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Protective Effect of Resveratrol | Study

Experiments Following Fluoride Exposure and Resveratrol Supplementation
Effect of resveratrol on fluoride-induced alteration of thyroid metabolic activity
The changes in tissue DNA and RNA level of rat thyroid have been represented in Figure 23 and 24. Resveratrol supplementation in fluoride-intoxicated rats restored the decreased DNA content of thyroid by 15.46% (p<0.001) as compared to the treated group. Vitamin C exhibited better beneficial effects than resveratrol to protect against fluoride-induced DNA damage in thyroidal tissue.

Figure 23: Effect of resveratrol on fluoride-induced alteration of DNA level in thyroid
[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]
RNA content of fluoride-exposed animals was also decreased significantly in thyroid gland of rat. The decrease was found to be 33.08% (p<0.001) from control value. Administration of resveratrol partially counteracted the decreased RNA content in thyroid gland by 33.98% (p<0.001) as compared to that of fluoride-intoxicated group. Vitamin C also exhibited partial (35.22%; p<0.001) ameliorative effects in restoration of RNA content in the thyroid gland.

Figure 24: Effect of resveratrol on fluoride-induced change in RNA level of rat thyroid
[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]

Figure 25: Effect of fluoride on thyroidal Na -K ATPase activity with or without resveratrol and vitamin C supplementation
[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]
Changes in the activity of Na -K -ATPase and 5′ deiodinase I are represented in Figure (25 and 26). Na -K -ATPase activity in the fluoride-exposed group decreased by 40.59% (p<0.001) as compared to the control group. Resveratrol along with sodium fluoride showed significant (p<0.001) ameliorative effect to restore the enzyme activity towards the control value. Similarly, vitamin C supplementation also counteracted the enzyme activity by 26.67% (p<0.001). Additionally, resveratrol checked the inhibitory effects of fluoride on 5' deiodinase I enzyme activity and counteracted its activity by 58.93% (p<0.001). Vitamin C also exhibited partial protective effect against fluoride-induced alteration of 5' deiodinase I enzyme activity.

Figure 26: Effect of fluoride on thyroidal 5′ deiodinase I activity with or without resveratrol and vitamin C supplementation
[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]
Table 26: Effect of co-administration of fluoride with resveratrol on thyroid parameters
Groups of animals
TPO
(?OD/min/mg of protein)
T3
(ng/ml)
T4
(µg/dl)
Control (6)
0.39±0.04
1.53±0.10
5.83±0.06
F-treated (6)
0.23±0.02, pa***
0.65±0.07, pa***
4.13±0.14, pa***
F-treated resveratrol (6)
0.34±0.02, pb***
0.97±0.09, pb***
4.91±0.19, pb***
F-treated vit. C (6)
0.33±0.03, pb***
1.1±0.07, pb***
5.09±0.16, pb***
[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]
Fluoride exposure significantly inhibited the activity of TPO in thyroid tissue by 42.06% (p<0.001) (Table 26). Resveratrol counteracted the TPO activity by 48.89% (p<0.001) as compared with the fluoride-intoxicated group, whereas vitamin C supplementation exhibited better protective effects than resveratrol against fluoride-induced decreased serum T3 level by restoring 43.7% (p<0.001) compared to that of fluoride intoxicated group.
Moreover, sodium fluoride significantly decreased T3 level in serum by 57.47% (p<0.001) (Table 26). Resveratrol counteracted fluoride-induced alteration of T3 level by 48.97% (p<0.001), whereas vitamin C supplementation exhibited better protective effect than resveratrol against fluoride-induced decreased serum T3 level by restoring 69.23% (p<0.001) as compared to that of fluoride-exposed group.
The thyroxine (T4) level was also reduced significantly in the serum samples of rats following exposure to fluoride (Table 26). The decrease was found to be 29.14% (p<0.01) compared to the control group. Administration of resveratrol appreciably counteracted fluoride-induced change in serum T4 level. Vitamin C supplemented group also exhibited 23.24% (p<0.01) restoration against fluoride-induced alteration of serum thyroxine level.
Table 27: Alkaline comet assay parameters (tail length, tail intensity and tail moment) in fluoride-exposed thyroid cells with or without resveratrol with the help of CASP
Assay Parameters
Control (6)
F-treated (6)
F-treated resveratrol (6)
F-treated vit. C (6)
Tail length (µm)
3.42±1.24
19.26±2.32
15.73±2.11
14.82±2.51
Tail intensity (µm)
0.35±0.05
1.71±0.52
1.46±0.49
1.39±0.62
Tail moment
0.04±0.006
0.21±0.04
0.18±0.006
0.17±0.009
[Values are Means ± S.D.]
Both qualitative and quantitative analyses of ethidium bromide stained comets on the basis of DNA damage are represented in Table 27. In comparison with the fluoride intoxicated group, there was a reduction in the tail length, tail intensity and tail moment of thyroid cells in the resveratrol supplemented group (Figure 27).

Figure 27: Comet assay micrograph
[A: undamaged and damaged thyroid cells of control, B: fluoride-treated, C: fluoride plus resveratrol-supplemented and D: fluoride plus vitamin C -supplemented group of animals. Cells were photographed under the fluorescence microscope using a 40x objective equipped with a 515-560 nm excitation filter and a 590 nm barrier filter.]

Figure 28: Histological changes in the rat thyroid after fluoride treatment with or without resveratrol
[(A) indicates normal architecture of follicular epithelial cell, (B) indicates flattened follicular epithelial cell in the enlarged follicle with over filling colloid, (C) and (D) indicate regaining of proper shape of follicular epithelia towards normalcy in “NaF Res.” and “NaF Vit.C” supplemented groups; Images were captured at 400 magnification]
Histological observations reveal that, the areas of thyroid follicles were significantly increased in NaF-intoxicated group. The follicular epithelia appeared flat in the enlarged follicles for being crushed over by filling colloid and hyperplastic nodules, consisting of thyroid parafollicular cells. Resveratrol markedly inhibited fluoride-induced damage on thyroid follicle and restored the changes near to the control group in respect of follicular size, colloidal material and epithelial tissue architecture (Figure 28).
Discussion The present study further elucidates protective effects of resveratrol on fluoride-induced alteration of thyroid metabolic activity in male Wistar rats. Fluoride exposure at the present dose and duration significantly decreased the gain in body weight of rats which is consistent with our earlier observations. Alteration in body weight is supposed to be due to less intake of food after fluoride exposure or increased breakdown of muscle and tissue proteins. Moreover, suppressed activity of growth hormone on overall body growth due to less free thyroxine level in fluoride-intoxicated animals as found in the present study as well as earlier (Sakamoto et al., 2001), may be regarded as another causative factor for decrease in whole body weight. Gain in organ (thyroid) weight in relation to body weight has been expressed in terms of organo-somatic index, which shows that fluoride exposure significantly increased the organo-somatic index. This may be due to enlargement of thyroid gland by fluoride either by hypertrophy or hyperplasia of thyroidal cells in experimental rats as thyroid gland has a strong capacity for absorbing and accumulating fluoride (Ge et al., 2005). Earlier reports revealed that structural alteration of thyroid follicle by fluoride has been associated with induction of cytoplasm reduction, karyopycnosis of follicular epithelial cells and also reduction in the number of microvilli on the cristae of epithelial cells leading to swelling of vacuoles in follicular epithelial cells (Bouaziz et al., 2005). The present findings reveal that sub-acute fluoride exposure significantly alters the histological architecture of thyroid gland characterized by larger follicles, compressed follicular epithelia, over filling of colloid and reduction in inter-follicular space (Figure 28). Resveratrol supplementation in fluoride-intoxicated rats almost completely restored the altered organo-somatic index by preventing the changes in thyroid tissue architecture caused by fluoride. Resveratrol is thus assumed to protect thyroid tissue from oxidative stress-mediated cell damage.
DNA damage by fluoride is found in various cell types and oxidative stress is recognized as a causative factor of it (Wang et al., 2004b). Over ingestion of fluoride may result in alteration of functional status of the hypothalamo-pituitary thyroidal system and adversely affecting the synthesis of cellular metabolites such as DNA and RNA in thyroidal cells. Marked increase in DNA damage of thyroidal cells due to high fluoride and low iodine uptake has been reported (Ge et al., 2005). In the present study, sub-acute exposure to fluoride caused damage to the cellular components like DNA, confirming the earlier observation. Fluoride may directly damage cells by altering membrane structure and induce rupture of DNA strands as indicated by increased tail length and tail intensity of DNA in thyroidal cells as evident from the comet assay (Figure 27). DNA damage is supposed to be one of the reasons for high morbidity rates among those afflicted with hypothyroidism goiter and subcretinism in high fluoride and low iodine areas (Yaming et al., 2005). Alteration of nucleic acid metabolism in thyroidal cells might be involved in fluoride-induced functional disorders of thyroid.
Moreover, fluoride exposure at the present dose and duration significantly reduced the RNA content in thyroid tissue. Fluoride-induced depression of RNA level in other tissue was reported earlier (Sarkar et al., 2014) which might be due to inhibition of nucleic acid synthesis and improper attachment of m-RNA to ribosome after fluoride exposure. Fluoride, being an inhibitor of calcium and magnesium, the co-factors of certain metalloenzymes involved in nucleic acid biosynthesis reduces their synthesis (Verma and Guna Sherlin, 2002). Disturbed nucleic acid metabolism in thyroid tissue upon fluoride exposure is indicative of transcriptional and translational imbalance and chromosomal abnormalities. Resveratrol supplementation in fluoride-exposed rats appreciably reversed the changes in nucleic acid contents that were perturbed due to fluoride toxicity and also checked DNA strand breaks in thyroid cells. It is assumed that beneficial effects of this antioxidant may be due to its detoxifying ability to eliminate toxic fluoride from the tissue and to check free-radical mediated damage of cellular components like DNA, RNA and proteins. Trans-resveratrol itself exerts antioxidant effects likely due to the redox properties of the phenolic hydroxyl groups in its structure and thereby imparts in scavenging highly toxic free radicals that can damage cellular components (Leonard et al., 2003).
Hormone synthesizing capability of thyroid was seriously affected by fluoride exposure at the present dose and duration. Disturbed synthesis and secretion of thyroid hormones by fluoride and its interference in activity of enzymes that catalyze the conversion of thyroxine (T4) to active triiodothyronine (T3) as observed presently were also previously reported (Bouaziz et al., 2005). The reasons for decreased level of T4 during fluoride intoxication might be due to inhibition of absorption of iodine through the interaction of fluoride and/or insufficient synthesis and secretion of thyroglobulin and oxidized iodides from the thyroid gland owing to the structural changes of the thyroid follicle injured by the excessive intake of fluoride. Earlier observation also suggested that fluoride inhibits the activity of TPO in the thyroid gland (Zhan et al., 2006c). TPO, the main regulatory enzyme for thyroid hormone biosynthesis showed positive correlation with T3 and T4 level in hypothyroid patients (Singla and Shashi, 2013). The inhibition of TPO-catalyzed reaction as evident from present findings results in decrease in serum level of thyroid hormones. The suggested mechanism of action for enzyme inhibition may involve the conversion of thyroid peroxidase to a free radical that reacts with resorcinol moiety to produce a flavonoid radical (Chandra et al., 2011) that could covalently bind to the catalytic amino acid residues on the enzyme, leading to enzyme inactivation (Chandra et al., 2011). Similar explanation can also be suggested for the present study where fluoride-induced conversion of TPO to free radical may cause inhibition of this enzyme activity. In animal model, Boas et al. (2012) reported that fluorinated compounds such as perfluorooctane sulfonate and perfluorooctanoic acid also inhibited TPO activity in rats, with reductions in T4 and T3. It is assumed that iodide groups on TPO molecule may attract fluoride that causes decrease in the free active site on TPO molecule, leading to inactivation of this enzyme. The present study further reveals that resveratrol counteracted fluoride-induced changes in serum T3 and T4 level as well as TPO activity towards normalcy. Resveratrol administration effectively elevated serum T3 and T4 level by increasing the activity of TPO. As resveratrol is suggested to scavenge toxic free radicals generated due to fluoride toxicity and may also eliminate those harmful metabolites from the tissue, thus reducing the adverse effects of fluoride on thyroid metabolic functions.
The present study further reveals that fluoride exposure inhibits Na -K -ATPase activity that plays an important role in active transport of Na and K ions across the plasma
membrane. This finding is in conformity with the earlier observation (Zhan et al., 2006c). The decreased activity of Na -K -ATPase could adversely affect the accumulation of iodide in the thyroid, which is opposite to stimulation of Na -K -ATPase induced by hypothyroidism (Le Grow et al., 1999). This effect might be due to accumulation of fluoride in the thyroid which directly inhibits the Na -K -ATPase (Murphy and Hoover, 1992) or the combined activity of fluoride and high TSH on activation of the protein kinase C, which decreases the activity of Na -K -ATPase (Bocanera et al., 2001). Fluoride may cause internal injury to the cell membrane and affect the activity of membrane bound enzymes like Na -K -ATPase by disturbing membrane fluidity and membrane integrity and thus altering its permeability (Chinoy et al., 1994). Both thyroidal and extra-thyroidal tissues, like liver and kidney, are the main site of generation of circulating T3, which is produced by peripheral deiodination of T4 to T3. 5?-monodeiodinase I enzyme is responsible for this deiodination reaction. Sodium fluoride exposure at the present dose and duration reduces significantly the activity of 5?-monodeiodinase I, suggesting that fluoride decreases the rate of conversion of T4 to T3. All of these adverse effects of fluoride may be due to interference with synthesis or secretion of thyroid hormones by various mechanisms such as blockage of iodine uptake by the thyroid follicular cells, by inhibiting Na -K -ATPase activity, organification defect due to inhibition of thyroid peroxidase and suppression of thyroid hormone release. The study further demonstrates that relative adverse effects of fluoride on thyroid functions are appreciably counteracted by resveratrol supplementation. Moreover, resveratrol elevates antioxidant power of the thyroidal cell by inducing the expression of a set of cytoprotective genes through an antioxidant responsive element (Iwasaki et al., 2013). Remarkable beneficial effects of resveratrol were found against fluoride-induced alteration of thyroid functions. Although no group of animals treated with resveratrol alone was included in the present investigation, studies by Duntas (2011) demonstrated that resveratrol influences thyroid metabolic function by enhancing iodide trapping and, by increasing TSH secretion via activation of sirtuins and the phosphatidylinositol-4-phosphate 5 kinase ? (PIP5K?) pathway. Resveratrol administration was also demonstrated to increase iodide trapping in thyroid cell line (Sebai et al., 2010), thus preserving the functional status of thyroid. These studies clearly indicate promising effects of resveratrol on thyroid metabolic functions. Resveratrol has been recognized as a powerful antioxidant and also used in earlier occasions to combat against oxidative stress-induced cytotoxicity (Iwasaki et al., 2013). Antioxidative effects of resveratrol have already been established against other environmental toxicants like arsenic trioxide (Zhang et al., 2008). The studies revealed that pre-treatment with resveratrol resulted in a significant increase in the activities of GSH, GPx, CAT and SOD in plasma, thus supporting the antioxidant property of resveratrol. The protective efficacy of resveratrol in fluoride induced thyrotoxicity has been demonstrated in in vivo rat model in terms of histological changes, activities of thyroid metabolic enzymes like TPO and 5?-monodeiodinase, iodide trapping mechanism by Na -K -ATPase and changes in thyroid hormone secretion.
Summary The present study elucidates the protective effect of resveratrol, a natural antioxidant against fluoride-induced alteration of thyroid functions, possibly mediated by the anti-oxidative property of resveratrol. Sub-acute fluoride exposure through drinking water imposes stress on biosynthetic machinery of thyroid hormones, as indicated by depletion of serum T3 and T4 and decreased activities of TPO, Na -K -ATPase and 5? deiodinase I enzymes. Additionally, perturbation and damage of the nucleic acids of the thyroid gland by fluoride are also observed in the present study. These metabolic changes were associated with tissue architectural alteration in fluoride-intoxicated thyroid gland. The efficacy of resveratrol to attenuate the adverse effects of fluoride on thyroidal metabolic function, DNA damage and ultra-structural disorganization could provide insight into whether abnormalities in metabolic function are linked to the development of early stages of fluoride induced thyroidal tissue damage. The present study thus establishes the new role of resveratrol as a promising protective agent against fluoride-induced thyroidal toxicity.

Effects of Vitamin B12 and Omega 3 on NGF, VEGF and HIF-1

1. Introduction Maternal micronutrient deficiencies are a cause of concern in low and middle-income countries (Salam et al., 2014) and have been shown to be associated with adverse pregnancy outcomes (Diaz et al., 2003). Reports have also established that maternal nutrition plays a key role in fetal brain development (Oliver et al., 2007; McMillen et al., 2008). In developing countries like India, which has a large population consuming a vegetarian diet suboptimal levels of both vitamin B12 (Pawlak et al., 2013) and omega-3 fatty acids (Muthayya et al., 2009) are common. Further reports also indicate that children born to mothers consuming a vegetarian diet may be at an increased risk of neurodevelopmental disorders (Larsen et al., 2014).
Animal studies carried out by us earlier have demonstrated lower levels of neurotrophins, which are important regulators of neural survival, development and function in the brain of the offspring as a consequence of maternal vitamin B12 deficiency (Sable et al., 2011, 2012, 2013). Studies indicate that neurotrophins regulate angiogenic markers like vascular endothelial growth factor (VEGF) in the brain (Nico et al., 2007; Turrini et al, 2002; Calza et al., 2001). VEGF is an angiogenic factor and recent studies have demonstrated its role as a stimulator of neurogenesis since vascular and nervous networks share common molecular mechanisms (Galvan et al., 2006). It is suggested that VEGF promotes endothelial cell migration towards a hypoxic area. During hypoxia, hypoxia inducible factor-1 (HIF-1) binds the regulatory region of the VEGF gene, thereby inducing its transcription and forming new blood vessels (reviewed by Ziello et al., 2007).
Apart from vitamin B12, omega-3 fatty acids especially docosahexaenoic acids (DHA) also play a crucial role in brain development and functioning by influencing multiple neuroprotective mechanisms. A recent study reports the protective effects of omega-3 fatty acid supplementation on post-stroke cerebral angiogenesis in transgenic fat-1 mice. (Wang et al., 2014). Studies carried out in our department in humans and animals have well demonstrated a link between micronutrients (folate, vitamin B12) and omega-3 fatty acids in the one carbon cycle (Khot et al., 2014a; 2014b; Sable et al., 2013; Kulkarni et al., 2011, Kale et al., 2010) (Sable et al., 2011, 2012, 2013). Though several plausible biochemical mechanisms for the individual effects of both vitamin B12 and omega-3 fatty acid supplementation are reported, the evidence from observational studies as well as RCTs is generally limited and too inconsistent to draw firm conclusions regarding the use of combined use of vitamin B and omega-3 fatty acids for brain development and functioning reviewed by van de Rest et al., 2012). Further, whether there exist any synergistic or antagonist effects between vitamin B12 and omega-3 fatty acids on brain angiogenic markers is unclear.
Therefore, the current study was undertaken to evaluate the effects of maternal vitamin B12 and omega-3 fatty acids given either individually or together on the levels of NGF, VEGF and HIF-1 alpha in the pup brain at birth.
2. Materials and methods 2.1. Animals and study design
The procedures carried out in the present animal study were approved by Institutional Animal Ethics Committee (IAEC) and were performed in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experimental Animals, (CPCSEA) Government of India.
The protocol for the study has been recently described by us in detail (Rathod et al., 2014; Khaire et al., 2013). Briefly, the dietary groups were as follows: Control (normal vitamin B12-25µg/Kg diet); vitamin B12 deficient group (BD); vitamin B12 deficient group supplemented with omega-3 fatty acids (BDO); vitamin B12 supplemented group (BS) (vitamin B12-50 µg/Kg diet); vitamin B12 group supplemented with omega-3 fatty acids (BSO). These dams were allowed to deliver normally on d22 of gestation and the offspring at birth were dissected to collect brain tissue. The study design is as shown in Fig.1.
The purified experimental diets were prepared in accordance with the AIN-93 guidelines (Reeves et al., 1993). The source of omega-3 fatty acid supplementation used in the present study was fish oil (MaxEPA, Merck Darmstadt, Germany) and was a combination of DHA (120 mg) and EPA (180 mg).
2.2. Tissue Homogenization
Brain tissue of the offspring was homogenized in chilled phosphate-buffered saline, at pH of 7.5 using a Teflon glass homogenizer and the process has been described by us earlier (Rathod et al., 2014). The homogenate was centrifuged at 10,000 rpm at 4ºC for 20 min after which the supernatant (lysate) was collected and used for total protein estimation. Total protein content of the lysates was estimated by Lowry method (Lowry et al., 1951).
2.3. VEGF Estimation
VEGF protein levels were measured from the brain homogenate (supernatant) using the VEGF Rat ELISA kit (Abcam, Cambridge Science Park in Cambridge, England). Relative absorbance was measured at 450 nm and the VEGF concentration was calculated using a standard curve. Values of VEGF were expressed as pg/mg protein.
2.4. HIF-1alpha Estimation
HIF-1 alpha protein levels were measured from the brain homogenate (supernatant) using HIF-1 alpha Rat ELISA kit (Uscn Life Science Inc. Wuhan). HIF-1 alpha protein levels in tissue homogenates were quantitated in the range of 0.156-10 ng/ml. Relative absorbance was measured at 450 nm. Values of HIF-1 alpha were expressed as ng/mg protein.
2.5. NGF Estimation
NGF protein levels were measured from the offspring brain homogenates using the NGF Emax immunoassay system (Promega, Madison, WI, USA) as described by us earlier (Sable et al., 2011). NGF levels in tissue homogenates were quantitated in the range of 3.9-250pg/ml. Values of NGF were expressed as pg/mg protein.
2.6. RNA isolation and cDNA synthesis
The total RNA from brain tissue was isolated using Trizol reagent (Invitrogen) and was quantified using Biophotometer (Eppendorf, Germany). One microgram of total RNA was reverse transcribed to cDNA using the High-Capacity cDNA reverse transcription Kit (Applied Biosystems, California, USA).
2.7. Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay
RT-qPCR for VEGF and NGF were performed using the TaqMan Universal PCR Master Mix (Applied Biosystems, California, USA) on the Applied Biosystems 7500 Standard Real Time PCR system and the protocol has been described by us earlier (Rathod et al., 2014). The relative expression level of the gene of interest was examined with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to normalize for variation in the quality of RNA and the amount of cDNA input. ΔCt values corresponded to the difference between the Ct-values of the genes examined and those of the GAPDH (internal control) gene. The RT-PCR reactions for each gene were performed in duplicate. To analyze the RT-PCR results, the average cycle number (Ct) of the reaction when it crossed a threshold valued was determined for each reaction. Relative expression levels of genes were calculated and expressed as 2ΔCt. The following TaqMan® assays (Applied Biosystems) were used in this study: GAPDH (Rn99999916_s1); VEGF (Rn01511601_m1); NGF (Rn01533872_m1).
2.8. Statistical Methods
Values are expressed in mean ± SD. The data was analyzed using SPSS/PC package (Version 20.0, Chicago, IL, USA). Mean values of the estimates of various parameters for the treatment groups were compared with those of control group at conventional levels of significance, using least significance (p<0.05) difference estimated from one way analysis of variance (ANOVA).
3. Results 3.1 Pup Brain VEGF mRNA Levels
Maternal vitamin B12 deficiency showed lower (p<0.01) VEGF mRNA levels as compared to that of control. Maternal omega-3 fatty acid supplementation to a vitamin B12 deficient group normalized VEGF mRNA levels. The VEGF mRNA levels in the maternal vitamin B12 supplemented were comparable to that of control group. Similarly, the VEGF mRNA levels did not alter in the vitamin B12 with omega-3 fatty acid supplemented group as they remained comparable to that of control group (Fig.2A).
3.2 Pup Brain VEGF Protein Levels
Maternal vitamin B12 deficient group showed lower (p<0.01) VEGF protein levels in the pup brain as compared to control. Maternal omega-3 fatty acid supplementation to a vitamin B12 deficient group also showed lower (p<0.01) levels of VEGF protein. The protein levels of VEGF in vitamin B12 supplemented group were comparable to the control group. Similarly, the group with both vitamin B12 and omega-3 fatty acid supplementation did not alter the levels of VEGF protein levels and remained comparable to that of the control group (Fig.2B).
3.3 Pup Brain HIF-1 alpha Protein Levels
Maternal vitamin B12 deficiency showed higher (p<0.05) HIF-1 alpha levels as compared to control while maternal omega-3 fatty acid supplementation to a vitamin B12 deficient diet normalized the HIF-1 alpha levels in the pup brain. The protein levels of HIF-1 alpha in the maternal vitamin B12 supplemented were comparable to control group. Similarly, in the vitamin B12 and omega-3 fatty acid supplementation group, the protein levels of HIF-1 alpha were comparable to control (Fig.3).
3.4 Pup Brain NGF mRNA Levels
Maternal vitamin B12 deficiency did not alter mRNA levels of NGF. Similarly, it was also comparable to that of control group in maternal omega-3 fatty acid supplementation to a vitamin B12 deficient diet. Maternal vitamin B12 supplemented group also showed comparable NGF mRNA levels to that of control while omega-3 fatty acid supplementation to the vitamin B12 supplemented group showed higher (p<0.01) NGF mRNA levels as compared to control as well as vitamin B12 supplemented group (Fig.4A).
3.5 Pup Brain NGF Protein Levels
Maternal vitamin B12 deficient group showed lower (p<0.05) NGF protein levels in the pup brain as compared to control while omega-3 fatty acid supplemented to vitamin B12 deficient diet showed higher NGF protein levels as compared to vitamin B12 deficient group. Maternal vitamin B12 supplementation did not alter the levels of these proteins as they remained comparable to that of control. Omega-3 fatty acid supplementation to a vitamin B12 supplemented group showed higher (p<0.01) NGF protein levels as compared to control and BS group (Fig.4B).
4. Discussion The present study reveals several novel and interesting key findings related to maternal vitamin B12 and omega-3 fatty acid status on neurovascular unit in the pup brain. The main findings of our study are (1) maternal vitamin B12 deficiency showed lower pup brain mRNA and protein levels of VEGF, higher HIF-1 alpha protein levels, lower NGF protein levels while NGF mRNA levels were not altered (2) maternal omega-3 fatty acid supplementation to a vitamin B12 deficient group normalizes VEGF mRNA levels, NGF protein levels and HIF-1 alpha protein levels (3) Levels of NGF, VEGF and HIF-1 alpha in the maternal vitamin B12 supplemented group were similar to that of control (4) Omega-3 fatty acid supplementation to vitamin B12 supplemented group showed higher NGF protein and mRNA levels while levels of VEGF and HIF-1 alpha protein were comparable to that of control.
The present study reports lower levels of VEGF in the pup brain at birth as a consequence of maternal vitamin B12 deficiency. The crucial role of VEGF in vascularization and neuronal cell migration has been well implicated in the developing brain (Acker et al., 2001; Schwarz et al., 2004). It has been reported that the developing brain requires a good vascular system for the delivery of oxygen and nutrients (reviewed by Mackenzie and Ruhrberg, 2012). The lower levels of VEGF in the present study may be indicative of hampered brain vasculature. Reduced VEGF levels are known to be associated with neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) in mice as well as in humans (Lambrechts et al., 2003). We have earlier reported high levels of maternal homocysteine as a consequence of vitamin B12 deficiency (Khaire et al., 2013) and likely to influence angiogenesis. It has been demonstrated that homocysteine inhibits angiogenesis through the inhibition of VEGF/VEGFR, Akt, and ERK1/2 mechanisms (Zhang et al., 2012). Hyperhomocysteinemia through the mediation of oxidative stress has been shown to be associated with changes in the structure and function of cerebral blood vessels (reviewed by Faraci and Lentz, 2004).
In the present study, pup brain HIF-1 alpha levels were higher in the maternal vitamin B12 deficient group. It has been demonstrated that higher HIF-1 alpha levels may be responsible for hypoxia-induced growth arrest and apoptosis (Goda et al., 2003). It has also been shown to regulating local brain hypoxia and influence and brain vascularization (reviewed by Giordano and Johnson, 2001). In the current study, VEGF levels were low although HIF-1 alpha levels were high suggesting that neuroprotective genes like VEGF may also be regulated by another transcription factor, independent of HIF-1alpha (Benderro et al., 2012). In the present study, omega-3 fatty acid supplementation to a vitamin B12 deficient group was able to restore HIF-1 alpha protein levels. Our results are in accordance with another recent study which demonstrates that omega-3 fatty acid consumption decreases the protein levels of HIF-1 alpha in subcutaneous adipose tissue of obese adolescents (Mejía-Barradas et al., 2014). However, the underlying mechanisms are not fully understood and needs to be explored.
In the current study, maternal vitamin B12 supplementation maintained the levels of VEGF, HIF-1 alpha and NGF in the pup brain as comparable to that of the control group. Similarly, the combined supplementation of vitamin B12 and omega-3 fatty acid group showed comparable levels of VEGF and HIF-1 alpha to that of control while NGF mRNA and protein levels were significantly higher as compared to control group. NGF is essential for the development and maintenance of sensory neurons and survival of neurons (reviewed by Berry, 2012). Apart from neurotrophic properties, recently NGF has been described as an important angiogenic molecule (Lazarovici et al., 2006), it is known to have a cross-talk with VEGF (Calza et al., 2001; Hansen-Algenstaedt et al., 2006) and is also known to promote endothelial cell proliferation and migration (Dolle et al., 2005; Moser et al., 2004).
We have recently reported that maternal vitamin B12 and omega-3 fatty acid supplementation increases brain derived neurotrophic factor (BDNF) and DHA levels in the brain of the offspring and also improves cognitive performance (Rathod et al., 2014). Vitamin B12 and omega-3 fatty acids may be involved in the regulation of neurovascular unit which requires proper network of molecules like angiogenic factors and neurotrophins which are involved in path finding, growth, migration and differentiation of neurons (Lee et al., 2009). Few other studies have also analyzed the effect of omega-3 polyunsaturated fatty acids on cerebral angiogenesis in several stroke models and shown to be protective against ischemic brain injury (Wang et al., 2014; Belayev et al., 2009; Zhang et al., 2010). Reports indicate that omega-3 fatty acid supplementation in transgenic mice has been associated with neurogenesis and oligodendrogenesis to improve post-stroke brain repair and long-term functional recovery (Hu et al., 2013). The process of neurogenesis has been shown to be associated with angiogenic microenvironment in the brain (Palmer et al., 2000),
5. Conclusion In conclusion, this is the first study which demonstrates the effect of maternal omega-3 fatty acids on a vitamin B12 deficient diet in influencing angiogenesis in the pup brain. Further it also demonstrates the effect of combined supplementation of vitamin B12 and omega 3 fatty acids on the pup brain NGF levels indicating the need for a combined maternal supplementation of these vital nutrients in improving the brain development and function in the offspring. The role of maternal nutrition in influencing the levels of neurotrophins and angiogenic factors will help to understand the putative mechanisms involved and may provide important clues to prevent early cognitive deficits and later neurobehavioral disorders in the offspring. Further investigation is required for better understanding the effect of vitamin B12 and omega-3 fatty acids on other molecules involved in the process of angiogenesis in the brain.

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