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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 62  |  Issue : 3  |  Page : 108-116

Additive and nonadditive effects of salmon calcitonin and omega-3 fatty acids on antioxidant, hematological and bone and cartilage markers in experimental diabetic-osteoarthritic rats


Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Nigeria

Date of Submission27-Dec-2018
Date of Decision11-Apr-2019
Date of Acceptance16-May-2019
Date of Web Publication25-Jun-2019

Correspondence Address:
Dr. Wale J Adeyemi
P. O. Box 6593, Ilorin
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_8_18

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  Abstract 

Reports on the coexistence of diabetes mellitus and osteoarthritis in human subjects dated back to the 1960s. However, there is no account in literature on the co-manifestation of these disease conditions in experimental animals. In our previous study, we reported for the first time, the effects of pharmacological agents on glucoregulatory indices, lipid profile, and inflammatory markers in experimental diabetic-knee osteoarthritic rat. However, in the present study, the effects of salmon calcitonin (Sct), and/or omega-3 fatty acids (N-3) were further investigated on other biomarkers. Forty-nine rats of seven animals per group were used for this study. Diabetes was induced by the administration of streptozotocin (65 mg/kg) and nicotinamide (110 mg/kg). Thereafter, knee osteoarthritis was induced by the intra-articular injection of 4 mg of sodium monoiodoacetate in 40 μl of saline. Nine days after the inductions, treatments started, and they lasted for 4 weeks. N-3 was administered at 200 mg/kg/day, while Sct was administered at 2.5 and 5.0 IU/kg/day. The results of the study indicated that the induced diabetes-knee osteoarthritis caused significant alterations in all the observed biomarkers. Sct showed a dose-specific effect and an additive action with N-3 in reducing malondialdehyde and lactate dehydrogenase, and in elevating total bilirubin and total antioxidant capacity. However, it largely demonstrated a nondose-specific effect and nonadditive action with N-3 on superoxide dismutase, catalase, glutathione peroxidase, total alkaline phosphatase, c-telopeptide of type-I collagen, collagen type-2 alpha 1, and hematological indices. In conclusion, the combined administration of Sct and N-3 proffer better therapeutic effects than the single therapy; therefore, they could be used in the management of diabetic-osteoarthritic condition.

Keywords: Calcitonin, diabetes, omega-3 fatty acids, osteoarthritis


How to cite this article:
Adeyemi WJ, Olayaki LA. Additive and nonadditive effects of salmon calcitonin and omega-3 fatty acids on antioxidant, hematological and bone and cartilage markers in experimental diabetic-osteoarthritic rats. Chin J Physiol 2019;62:108-16

How to cite this URL:
Adeyemi WJ, Olayaki LA. Additive and nonadditive effects of salmon calcitonin and omega-3 fatty acids on antioxidant, hematological and bone and cartilage markers in experimental diabetic-osteoarthritic rats. Chin J Physiol [serial online] 2019 [cited 2019 Sep 15];62:108-16. Available from: http://www.cjphysiology.org/text.asp?2019/62/3/108/261313


  Introduction Top


Early evidence on the association of diabetes mellitus and osteoarthritis dated back to the 1960s. A higher occurrence of radiographic osteoarthritis has been reported in diabetic individuals than in nondiabetic subjects.[1] Specifically, a significant increase in radiographic hip osteoarthritis was observed in the bilateral joints of diabetics receiving hip arthroplasty.[2] Moreover, epidemiological studies reveal that diabetes has an independent role in the pathogenesis and advancement of osteoarthritis.[3],[4] On the other hand, there are indications that osteoarthritis could precipitate diabetes mellitus.[5]

The hyperglycemic symptom in diabetes triggers the formation of advanced glycation end products,[6] which binds to their receptors and hence instigates pro-inflammatory and pro-oxidant responses.[7] As in diabetes, osteoarthritis is also characterized by diminution in the activities of endogenous antioxidant enzymes, and in the level of nonenzymatic antioxidants,[8],[9] resulting to oxidative stress, and subsequently the progression of the disease process.[10]

Apart from the imbalance in the antioxidant/pro-oxidant status in diabetes, type 2 diabetes is characterized by decline in the number of osteoblast,[11] and hence diminution in bone formation process. However, there are inconsistent reports in literature about the effect of diabetes on bone resorption.[12],[13] Nevertheless, in animal studies, diabetes has been associated with an increased activity of osteoclast.[14] Similarly, osteoarthritis is marked by elevated cartilage degradation[15] and increased subchondral bone formation and degradation.[16] While the optimum therapy for the management of diabetes has been a controversial issue,[17] a combined therapeutic approach is considered favorable for the treatment of osteoarthritis.[18]

Salmon calcitonin (Sct) is the most widely used calcitonin preparation in medical practice.[19] The hormone has favorable effects on bone and cartilage integrity.[20],[21] Like calcitonin, omega-3 fatty acids (N-3) affects bone and cartilage integrity.[22],[23] However, not all studies concluded that dietary supplementation with N-3 is beneficial in the management of osteoarthritis.[24] In diabetic state, N-3 have been reported to favor,[25] worsen[26] or have no effect[27] on the disease process.

As there is no report in literature about the effect of Sct and/or N-3 in experimental diabetic-osteoarthritic (DOA) state, the present study investigated the effects of single or combined administration of these therapies on selected biomarkers in induced DOA male Wistar rats.


  Materials and Methods Top


Chemicals and drugs

Sct, sodium monoiodoacetate (MIA), streptozotocin (STZ), and nicotinamide (NAD) were purchased from Sigma-Aldrich, St. Louis, MO, USA, while N-3 were purchased from Gujarat Liqui Pharmacaps Pvt. Ltd., Vadodara, Gujarat, India. In addition, sodium pentobarbital was procured from Nicholas Piramal Ltd., Thane, Maharashtra, India.

Experimental animals and care

Forty-nine adult (10–12 weeks old) male Wistar rats weighing between 180 and 220 g were used for this study. They were acquired from trusted commercial breeders. The rats were kept in wooden cages at about 30°C and photoperiodicity of 12 h light/12 h dark. After 1 week of acclimatization, but before the administration of various chemicals and drugs that were used in this study, the rats were randomly allotted to separate groups. They were given standard pelletized diet and water ad libitum daily and were weighed weekly.

The rats received humane care in accordance with the standards outlined in the “Guide for the Care and Use of Laboratory Animals” documented by the National Academy of Sciences,[28] and sanctioned by the Ethical Committee of the resident university of the authors.

Experimental design

The 49 rats that were used in this study were divided into seven groups, which included: Control (untreated); DOA untreated; DOA + N-3; DOA + Low dose of Sct (Sct. Lw); DOA + High dose of Sct (Sct. Hi); DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi.

Omega fatty acids (eicosapentaenoic acid and docosahexaenoic acid-3:2) were administered at 200 mg/kg body weight (b.w.)/day (p.o.), while Sct was administered at 2.5 and 5.0 IU/kg b.w./day (i.m.).[29] Treatments with N-3 and Sct started 9 days after the induction of diabetes and osteoarthritis, and they lasted for 4 weeks.

Induction of diabetes mellitus

Diabetes mellitus was induced in overnight fasted rats by the administration of STZ (65 mg/kg b.w., i.p.) and NAD (110 mg/kg b.w., i.p.). STZ was dissolved in citrate buffer (pH 4.5), and was administered 15 min after the administration of NAD in physiological saline.[30] Three days after the induction of diabetes, the blood glucose of the rats was examined using Accu-Check Active glucometer (Roche Diagnostics, Pvt. Ltd., India), and the rats with a blood glucose concentration of more than 250 mg/dl were considered diabetic and were used for the study.

Induction of knee osteoarthritis

Knee osteoarthritis was induced with 4 mg of sodium MIA in 40 μl of sterile saline. The solution was injected (using a 27-gauge needle) intra-articularly through the patellar ligament of the rats' left knee joints while they were under sodium pentobarbital (40 mg/kg, i.p.) anesthesia.[31] Under the same procedure, the rats in the control group were injected intra-articularly with 40 μl of sterile saline. It should be noted that osteoarthritis was induced 12 h after the induction of diabetes.

Preparation of injectableform of salmon calcitonin

Sct powder was dissolved in 0.9% NaCl solution to obtain the desired doses.[32] The solution was stored in a refrigerator at 2°C–8°C to ensure the viability of the hormone.

Biochemical and hematological analyses

Twelve hours after administration on the 28th day of the experiment, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.m.). Afterward, they were dissected to collect blood by cardiac puncture. The whole blood was collected into heparinized sample bottles and was centrifuged at 4000 revolutions per minute for 15 min, at −4°C using a cold centrifuge (Centurion Scientific Ltd., Chichester, West Sussex, UK). The supernatant plasma samples were collected into separate plain bottles before the analyses.

Diagnostic kits for the determination of malondialdehyde, lactate dehydrogenase, superoxide dismutase, glutathione peroxidase, catalase, total bilirubin, and total antioxidant capacity were obtained from Fortress Diagnostics Limited, United Kingdom. Analytic kits for the determination of total alkaline phosphatase (TALP), c-telopeptide of type 1 collagen (CTX-1), and collagen type 2 (C2M) alpha-1 were purchased from Elabscience Biotechnology Company Ltd., Wuhan, Hubei, China. The analyses were performed according to the manufacturers' instruction.

As for the hematological analysis, hemocytometer method[33] was used for the determination of erythrocyte and total leukocyte counts. Microhematocrit[33] and cyanmethemoglobin[34] methods were used for the determination of packed cell volume (PCV) and hemoglobin (HB) concentration, respectively. In addition, the differential white blood cell (WBC) count was determined by the battlement counting method.[35]

Data analysis

Data were analyzed using statistical package for social sciences version 20.0 (SPSS Inc., Chicago, Illinois, USA). Statistical evaluations of the differences between the group mean values were tested by one-way analysis of variance following the least significant difference post hoc test. The results were expressed as mean ± standard error of mean (SEM), and statistical significance was considered at P < 0.05.


  Results Top


Effects of salmon calcitonin and/or omega-3 fatty acids on malondialdehyde, lactate dehydrogenase, superoxide dismutase, catalase, glutathione peroxidase, total bilirubin, and total antioxidant capacity in experimental diabetic rats

Relative to the control group, there was a significant (P = 0.01) increase in MDA level in DOA untreated group, but, a significant (P = 0.04) decrease in DOA + N-3+ Sct. Hi group [Figure 1]a. Compared to DOA untreated group, there were statistically significant (P < 0.02) decreases in MDA level in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. There was a significant (P < 0.05) increase in the activity of LDH in DOA untreated, DOA + Sct. Lw, DOA + Sct. Hi, and DOA + N-3+ Sct. Lw groups, relative to the control group [Figure 1]b. Compared to DOA untreated group, there was significant (P < 0.05) decrease in the activity of LDH in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi group. In addition, there was a significant (P = 0.032) increase in LDH activity in DOA + N-3+ Sct. Hi group, compared to DOA + Sct. Hi group. Significant (P < 0.05) decreases in SOD [Figure 1]c and CAT [Figure 1]d activities were recorded in DOA-untreated group, compared to the control group. Relative to the former, there were significant (P < 0.04) elevations in SOD and CAT activities in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Compared to the control group, there were significant (P < 0.03) decreases in GPx activity in DOA untreated, DOA + Sct. Lw, and DOA + Sct. Hi groups [Figure 1]e. However, significant (P < 0.05) elevations were recorded in DOA + N-3, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Relative to DOA-untreated group, there were significant (P < 0.05) elevations in GPx activity in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Moreover, significant increases in GPx activity were recorded in DOA + N-3+ Sct. Lw group, relative to DOA + Sct. Lw group (P = 0.01), and in DOA + N-3+ Sct. Hi group, compared to DOA + Sct. Hi group (P = 0.02). There were significant (P < 0.01) reductions in TB level in DOA untreated, DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups, relative to the control group [Figure 1]f. Compared to DOA untreated group, significant (P < 0.05) elevations in TB level were recorded in DOA + Sct. Lw, DOA + Sct. Hi, and DOA + N-3+ Sct. Hi groups. Relative to the control group, there was a significant (P = 0.042) decrease in TAC in DOA untreated group. However, a significant (P = 0.036) increase was recorded in DOA + N-3+ Sct. Hi group [Figure 1]g. Compared to DOA-untreated group, there were significant (P < 0.05) increases in TAC in DOA + Sct. Lw, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Moreover, there was a significant (P < 0.032) increase in TAC in DOA + N-3+ Sct. Hi group, relative to DOA + N-3 and DOA + Sct. Hi groups.
Figure 1: Effects of Sct and/or N-3 on malondialdehyde level (a); lactate dehydrogenase activity (b); superoxide dismutase activity (c); catalase activity (d); glutathione peroxidase activity (e); total bilirubin level (f), and total antioxidant capacity (g). Values (n = 7) are expressed as mean ± Standard error of mean. *P < 0.05 is significant compared to control group;#P < 0.05 is significant compared to DOA untreated group;bP < 0.05 is significant – DOA + N-3 versus DOA + N-3+ Sct. Hi;dP < 0.05 is significant– DOA + Sct. Lw versus DOA + N-3+ Sct. Lw;eP < 0.05 is significant – DOA + Sct. Hi versus DOA + N-3+ Sct. Hi. DOA: Diabetic–Osteoarthritic, N-3: Omega-3 fatty acids, Sct: Salmon calcitonin, Sct. Lw: Low dose of salmon calcitonin, Sct. Hi: High dose of salmon calcitonin

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Effects of salmon calcitonin and/oromega-3 fatty acids on total alkaline phosphatase, c-telopeptide of type 1 collagen, and collagen type 2 alpha-1 in experimental diabetic rats

Significant increases in TALP activity were recorded in DOA untreated (P = 0.001) and DOA + Sct. Hi (P < 0.05) groups, compared to control group [Figure 2]a. Relative to DOA untreated group, there were significant (P < 0.05) decreases in TALP activity in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Moreover, there was a significant elevation in TALP activity in DOA + Sct. Hi group, relative to DOA + Sct. Lw (P = 0.043) and DOA + N-3+ Sct. Hi (P = 0.038) group. In addition, there was a significant (P = 0.045) diminution in TALP activity in DOA + N-3+ Sct. Lw group, compared to DOA + Sct. Lw group. There was significant (P = 0.01) increase in CTX-1 [Figure 2]b and C2M [Figure 2]c levels in DOA-untreated group, compared to control group. Relative to the former, significant (P < 0.05) decreases in the plasma level of CTX-1 and C2M were recorded in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups.
Figure 2: Effects of Sct and/or N-3 on total alkaline phosphatase activity (a); c-telopeptide of type I collagen (b), and collagen type 2 alpha 1 level (c). Values (n = 7) are expressed as mean ± Standard error of mean. *P < 0.05 is significant compared to control group;#P < 0.05 is significant compared to DOA untreated group;cP < 0.05 is significant – DOA + Sct. Lw versus DOA + Sct. Hi;dP < 0.05 is significant – DOA + Sct. Lw versus DOA + N-3+ Sct. Lw;eP < 0.05 is significant – DOA + Sct. Hi versus DOA + N-3+ Sct. Hi. DOA: Diabetic–Osteoarthritic, N-3: Omega-3 fatty acids, Sct. Lw: Low dose of salmon calcitonin, Sct. Hi: High dose of salmon calcitonin, Sct: Salmon calcitonin

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Effects of salmon calcitonin and/or omega-3 fatty acids on hematological indices in experimental diabetic rats

There were significant diminutions in HB level in DOA untreated, DOA + Sct. Lw, and DOA + Sct. Hi groups (P = 0.032, 0.047, and 0.045, respectively), compared to the control group [Table 1]. Relative to DOA-untreated group, significant elevations in HB level were recorded in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups (P = 0.030, 0.037, 0.039, 0.028, and 0.031, respectively). In addition, there was a significant (P = 0.046) decrease in HB level in DOA + Sct. Lw group, compared to DOA + N-3+ Sct. Lw group, and in DOA + Sct. Hi group, relative to DOA + N-3+ Sct. Hi group (P = 0.045). Relative to the control group, there were significant (P = 0.024, 0.047, 0.045, 0.045, and 0.046, respectively) decreases in PCV in DOA untreated, DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, and DOA + N-3+ Sct. Lw groups [Table 1]. Compared to DOA-untreated group, significant (P = 0.040, 0.043, 0.043, 0.038, and 0.036, respectively) elevations in PCV were recorded in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Compared to the control group, there were significant (P < 0.05) reductions in red blood cell count (RBC) in the other experimental animal groups [Table 1]. Relative to DOA-untreated group, significant (P = 0.041, 0.043, 0.043, 0.037, and 0.032, respectively) increases in RBC count were recorded in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Moreover, there was a significant (P < 0.040) increase in RBC count in DOA + N-3+ Sct. Hi group, compared to DOA + N-3 and DOA + Sct. Hi groups. A significant (P = 0.033) increase in total WBC count was recorded in DOA-untreated group, compared to control group [Table 1]. Relative to the former, there were significant (P = 0.043, 0.045, 0.046, 0.041, and 0.038) decreases in the WBC count in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. Relative to control group, there were significant (P < 0.05) elevations in the platelet [Table 1] and neutrophil [Table 2] counts in DOA-untreated and DOA + Sct. Lw groups. Compared to DOA-untreated group, significant (P < 0.05) reductions were recorded in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. There were significant (P = 0.041 and 0.043, respectively) increases in lymphocyte count and neutrophil/lymphocyte ratio in DOA untreated groups, relative to control group [Table 2]. Compared to the former, there were significant (P < 0.05) diminutions in lymphocyte count and neutrophil/lymphocyte ratio in DOA + N-3, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups. A significant (P = 0.042) increase in platelet/lymphocyte ratio was recorded in DOA-untreated group, compared to control group [Table 2]. Relative to the former, there were significant (P < 0.044) decreases in platelet/lymphocyte ratio in DOA + N-3, DOA + Sct. Lw, DOA + Sct. Hi, DOA + N-3+ Sct. Lw, and DOA + N-3+ Sct. Hi groups.
Table 1: Effects of salmon calcitonin and/or N-3 on hematological indices

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Table 2: Effects of Sct and/or N-3 on hematological indices

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  Discussion Top


Diabetes has been implicated in the initiation and advancement of osteoarthritis. The detrimental effects of diabetes on osteoarthritis are often manifested by biochemical and biomechanical alterations.[36] Adeyemi and Olayaki reported on the additive and antagonistic effects of the induction of diabetes and/or osteoarthritis on endogenous biomarkers.[37] The authors also affirmed that in the presence of diabetes, osteoarthritis worsens glycemic state. Moreover, in our previous research, the effect of Sct and N-3 on glucoregulatory indices, lipid profile, and inflammatory markers in experimental diabetic-knee osteoarthritic rat was reported.[29] However, in the present study, the effect of Sct and/or N-3 was further evaluated on other biomarkers in the same experimental animal model.

The pathogenesis and progression of diabetes and osteoarthritis have been associated with the disturbance of the antioxidant system,[20],[38] which scavenges free radicals and hence inhibits oxidative stress, lipid peroxidation, and possible cell death. Therefore, any pharmacological agents that would boost the efficiency of the antioxidant system, and as such, redress the imbalance in the endogenous antioxidant/pro-oxidant status, would be beneficial in the management of these chronic diseases.

In the present study, the co-induction of diabetes and osteoarthritis were accompanied by peroxidation of membrane lipids, indicated by the significant increase in the plasma level of malondialdehyde. However, Sct and N-3 significantly reversed this effect. Sct has been reported to significantly reduce the level of malondialdehyde in erythrocytes.[39] However, there are incongruent reports in literature on the effect of N-3 on peroxidation of lipids. N-3 have been documented to have no effect on indices of lipid peroxidation.[40] However, other researchers indicated that N-3 have anti-lipid peroxidative effect.[41]

A positive association was observed between malondialdehyde level and the activity level of lactate dehydrogenase, which is considered as a marker of acute or chronic cellular damage.[42] The significant increase in the activity of lactate dehydrogenase as a result of the co-induction of diabetes and osteoarthritis could be attributed to the probable increase in reactive species, which eventually lead to the precipitation of lipid peroxidation, and hence damaged cellular integrity or cell death. Increased activity of lactate dehydrogenase characterizes the osteoarthritic state;[42] however, there are inconsistent reports on the level of activity of this enzyme in diabetics. Investigators have documented an elevated lactate dehydrogenase activity in diabetic subject,[43] yet, other researchers reported a decrease[44] or no change[45] in the activity of this enzyme. The free radical scavenging effects of Sct and N-3, evident by their anti-lipid peroxidative actions, explain the significant reductions in lactate dehydrogenase activity, following the single or combined administration of these pharmacological agents. Although there was a slight evidence of the additive effect of these therapies on malondialdehyde level, this was not observed in the determination of lactate dehydrogenase activity.

The aforementioned lipid peroxidation and possible cell damage or death that accompanied the induced diabetes-knee osteoarthritis was inversely related to the level of activities of superoxide dismutase, catalase, and glutathione peroxidase. Both diabetes and osteoarthritis are marked by the diminution of the antioxidant profile.[9],[10],[46] The observed antioxidant effect of N-3,[47] could be linked to the anti-inflammatory action of its metabolites.[48] This is because oxidative stress has been considered as one of the outcomes of pro-inflammatory disease conditions.[49] The reported lipid peroxidative effect of Sct,[39] and its anti-inflammatory action,[50] could explain the significant increases in the activities of superoxide dismutase, catalase, and glutathione peroxidase following the administration this therapy. Although both Sct and N-3 were found to significantly increase the activities of these enzymes, their effects were not additive. Unlike the observed nondose specific effects of Sct on superoxide dismutase, catalase, and glutathione peroxidase, in the determination of total antioxidant capacity, there was an indication of the dose-specific action of Sct. Apart from the conventional and well-known constituents of the antioxidant system, bilirubin has also been identified to have anti-oxidative property.[51] Similar to what was observed in the determination of total antioxidant capacity, the plasma level of total bilirubin was significantly reduced, following the induction of diabetes and osteoarthritis. N-3 had no significant effects on total bilirubin and total antioxidant capacity, even though they significantly increased the activities of superoxide dismutase, catalase, and glutathione peroxidase. Therefore, it could be suggested that N-3 have favorable effects on certain endogenous antioxidants and not on others. Hence, their overall effect on the antioxidant system might not necessarily be significant. This could explain why N-3 have been reported to have no effect on reactive oxygen species,[52] and also on indices of lipid peroxidation.[40]

Diabetes results in reduced bone formation,[11] and increased degradation of bone and cartilage tissues.[14],[53] On the other hand, osteoarthritis is accompanied with elevated bone formation,[54] increased cartilage degradation,[15] and decreased,[55] or increased[56] bone turnover. In the present study, the co-induced diabetes and osteoarthritis showed a significant elevation in bone formation marker-TALP and increased degradation of bone and cartilage tissues, indicated by the determination of the plasma levels of CTX-1 and C2M alpha-1, respectively. Although both Sct and N-3 brought about significant reductions in the plasma level of these markers, their effects were not additive. Furthermore, the effects of Sct on CTX-1 and C2M alpha-1 were largely found to be nondose specific. The observed favorable action of Sct on bone formation in the present study could be dose related, as high doses of calcitonin seem to promote bone formation, an effect that has been noted to exacerbate osteoarthritis disease process.[16],[57] Furthermore, N-3 have been reported to suppress bone formation process, and bone and cartilage degradation.[23],[58] Like N-3, calcitonin reduces bone and cartilage degradation.[59],[60]

In this study, the co-induced diabetes and osteoarthritis were accompanied by significant reductions of HB, RBC count, and PCV. However, a significant elevation in the total WBC count, platelet/lymphocyte ratio, and neutrophil/lymphocyte ratio. Increased WBC count, platelet/lymphocyte ratio, and neutrophil/lymphocyte ratio have been considered as a useful index of systemic inflammation.[61],[62],[63] Classically, OA is considered as a non-inflammatory disease condition.[64] However, increasingly, it is accepted to have modest inflammatory component.[65] On the other hand, hyperglycemia in diabetes triggers a low-grade systemic inflammation.[66] WBCs produce cytokine, for example, interleukin-6, interleukin-1β, among others.[67] Interleukin-6 promotes the synthesis of hepcidin,[68] which binds to ferroportin, an iron efflux protein.[69] This could result to inhibition of the release of iron recycled from senescent RBCs,[70] and hence compromised HB synthesis, reduced RBC count, and PCV that were recorded in this study. Asides, since both diabetes and OA are characterized by oxidative stress; the cumulative-free radical producing consequences of the two ailments could have compromised the integrity of RBC membrane, resulting to the reported anemia. The administration of Sct and/or N-3 was accompanied by significant increases in HB level, RBC count, and PCV. However, significant reductions in platelet/lymphocyte ratio and total WBC count. Both therapies were found to have additive effects on hemoglobin level and RBC count, and not on the other hematological indices. In addition, the effects of Sct on these parameters were nondose specific. The observed actions of Sct and N-3 on HB level, RBC count, and PCV could be related to their free radical scavenging properties, as free radicals promote osmotic fragility of erythrocytes,[71] and hence, reduced their life span. Docosahexaenoic and eicosapenatenoic acids serve as substrates for the synthesis of anti-inflammatory compounds,[72] and at the same time inhibit the activation of nuclear factors that causes inflammation.[73] As a result, N-3 could attenuate the possibility of pro-oxidative responses as a result of pro-inflammatory reactions.[74] Like N-3, the favorable effects of Sct on the hematological profile could be related to its anti-inflammatory[50] and antioxidant[39] actions.


  Conclusions Top


The combined administration of Sct and N-3 proffer better therapeutic effects than the single therapy. Therefore, they could be used in the management of DOA condition.

Acknowledgment

The authors would like to acknowledge Mr. Adebowale Olabanji of Bridge Scientifik Enterprises for his technical assistance during the biochemical assays.

Financial support and sponsorship

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest

There are no conflicts of interest.

Authors' contribution

WJA was a doctoral student under LAO, therefore, he was responsible for the study design, funding of the research work, data collection, statistical analyses, data interpretation, literature search, and the drafting of the manuscript. LAO supervised the research work, corrected and approved the final draft of the manuscript.



 
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