Effect of 6 Weeks Aerobic Training with Nanocurcumin Consumption on IL1β, Nitric Oxide and Depression in Women with Syndrome Metabolic


 Backgrounds and Objectives: The use of anti-inflammatory supplements is important in improving the function of obese people. The aim of this research was to investigate the effect of 6 weeks aerobic exercise with moderate intensity and consumption of nanocurcumin on IL1β, nitric oxide and depression in 60-65 years old women with syndrome metabolic. Materials and Methods: 44 women with metabolic syndrome (MetS) were randomly selected and divided into four groups of 10, including of MetS exercise+ Curcumin (MECU), MetS exercise (ME), MetS NanoCurcumin (MCU), MetS control (MC Training groups performed an exercise protocol of aerobic exercise for 6 weeks (three sessions per week). Blood samples were obtained before and after training period for antioxidant indicators and lipid degradation measurement. Also, Beck anxiety questionnaire used for evaluating levels of anxiety. T-test and one-way analysis of variance were used for the evaluation of within-group and between-group differences, respectively.Results: There was a significant difference in IL1β, NO and depression before and after exercise in all three experimental groups (p≤0.05). Also, the results showed a significant difference in the level of depression, NO and depression in the Reaserch groups. The highest decrease in these variables was observed in the aerobic training group and training with nanocurcumin consumption group (p≤0.05).Conclusion: These findings indicated that six-week nanocurcumin supplementation with aerobic training a suitable method for reducing IL1β and NO and depression preventing metabolic, cardiovascular and inflammatory diseases in women with syndrome metabolic.Name of the registry: IR.SEMUMS.REC.1396.107Trial registration number: IRCT2017082335857N1


Introduction
Aging is an inevitable fact, the increase in the elderly population around the world is so important, The World Health Organization estimated the total number of elderly people in the world in 2006 at about 700 million and And it will double in the next 40 years, In Iran, the population over the age of 60 will reach about 10 million by 2020 and more than 26 million by 2050 (1). Depending on the social and family circumstances of the elderly, 12 to 16% of the elderly become depressed (1). Depression reduces the quality of life of the elderly and increases their dependence on others (1). Depression increases drug use, length of hospital stay, and care costs (1). Its prevalence is also expected to increase to more than 15-20% (1,2). Symptoms of depression: sadness, irritability, decreased interest in fun activities, signi cant weight gain and loss, insomnia or excessive sleep, decreased memory, decreased libido, fatigue, loss of energy, worldly worthlessness, excessive guilt, decreased The power of concentration and increased suicidal ideation (2)(3)(4). Biologically, depression is a motivational disorder caused by a lack of biogenic amino acids. Biogenic amines are neurochemicals that facilitate neurotransmission and play an important role in the pathology of mood disorders. Speculation about the neurochemical basis of depression is often about reduced serotonin and norepinephrine (5). Serotonin levels are related to the amount of tryptophan in the blood. Researchers have shown that 10% of tryptophan is freely present in the blood, with free tryptophan entering the brain and the rest bound to albumin. The binding site of tryptophan and fatty acids to albumin is the same. Some researchers have shown that long-term aerobic exercise increases the recall of fatty acids and therefore the amount of free tryptophan in the blood increases due to the competitive binding of fatty acid and tryptophan to albumin binding. The rate of serotonin synthesis increases in the brain (6)(7)(8). Curcumin was used in China and India to treat some diseases (9). Fundamental studies on the medicinal properties of curcumin have shown its effect on lowering blood sugar (10)(11)(12), anti-in ammatory and anti-in ammatory effects (2) and antioxidant properties (13) of turmeric (2). Aging and metabolic syndrome are associated with increased chronic in ammation (1). Due to lifestyle changes, the prevalence of metabolic syndrome has increased in today's society (1). Metabolic syndrome refers to the presence of at least 3 of 5 risk factors. These factors include: waist fat greater than 94 cm, triglyceride higher than 150, HDL level less than 40, hyperglycemia (glucose higher than 110) and blood pressure higher than 85/130 (1). Some researchers have suggested a decrease in serotonin in connection with an increase in in ammation (14,15). The exercise and curcumin supplements both reduce in ammation, exercise and curcumin supplementation can be used as a treatment strategy to improve severe depression (5,(16)(17)(18). In this regard, research has been done, which we will brie y mention. Osali et al. (2017) expressed the positive effect of exercise and curcumin consumption on reducing in ammatory factors and depression. Kulkarni et al. (2008) reported curcumin supplementation as effective in reducing depression (16). Legrand et al. (2009) showed the effect of regular walking on reducing depressive symptoms in women (19). Mokhtari et al. (2013) also reported the effect of 12 weeks of aerobic exercise on reducing depression (20). Dey et al. (1994) stated that 4 weeks of moderate-intensity swimming is effective in improving the behavior of depressed people (21). Donohue et al. (2004) also reported the lack of effect of exercise training on depression in young athletes (22). NO has multiple biological actions and can regulate physiology acutely or lead to long-term changes in cell function. The pleiotropic roles of NO include the regulation of long-term synaptic transmission, learning, memory, platelet aggregation, leukocyte-endothelial interactions, immune function, and angiogenesis and arteriogenesis. However, NO is most well known as a potent regulator of blood ow and was originally termed endothelial-derived relaxing factor (EDRF). The story unfolded from Furchgott and Zawadzki's initial discovery that endothelial cells control acetylcholine-induced relaxation of smooth muscle. A few years later, NO was identi ed as the key endothelium-derived molecule promoting vasodilation: NO synthesized by NOS in the endothelium diffuses into the vessel wall where it activates sGC in vascular smooth muscle; this leads to a rise in cyclic GMP (cGMP) and elicits relaxation of the vessel. However, it readily became apparent that different isoforms of NOS have different physiological functions. For example, NOS3 and NOS1 were found to have distinct roles in regulating microvascular tone; Overall, NO derived from the integration of NOS3 and NOS1 activities play key roles in regulating systemic blood pressure and acutely regulating organ blood ow, whereas NOS2-derived NO species are most well recognized for their impact on pathogen killing and in ammatory processes (23).
The absorption of curcumin in turmeric is very low due to its lipophilic nature from the digestive system.
In fact, there is a layer of water on the surface of intestinal epithelial cells. In order for a drug to be absorbed from the intestine after oral administration, it must have a minimum solubility in water to pass through this layer after dissolving in water. In other words, small amounts of curcumin in the gut (such as lipophilic substances) are converted to mesylate and absorbed. The curcumin capsules contain nanocurcumin that its size is 10 nm and its solubility on the water is 100 times more than turmeric powder and this factor causes it to absorb. After oral consumption soft gel capsules which contain nanocurcumin opend in less than 15 minutes in acidic gastric medium and distribute in all of gastric. These Nano Curcumin in acidic gastrit conditons are constant at least 6 hours and they change the intestine intake manner. With regard to the increasing of curcumin absorbtion when using it as a Nano_ drug, this question raised out that: Dose the 80 miligram consumption of nanocurcumin cause the signi cant decrease depression, NO and IL1β?

Methodology
The method of quasi-experimental study is applied studies. The research design included pre-test and post-test with one control group and three experimental groups All stages of the research were approved by the ethics committee of Semnan University of Medical Sciences with the license number IR.SEMUMS.REC.1396.107 Also, this research has been registered in the Clinical Trial Registration Center of Iran under the number IRCT2017082335857N1.
The statistical population of the present study was 250 wives of martyrs aged 50 to 65 years in Zanjan.
Sampling is purposeful. After distributing an advertisement in the General O ce of the Martyr and Veterans Affairs Foundation of Zanjan Province and sending an invitation to the statistical community, at the beginning of the research, 65 people volunteered to obtain a permit to participate in the physical activity considered in the study. Subjects were examined by a physician for a history of illness and physical distress, psychological problems, sleep, and blood pressure, and if necessary, some of them underwent heart health tests. None of the subjects had a history of regular physical activity in the past year. It should be noted that in this study, the ATPIII (Adult treatment panel iii) criterion was used to identify metabolic risk indicators, which were considered in the presence of three of the ve indicators (waist circumference more than 94 cm, blood triglyceride more than 150 mg / dL, blood HDL less than 40 mg / dL, blood pressure more than 130.85 mm Hg and fasting blood glucose higher than 110 mg / dL). In other words, volunteers with three or more metabolic risk indices based on ATPIII criteria were considered as subjects with metabolic syndrome (1).

Training protocol and supplementation
Subjects were allowed to practice from 9 a.m. to 12 p.m. The training period was six weeks. They practiced 3 days a week. Each session, the exercises were performed in the form of three consecutive sets with a break interval of 5 minutes between sets. The training set time in the rst week was 12 minutes, and with the passing of each week, one minute was added to the duration of the training sets, so that in the sixth week of training, it reached three sets of 17 minutes. Exercise was performed at an intensity of 65 to 75% of the reserve heart rate. Resting heart rate was checked weekly and the intensity of the exercise program was adjusted using a Polar (Finland) pacemaker. The whole training session started with 5 minutes of warming up ( exibility and stretching exercises) and ended with 5 minutes of cooling down. The control group avoided regular, regular physical activity during this six-week period. The stored heart rate was calculated using the Carvonen formula (1). 60 to 70% reserve heart rate= (Maximum heart rate -Resting heart rate× [(60 70 %))]+ Resting heart rate Heart rate when waking up and lying down before getting out of bed = resting heart rate Amount and method of receiving nanocurcumin Subjects received an 80 mg nanocurcumin tablet each morning.

Measurement of biochemical parameters
All subjects underwent fasting blood sampling at 9 am (to measure plasma levels of IL1β and serum glucose, triglyceride, plasma doped lipoprotein) in two stages including pre-test and post-test (after six weeks). In order to eliminate the acute effects of exercise such as delayed contusion and small possible injuries to the muscle structure on the serum level of IL1β, blood sampling was performed in the post-test phase four days after the last training session (23,24). At each blood draw, blood vessels of the brachial vein were collected in tubes without EDTA anticoagulant. After centrifugation (12 minutes at 3000 rpm) and separation of serum, serum IL1β levels were measured by ELISA by a special kit for measuring IL1β (eBioscience, Vienna, Austria) with a sensitivity of 0.05 pg / mL. Blood glucose was measured by glucose oxidase and fat levels were measured by standard enzymatic method (Pars Kit test, Karaj, Iran) using Kubas Mira biochemical AutoAnalyzer . The coe cient of variation of this kit in each assay and between different assays (inter-assay variation) is equal to 1.82% and 1.6% for triglycerides, 1.74% and 1.19% for blood sugar, respectively, and for HDL was equal to 2.15% and 1.28%, respectively. Nitric Oxide (NO) is extremely unstable and undergoes rapid oxidative degradation to stable nitrite (NO2-) and nitrate (NO3-), which react with the colorant and produce azo-pink composition and is quanti ed spectrophotometrically [27]. Serum levels of metabolites were measured by colorimetric Griess assay. During the colorimetric assay, the nitrite concentration was determined by measuring the absorbance at 450 nm.

How to calculate body fat percentage
The percentage of body fat of the subjects was calculated by the pneumatic composite device model OMRON BF500 made in Germany.
How to calculate calorie intake Subjects recorded daily food intake in the notebook before starting the training protocol (beginning, middle and end of the week) and then the calories of food consumed for breakfast, snacks, lunch and dinner were calculated by N4 software. The results showed no signi cant difference in the amount of calories consumed between the groups (1).

Statistical analysis
Data were presented as mean ± SD. Levene's test was used to test the homogeneity of variances in the pre-test. The Kolmogorov-Smirnov test was used to ensure the distribution of the variables was normal.
After showing the normality of data, to analyze the data,paired sample t-test was used to compare the pre-test and post-test means within the group, and One-way analysis of variance (difference between test and post-test) was used to compare the groups. Also, in the case of signi cant ndings, Bonferron's post hoc test was used. All data analysis was performed using SPSS software version 24 and at a signi cance level of p≤0.05.

Results
The physical qualities of subjects were measured twice: at the rst and the last sessions. The measures are as follows in the last session: expressed as mean ± standard deviation [SD]: age, 62.3 ± 1.23 years; height, 164 ± 7 cm; body weight, 81.2 ± 2.4 kg; and body mass index (BMI), 29.5 ± 1.2 kg/m 2 . The results of group comparison of metabolic syndrome, weight, fat percentage, BMI, IL1β, NO, depression are listed in Tables (1, 2). The results of paired sample t-test showed that IL1β levels in the training group (p=0.003) nanocurcumin consumption (p = 0.05) and training with nanocurcumin (p=0.002) decreased signi cantly in posttest compared to pretest. IL1β did not change signi cantly in control group compared to pre-test time (p=0.8). Also, NO levels in training group (p=0.002), nanocurcumin consumption (p=0.002), and training with nanocurcumin consumption (p = 0.002), decreased signi cantly in posttest compared to pretest. NO did not change signi cantly in comparison with the pre-test time in the control group (p=0.5) ( Table 2). The results of one-way ANOVA showed that levels of IL1β in the training, nanocurcumin and training with nanocurcumin groups were signi cantly lower than the control group (p=0.002) ( Table 2). The results of the Bonferroni post-hoc test of IL1β showed no difference between training and training with nanocurcumin groups (p=0.1), but there was a difference between nanocurcumin and training groups (p=0.000), training and control groups (p=0.000), training with nanocurcumin and nanocurcumin groups (p=0.002), nanocurcumin and control groups (p=0.000), and training with nanocurcumin and control groups (p=0.002). Also, levels of NO in training, nanocurcumin and training with nanocurcumin groups were signi cantly lower than the control group (p=0.002). The results of Bonferroni post hoc NO test showed no difference between training and nanocurcumin groups (p=0.1). But differences were shown between training and training with nanocurcumin groups (p=0.006), training and control groups (p=0.02), nanocurcumin and training with nanocurcumin groups (p=0.001), nanocurcumin and control groups (p=0.007), training with nanocurcumin groups with control groups (p=0.002). In comparison between groups, depression in the exercise + supplement group was higher than the exercise and supplement groups, and this difference is statistically signi cant.   2004) showed that young people who exercised regularly were more likely than other non-athlete youths to have impulsive, neurotic, and psychotic disorders. Considering the age of the subjects and the purpose of exercise in this age period, it can be said that the attitude towards exercise itself is effective in reducing and increasing depression and behavioral disorders. In the present study, exercise was performed to treat and reduce depression, which showed a signi cant reduction in blood pressure, body fat percentage, weight, waist size, triglyceride. But in the study of Donohue et al. (2004), exercise was aimed at achieving results (championships), which increases behavioral disorders. Differences in age of subjects, body composition and purpose of exercise are among the reasons for inconsistent results. The mechanism of effect of exercise and nanocurcumin consumption on reducing depression in women aged 65-60 years with metabolic syndrome can be investigated through several pathways. The rst pathway is the effect of exercise and nanocurcumin consumption on IL1β reduction. In the present study, the reason for the decrease in IL1β can be attributed to a signi cant decrease in blood pressure, triglyceride, waist size and a signi cant increase in HDL. As Zorilla et al. (2001) and Mossner et al. (2007) pointed out that the increase in in ammatory factors is associated with a decrease in serotonin (14,15). The cause of reduced depression can be attributed to a decrease in IL1β and a decrease in IL1β is effective in increasing serotonin (17).
The second pathway is the effect of exercise on increasing tryptophan levels. Tryptophan is a precursor to the synthesis of serotonin, which is increased by prolonged aerobic exercise. The mechanism of increase of tryptophan: Fatty acids and tryptophan are transported in the blood by albumin, both of which have the same binding site on albumin, In training, the amount of fatty acid recall increases, This increase leads to a decrease in the albumin site for the binding of tryptophan to albumin. Increased free tryptophan in the blood leads to more tryptophan entering the brain and the presence of tryptophan in the central nervous system leads to more synthesis of serotonin and relief of depression (6)(7)(8). Curcumin also inhibits both monoamine oxidase A and B. Monoamine oxidases A and B are oxidizers of dopamine, norepinephrine and serotonin, which curcumin reduces their oxidation and consequently reduces the levels of dopamine, norepinephrine and serotonin (2,16,27). Previous studies concerning serum or plasma cytokine levels and their concentrations have evaluated the acute effects of exercise . Based on the ndings reported in previous as well as the present study, it could be hypothesized that major weight loss connected to exercise training is necessary to modulate in ammatory indicators levels. Also, we found a signi cant reduction of IL-6 and CRP in women with metabolic syndrome. In this regard, some of the previous studies have shown signi cant reductions in C-reactive protein concentration which is another important indicator of systemic in ammation, following nano-curcumin intake and supplementation (28). Increased expression of in ammatory factors causes the activity of nuclear factor kappa B (NF-κB). NF-κB binds to DNA to block the expression of BDNF (brain-derived neurotrophic factor). Curcumin reduces IL1β expression and NF-κB transcription, which in turn increases BDNF expression (2,17). BDNF is a neurotrophic factor that causes both neurogenesis and neuroplasticity (28)(29)(30). BDNF is a neurotrophic factor that causes both neurogenesis and neuroplasticity (28)(29)(30). Due to the fact that neurotransmission is biologically impaired in depressed people (5) Signi cant decrease in waist circumference, blood pressure, triglyceride and increase in HDL due to two months of moderate-intensity aerobic exercise signi cantly reduced IL1β and decreased IL1β decreased NF-κB activity and binding to DNA. This process leads to increased BDNF expression and increased BDNF also improves neurotransmission and relieves depression (2,31). The present study showed that the exercises for 6 weeks with nanocurcumin supplementation had signi cant effect on NO levels in women 60-65 years with metabolic syndrome. Chematic of changes in NOS3 or NO: (A) Decreased NOS3 expression commonly occurs in obese and diabetic states. The mechanisms proposed for diminished expression include TNF-α-mediated destabilization of NOS3 mRNA, which may involve eEF1A1. High levels of NO may regulate NOS3 abundance through cGMP-mediated or via NF-κB-SNO feedback regulatory pathways. A small 27-nt RNA regulates NOS3 expression also, although it is not known whether this mechanism is invoked in obesity or diabetes. (B) Decreased NOS3 activity in obesity and diabetes is largely attributed to insulin resistance, which may be mediated by free fatty acid (FFA)-induced activation of TLR2, TLR4, and NF-κB. In addition, activation of PKCβII may diminish Akt signaling, which normally promotes phosphorylation of NOS3 on Ser1177. Phosphorylation at this site increases NO output by the enzyme. Hyperglycemia may also lead to increased O-GlcNAcylation of NOS3, which decreases Ser1177 phosphorylation and inhibits its activity. In addition, conditions leading to obesity promote upregulation of Cav-1, which is a negative regulator of NOS3, and ceramide accumulation disrupts the NOS3-Akt-HSP90 complex, diminishing activity of the enzyme. (C) NOS3 may also be uncoupled or NO quenched in obese and diabetic states. Diminished levels of substrates and cofactors, such as L-arginine or tetrahydrobiopterin (BH4), lead to uncoupling of the enzyme, which is commonly associated with the presence of NOS3 monomers rather than dimers and can produce superoxide instead of NO. Endogenous inhibitors of NOS3 such as ADMA are also increased in obese conditions and can promote NOS uncoupling. Elevated production of reactive oxygen species such as superoxide can quench NO and result in its oxidation to highly reactive peroxynitrite, which damages biomolecules and can oxidize BH4 to BH2 (23).

Conclusion
Due to the decrease in IL1β, NO and depression the improvement of metabolic syndrome due to six weeks of moderate-intensity training and nanocurcumin consumption, the cause of reduced depression can be attributed to them. And suggested aerobic exercise with nanocurcumin as a non-invasive strategy in the treatment of depression in women 60-65 years with metabolic syndrome. Finally, it is suggested to conduct research with different intensity and duration of training period on the target community.

Consent for publication
All participants were aware during the informed consent process that the results of this study may be published.

Availability of data and materials
Not applicable. Conclusions of the manuscript are based on relevant data sets available in the manuscript.