nuclear factor Kappa-b ligand" (RANK-L) levels in the blood, in addition to histological examination of the trabecular structure of femur bone. The results show a significant (p≤0.001) increase in serum RANK and RANK-L levels of methionine treated group in comparison with the control group. The histological examination of the trabecular structure of femur bone shows an increase in osteoclasts percentage, activity, and large resorption pits in the methionine treated group. The HHcy that was induced by methionine overload, caused an increase in osteoclast activity and numbers in male rabbits suggested a mechanistic role for bone resorption by Hcy. Future research clarifying the mechanistic function of elevated concentrations of Hcy in osteoporosis may have important therapeutic implications.

 

 

INTRODUCTION:

Methionine is essential amino acids that contain sulfur, it is found in eggs, fish, meat, cheese, and rice¹, and it is a source of Hcy in blood, that is formed in the metabolic pathway between methionine and cysteine². Methionine excess contributes to higher Hcy levels that cause a status of Hyperhomocysteinemia (HHcy)³, It also happens either because of a co-factor deficiency "folic acid, vitamins B6, vitamins B9" that has a major role in methionine metabolism⁴ or because of genetic defects of the transcription of enzymes responsible for the metabolism of Hcy, particularly the polymorphisms of the major enzymes involved in Hcy metabolism such us Methylenetetrahydrofolate reductase and Cystathionine-β-synthase^5,6.

 

Homocysteine level decreases but not significantly after treatment with telmisartan whereas its level increases significantly after treatment with amlodipine⁷. The Hcy is considered one of the strongest oxidant agents in the living cells⁸, Which has the potential to induce: inflammation, oxidative stress, endothelium dysfunction, and endoplasmic reticulum stress have been considered to play the main role in the pathogenesis of several diseases including atherosclerosis^9,10, myocardial infarction, cardiovascular disorders, stroke, Parkinson's disease dementia, epilepsy, multiple sclerosis, eclampsia, cancer development, autoimmune disease, kidney disease, and social life^11-17. A recent study has suggested HHcy as a newly recognized risk factor for osteoporosis¹⁸. It has been reported that the ethanolic extract of Rhizophora mucronata poir has significant antihyperhomocysteinemic and procoagulant factors lowering potential in hyperhomocysteinemic rats¹⁹.

 

Osteoporosis: is a major health problem characterized by deterioration of bone microarchitecture, low bone mineral density, and increased fracture risk. Osteoporotic fractures are related to increased morbidity and mortality levels and with substantial economic costs ²⁰. The homeostasis of the bone depends on bone resorption by osteoclasts and osteoblast formation of the bone. Imbalance of this closely connected process can cause diseases such as osteoporosis^21,22. Osteoblasts: are cells that synthesize new bone, whereas osteoclasts: are specialized cells that resorb bone. The receptor activator of nuclear factor-kappa-b ligand (RANK-L) a protein expressed by the osteoblasts has a major role in the osteoclasts development function and survival through its interaction with receptor activator of nuclear factor-kappa-b" (RANK) on the osteoclast^22-24. Bone resorption and turnover rate among individuals with HHcy have been reported to increase²⁵. The molecular mechanisms of how Hcy disrupted bone remodeling, or how bone resorption increased, are still unclear²⁶. Recently abundant research on the interaction of Hcy and the bone has been published. And multiple mechanisms and theories about this role of Hcy in bone pathology have been suggested. Some of these studies have been effective in finding a correlation between Hcy and bone remolding and others have failed^27-31, a study by Herrmann et al Proposed four mechanisms by which Hcy improves osteoclastic activity to alter bone remodeling: a- reduction in osteoblastic activity, b- increase in osteoclast activity, c- reduction in blood flow in bone and, d- direct interaction of Hcy with bone matrix³².

 

Therefore, this study aimed to investigate the effect of Hcy that on osteoclast activity by measuring the levels of RANK and RANK-L after methionine overload and compared to the control group, also, to study the histological changes that occur in bone tissues.

 

MATERIALS AND METHODS:

This study was carried out from October 2019 to January 2020 in the Department of Physiology, Biochemistry and pharmacology/college of Veterinary Medicine of Kerbala University.

 

In this study, we recruited 20 males of New Zealand rabbits at 6 months of age that divided randomly (10/group) the control group and treated group with methionine, were housed in a typical housing with regulated temperature (25±3°C), with maintained on a 12-h:12-h light: dark cycle, besides the free access to the food and water, after two weeks of adaptation, we intubated (100mg/kg of BW) L- methionine to the treated group daily for 12 weeks.

 

The two groups were anesthetized by using a single dose of Ketamine (50mg/kg) administered slowly through the (lateral marginal vein) with the administration of xylazine (0.6 ml) inter Muscular³³. The autopsy, blood samples collected, and bones were done in the research laboratory of the College of Veterinary Medicine, University of Kerbala.

 

Biochemical study:

A blood sample was taken to undergo serum RANK and RANK-L estimates with the use of a commercial enzyme-linked-immunosorbent-assay (ELISA) test: "Rabbit Tumor Necrosis Factor Receptor Superfamily Member 11A" ELISA Kit and "Rabbit Receptor Activator of Nuclear Factor Kappa B Ligand" ELISA kit respectively. Both kits were manufactured by bioassay technology laboratory shanghai – China.

 

Histological study:

For the specimen preparation for the light microscope examination, during the autopsy process, one femur from each subject was separated from the carcass. Remove the fur and flesh, and preserved in formaldehyde (10%) for two days and after that, it was sent to the laboratory for histological examination. Gross dissection and paraffin block preparation done after one day (24 hr) decalcification by 12% nitric acid, thereafter automated processing of the sample using Thermoscientific Fischer pathology system/UK, formed by an automated processor, paraffin embedding system, and automated microtome used to prepare slides after paraffin blocking, Each slide stained with hematoxylin and eosin stain³⁴.

 

Statistical analysis:

Data were presented in mean ±Standard Deviation (SD), the data were analyzed by using SPSS-25 by measuring "independent samples T-test" after being coded. The comprised of significant P-value in any test were significant difference P<0.05 and Non-Significant difference P>0.05 ³⁵.

 

RESULTS:

Table (1) shows the effect of daily oral intubation of methionine (100mg/kg of BW) for 12 weeks on serum "Receptor Activator of Nuclear factor Kappa-b (RANK)" concentration (ng/ml). This table shows a significant (p≤0.001) increase in the serum RANK (ng/ml) concentration in the methionine treated group when compared with the control group.

 

Table 1: The effect of daily oral intubation of methionine on serum RANK concentration.

Groups	Parameter	M ± SD	P-value
Control	RANK	2.84±0.75	0.001
treat		5.32±0.66

Values are expressed as mean (M) ±(SD). N = 10/group, statistically highly significant difference between the treated and control group.

 

Table (2) illustrates the effect of daily oral intubation of methionine (100mg/kg of BW) for 12 weeks on serum "Receptor Activator of Nuclear factor Kappa-b ligand (RANK-L)" concentration (ng/l), this table shows a highly significant (p≤0.001) increase in the serum RANK-L (ng/l) concentration in methionine treated group when compared with the control group.

 

Table 2: The effect of daily oral intubation of methionine on serum RANK-L concentration

Groups	parameter	M ± SD	P-value
Control	RANK-L	69.18±18.67	0.001
treat		123.85±15.44

Values are expressed as mean (M) ±(SD). N = 10/group, statistically highly significant difference between the treated and control group.

 

Histological examination:

Figure (1) Shows the histological section of the femur head under a light microscope of control animal note: - normal bone trabecula, normal thickness, normal density, and very few osteoclasts, with no surface resorption pits.

 

Figure 1: Histological section of the femur head under a light microscope of the control group appeared the normal composition of bone trabeculae (arrow), (H&E X200).

 

Figure (2) shows the histological section of the femur head under a light microscope of animal treated with methionine (100mg/kg BW) daily for 12 weeks note: - very thin and disconnected bone trabeculae.

 

Figure 2: Histological section of the femur head under a light microscope of the treated group appeared the thin and disconnected bone trabeculae (arrow), (HandE X100).

Figure (3a,b) clarify the histological section of the femur head under a light microscope of animal treated with methionine (100mg/kg BW) daily for 12 weeks note: bone trabecula surface with an increase in osteoclasts and resorption pits.

 

Figure 3a,b: Histological sections of the femur head under a light microscope of the treated group. a: The osteoclast, it is on the surface of bone trabeculae (arrow) (H&E X200). b: Increasing in the resorption pits in bone trabecula(arrow) (H&E X400).

 

DISCUSSION:

Our study's key finding was that osteoclast activity significantly increased in the group treated with methionine. The result of the current study shows that daily intubation of methionine for 12 weeks caused a significant (p≤0.001) increase in serum RANK of methionine treated group when it's compared to the control group (table 1).

 

This rise in serum RANK can be due to Hcy's excess production of reactive oxygen species (ROS) after methionine overload, where several previous studies have shown that "ROS" is essential for the process of osteoclasts differentiation through stimulation of RANK production^26-28, where any increase of the ROS level may lead to bone disorder and destruction³⁹.

 

A study conducted by⁴⁰ showed up the role of M-CSF in the differentiation of hematopoietic precursor cells into osteoclasts by increase the intracellular level of ROS through NADPH oxidase and induced RANK production. Increase serum Hcy leads to increases in the levels of ROS through activation of NADPH oxidase, where several studies show that enzymes NADPH oxidase are very important sources of ROS^38,41,42. It was found that Hcy stimulates phosphorylation of "NADPH oxidase subunits that involved p47phox, p67phox" in addition to the up-regulation of NADPH oxidase 2, and this stimulation, in turn, will raise the ROS levels^43-45.

 

Thus, the role of Hcy is similar the role of M-CSF in ROS production but with more harmful effect, because the high levels of Hcy will contribute in the elevation of ROS production¹², and this subsequently lead to increases RANK expression and production, thus, enhancing osteoclast differentiation and activation by RANK-L⁴⁶, and this in turn negatively reflected on bone health²⁴.

 

In the table (2), the result showed a significant (P≤0.001) increase in serum RANK-L when compared to the control group. this result was consistent with^47,48. Their interpretation of high RANK-L levels was attributed to the oxidative stress induced by Hcy that "deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG (a natural inhibitor of RANKL) and increase RANKL synthesis in osteoblast cultures", where they found 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 Hcy-treated osteoblast cultures". Conversely, Hcy increased RANK-L synthesis in osteoblasts through c-Jun/JNK MAPK signaling mechanisms independent of FOXO1. The HHcy after high methionine diet shows the reduced expression of FOXO1 and OPG and increased synthesis of pro-resorptive and inflammatory cytokines, such as RANKL, M-CSF, GM-CSF, MIP-1α, IL-1α, IL-1β, G-CSF, and TNF-α. These findings stress that HHcy alters the osteoblast's redox regulatory mechanism by activating PP2A and deranging FOXO1 and MAPK signaling cascades, ultimately shifting the OPG: RANK-L ratio toward decreased bone quality and increased osteoclast activity⁴⁷.

 

This raise in serum RANK and RANK-L may also be attributed to the role of prostaglandin E2 (PGE2) that is mediated by Hcy. Where, several studies have suggested that one of the RANK, RANK-L stimulation factors is PGE2^49,50, and according to a study conducted by⁵¹ they found that Cyclooxygenases 2(COX2) and PGE2 stimulate osteoclastogenesis through stimulation of RANK-L production by osteoblasts, increase of RANK production by osteoclasts and inhibition of OPG secretion, the PGE2 also stimulated Interleukin 6 (IL-6) production.

 

IL-6: serves as modulators of osteoclast differentiation and encourages osteoclastogenesis and can cause excessive osteoclastic activity, the "IL-6" is tied to excessive promotion of RANK-L, Makes preosteoclasts more sensitive to stimulation RANK-L, up-regulation of RANK expression and inhibition of OPG, The IL-6 has been linked to many of bone disease including bone-metastatic cancers, osteoporosis, osteolysis and rheumatoid arthritis^52-55.

 

The effect of methionine overload on histological section:

The microscopic examination of histological sections of the femur head for the control group shows normal bone trabeculae with normal thickness and density in addition to very few osteoclasts with no surface resorption pits figure 1. The microscopic examination of histological sections of the femur head for methionine treated group shows very thin and disconnected bone trabecular (figure 2), increases in osteoclasts, and resorption pits on the surface of trabecular bone as shown in figure (3 a,b). These changes in bone tissues of methionine treated group attributed to an increase in osteoclasts number and activity due to increase in RANK and RANK-L levels after methionine overload and occurrence of HHcy, in brief, the binding of RANKL (produce by osteoblast) to RANK, located on the surface of osteoclast precursors, Recruit the cytoplasmic adapter protein TRAF6, resulting in (NF-κB) activation and nucleus translocation; in the nucleus, the translocated (NF-κB) increases the expression of c-Fos; the interaction of (c-Fos) with (NFATc1) leads to increased expression of various osteoclastogenic genes; consequently, osteoclast formation is increased, thereby resulting in increased bone resorption⁵⁶.

 

CONCLUSION:

Our study showed that HHcy led to significant structural changes in the femur trabecular bone in male rabbits. These changes confirmed the association between increased Hcy level and an increase in osteoclast activity that plays a major role of occurrence osteoporosis, which is considered an important factor of the risk of bone fractures.

 

RECOMMENDATION:

Future studies clarifying the mechanistic role of increased Hcy concentrations in osteoporosis could have interesting therapeutic implications because Hcy lowering therapy could be beneficial for osteoporotic patients.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 27.08.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5443-5448.