Protective effect of aerobic training and micro-curcumin supplementation on motor dysfunction in offspring mice exposed to gestational lead

This study aimed to evaluate the effect of maternal aerobic training and curcumin (Cur) supplementation on protecting motor function (e.g., muscle strength and muscle relaxant activity) from the harmful effects of maternal lead nitrate (Pb[NO3]2) exposure in female and male offspring BALB/c mice. The experimental groups included a healthy group, intraperitoneally Micro-Cur-treated group (50 mg/kg of body weight), aerobic training group (received training once a day during 8 weeks of experiments), aerobic training group exposed to Pb(NO3)2 (2 mg/kg) through drinking water during breeding and pregnancy, and aerobic training group treated with Micro-Cur and exposed to Pb(NO3)2 through drinking water during breeding and pregnancy. The pre-pregnancy training program was performed 5 times a week for 4 weeks at a speed of 10 to 12 m/min (75% VO2 max). All offspring were grouped based on the maternal groups, and the motor function was evaluated using an inclined plane, linear wire hang, and grip strength tests. Our results indicated that maternal Pb(NO3)2 exposure significantly decreased muscle strength and muscle relaxant activity, especially in males. These adverse effects were compensated by maternal aerobic training and Cur supplementation. The maximum of this compensation was observed in the maternal aerobic training group treated with Cur supplementation. Moreover, maternal aerobic training and Cur supplementation synergistically protected motor function in offspring mice from the adverse effects of maternal Pb(NO3)2 exposure, probably through different physiological mechanisms.


Introduction
Lead (Pb) is a common environmental contaminant and potentially toxic element.This contaminant can be absorbed by the body and accumulated in bones and blood [1].Both acute and chronic Pb exposure can have negative health effects, because the mammalian body poorly excretes Pb [1].Although the harmful effects of parental Pb exposure on offspring development are well documented, its biological mechanism is unknown [2].After maternal exposure to Pb, it will accumulate in bones, which can then be released during the increased bone turnover in pregnancy [3].Pb can cross the placental barrier, resulting in the impairment of fetal development [4].The accumulation of Pb in the fetus of humans and experimental animals is initiated from the second trimester [5,6].Besides, this accumulated Pb in the mother's body is excreted into the milk and thus poses a risk for the newborn during lactation [7].
A few papers have indicated that body skeletal muscle could be targeted by Pb exposure, leading to proximal muscle weakness [8,9] or asymmetric motor neuropathy [10].The presence of Pb in muscles can increase the activity of the Na-Ca exchange pump and interfere with Na-K adenosine triphosphatase (ATPase) activity [11], resulting in the disturbance of muscle function.Moreover, Pb exposure can produce free radicals in the body [12], mediating skeletal muscle dysfunction [13].
In line with animal and human studies, epidemiological studies have indicated that exercise during pregnancy is useful for maternal and neonatal health [14][15][16][17].As a result, the effects of maternal exercise on fetal development, particularly skeletal muscle development, are still unknown.Skeletal muscle is the most significant tissue for energy use and accounts for 30-40% of body weight [18].Maternal obesity/ malnutrition or metabolic dysfunction of muscle in progeny could result in impaired development of fetal muscle.This can cause obesity and metabolic diseases in offspring [19].Also, poor skeletal muscle performance is associated with exercise fatigue [20].This can worsen obesity and metabolic syndromes in offspring, causing reduced quality of life and poor physical movement.By shifting muscle fiber types from glycolytic to oxidative, maternal exercise can improve the oxidative capacity of offspring muscle without changing muscle weight.This can be related to improved mitochondrial biogenesis [21].In line with these results, we observed that maternal exercise enhanced the endurance capacity of offspring muscle.
Some papers have indicated that exercise during pregnancy can improve metabolism and skeletal muscle development in offspring [22].Aerobic training can increase insulin sensitivity by upregulating the expression of glucose transporter type 4 (GLUT4) in skeletal muscle in mother and offspring's bodies [23].However, the protective effect of exercise training during pregnancy, which may improve skeletal muscle dysfunction after Pb exposure, has not been examined so far.
Nowadays, an ingredient in the spice turmeric called curcumin (Cur) has caught the eye of researchers due to its ability to alleviate disease pathologies through its antioxidant, anticarcinogenic, and anti-inflammatory properties [24].Therefore, Cur can alter the cellular redox status and directly quench free radicals, which could be beneficial in protecting muscle from the adverse effects of Pb exposure [25].Although some studies have shown that Cur could prevent skeletal muscle atrophy and upregulate NF-E2-related factor 2 (Nrf2) in skeletal muscle, no study (to our knowledge) has evaluated its ability to prevent the adverse effects of heavy metals on motor function (e.g., muscle strength and muscle relaxant activity).
On the other hand, Micro-Cur has some characteristics making it suitable for this case, including the best bioavailability, biological half-life, and tissue distribution [26].Accordingly, this research critically assessed the effectiveness of maternal Micro-Cur supplementation and aerobic training during pregnancy in protecting motor function (e.g., muscle strength and muscle relaxant activity) against the harmful effects of maternal Pb exposure in male and female offspring.

Animals
Male and non-pregnant female BALB/c mice aged 6 weeks (weighing 25-30 g) were used in the experiments.This study was registered and approved by the Committee of Animal Investigations, Khorasgan Islamic Azad University of Iran (code: IR.IAU.KHUISF.REC.1398.038).The international guidelines for the care and use of animals in the laboratory approved all aspects of this experimental protocol.The animals had free access to food and water.They were maintained in a low-stress environment on a 12-h light/dark cycle (lights on at 07:00).

Mice mating
After 5 weeks of taking lead nitrate (Pb[NO 3 ] 2 ) and participating in aerobic training groups in the exercise program, 2 female mice were selected and placed in PVC cages with 1 male mouse, and the vaginal plaque was examined the next morning.Then, the pregnant female rats were separated from the male, and the gestational day 0 (GD0) was considered for them.
During pregnancy, Pb(NO 3 ) 2 dissolved in water was available to animals through drinking water [27].
Pregnant animals in the aerobic training groups continued their training program.The Micro-Cur and combination groups were injected intraperitoneally 5 days a week from the first day of pregnancy to delivery.Six weeks after delivery, male and female offspring's muscle strength and muscle relaxant activity were evaluated.

Experimental design
First, the animals were acclimated to a control diet for 7 days and exercise sessions on a motorized treadmill (electrical drive) at a speed of 5-8 m/min (0% grade) for 10 min 5 days per week [19].After that, the rats were randomly divided into 5 experimental groups (6 mice in each group), including the healthy control group, Pb(NO 3 ) 2 control group, Pb(NO 3 ) 2 + Micro-Cur group, Pb(NO 3 ) 2 + aerobic exercise group, and Pb(NO 3 ) 2 + Micro-Cur + aerobic exercise group [28].
The Pb(NO 3 ) 2 group exposed to 2% Pb(NO 3 ) 2 through the water once a day during 8 weeks of experiments was treated intraperitoneally with deionized water and ethanol solution (200 µL) 5 days a week from GD 1-14 [29].The Pb(NO 3 ) 2 + Micro-Cur supplementation group (PbMcur group) included animals exposed to 2% Pb(NO 3 ) 2 through the water once a day during 8 weeks of experiments was treated with Micro-Cur supplementation dissolved in ethanol plus distilled water by intraperitoneal injection for 5 days a week at a dose of 50 mg/kg of body weight from GD 1-14 [30].The Pb(NO 3 ) 2 + aerobic training group (PbExr group) was exposed to 2% Pb(NO 3 ) 2 through the water once a day during 8 weeks of experiments and 8 weeks of aerobic training.Finally, Pb(NO 3 ) 2 + Micro-Cur + aerobic training group (PbExrMcur group) was exposed to 2% Pb(NO 3 ) 2 through the water once a day during 8 weeks of experiments and treated with 8 weeks of aerobic training; they were treated with Micro-Cur supplementation dissolved in ethanol plus distilled water (50 mg/kg of body weight) by intraperitoneal injection 5 days a week from GD 1-14.

Consumption of Pb(NO 3 ) 2
Pb(NO 3 ) 2 (Merck Company, Germany) was dissolved in distilled water at a concentration of 2% and was made available to the animals as drinking water during the 8 weeks of the study.

Familiarization of animals with the treadmill and training protocol
Before the beginning of pregnancy, the animals in the training groups were familiarized with the treadmill for 1 week.They trained for 10 min and 5 times a week at a speed of 5-8 m/min.After that, the mice received 4 week training before pregnancy.The pre-pregnancy training program was performed 5 times a week for 4 weeks at a speed of 10 m/ min for 20 min and up to a speed of 12 m/min (average intensity equal to 75% VO 2 max) [31,32].After pregnancy, the mice received training similar to the last week before pregnancy (i.e., at a speed of 12 m/min for 40 min) and continued until the last week of pregnancy [33].The diagram of the animal study is shown in Fig. 1.

Inclined plane test
The offspring mice were tested at a postnatal age of 40 days.To evaluate the motor function, an inclined plane device was used for all offspring experimental animals [34,35].This test evaluates the ability of mice to prevent themselves from falling and determines the strength and endurance of the lower limbs.First, the lower limbs of the mice were allowed to clutch the hook side of a Velcro fabric, while the upper limbs remained on the acrylic surface of a testing plane inclined at a 25° angle.Then, the angle of the inclined plane was increased until the lower limbs were detached from the Velcro, and the mice slid down.The maximum angle was recorded.

Linear wire hang test
The offspring mice were tested at a postnatal age of 42 days.The linear wire hang test was conducted based on a previously described method [36].This test is useful for evaluating movement abnormalities, measuring coordination, endurance, strength, and balance in small laboratory animals.The offspring mice were placed in the center of the wire with all 4 paws.When the animals fell off the wire, the time was recorded.Three separate hang times were recorded with a 30 s rest.There was no limitation on the duration of each hanging.

Grip strength test
A grip strength meter (GPM-100, Melquest, Toyama, Japan) was used to evaluate forelimb grip strength in offspring based on a previously described method [37].Briefly, the animal was allowed to grasp the bar mounted on the force gauge.Then, the gauge was reset to 0 g after stabilization, and the animal's tail was slowly pulled back by an inspector.When the mouse released its forepaws from the bar, the tension was recorded.The test was performed at a postnatal age of 45 days.

Statistical analyses
The data normality was tested using Shapiro-Wilk and Levene's tests.Quantitative data are shown as mean ± SD.Differences between experimental groups were analyzed by 2-way repeated analysis of variance (ANOVA), followed by Tukey's correction post hoc tests.The statistically significant level was 5%.All statistical analyses were performed using OriginPro, 2016, b9.3.226(OriginLab Corporation, USA).

Inclined plane test
The findings of the inclined plane test showed that maternal exposure to Pb(NO 3 ) 2 and treatment with Micro-Cur, as well as maternal exposure to Pb(NO 3 ) 2 with aerobic training, significantly increased the maximum angle compared to controls only in male offspring (Fig. 1; P < 0.05).A significant increase in the maximum angle was observed in male offspring maternally exposed to Pb(NO 3 ) 2 and treated with Micro-Cur and aerobic training.There was no change in the angle (degree) in female offspring (P > 0.05; Fig. 2).

Linear wire hang test
Maternal Pb(NO 3 ) 2 exposure significantly decreased latency to fall in male and female offspring compared to the control group (Fig. 2; P < 0.05).In both female and male offspring, maternal exposure to Pb(NO 3 ) 2 and treatment with Micro-Cur supplementation, maternal exposure to Pb(NO 3 ) 2 with aerobic training, and maternal exposure to Pb(NO 3 ) 2 and treatment with Micro-Cur supplementation and aerobic training significantly increased latency to fall compared to the maternal Pb group (P < 0.05; Fig. 3).

Grip strength test
The results of the grip strength test showed that maternal exposure to Pb(NO 3 ) 2 in male offspring significantly decreased forelimb grip strength compared to the control group (Fig. 3; P < 0.05).In male offspring, maternal exposure to Pb(NO 3 ) 2 and treatment with Micro-Cur supplementation, maternal exposure to Pb(NO 3 ) 2 and treatment with aerobic training, and maternal exposure to Pb(NO 3 ) 2 and treatment with Micro-Cur supplementation and aerobic training significantly increased forelimb grip strength compared to the maternal Pb group (Fig. 3; P < 0.05).On the other hand, in female offspring, maternal exposure to Pb(NO 3 ) 2 and treatment with aerobic training and maternal exposure to Pb(NO 3 ) 2 and treatment with Micro-Cur supplementation and aerobic training significantly increased forelimb grip strength compared to the other groups (Fig. 4; P < 0.05).

Discussion
The main objective of this paper was to document the protective role of maternal aerobic training and Cur supplementation on motor dysfunction of offspring (e.g., muscle strength and muscle relaxant activity) resulting from maternal exposure to Pb(NO 3 ) 2 .All behavior tests in male offspring indicated that maternal exposure to Pb(NO 3 ) 2 caused motor dysfunction, whereas maternal Pb(NO 3 ) 2 exposure in female offspring only decreased muscle strength.These observations indicated that maternal exposure to Pb(NO 3 ) 2 might have more influence on motor functions in male offspring than in female offspring.Previous papers have reported that chronic Pb toxicity caused symmetrical distal wasting and weakness of muscle and upper or lower motoneuron involvement [10].
Exposure to Pb during pregnancy caused the accumulation of this heavy metal inside the mother's body, resulting in chronic exposure to Pb via the placenta and milk in the offspring [38].Chronic exposure to Pb can have harmful effects on the motor cortex and reduce the number of neurons and astrocytes by oxidative stress, which consequently leads to alterations in motor skills [39].Besides, chronic exposure to Pb is able to direct changes in energy metabolism and redox reaction in muscles, inducing muscle fiber transformation and activation [40].
Studies have indicated that Pb results in decreased brain gray matter volume, decreased brain volume, reduced executive function [41,42], cognitive and long-term behavioral disorder [43], reduced attention, neurological deficits [44], and impairment in learning and planning rules (mainly in males) [45].Previous studies have indicated that Pb has sexspecific neurotoxic effects, in which both genders are equally negatively affected, except for hyperactivity in males.The literature shows that the brain is less vulnerable to toxic exposures in females than in males.This is because of reduced vulnerability to oxidative stress in females, greater sulfate-based detoxification capacity in females, greater glutathione availability in females, greater neuroinflammatory response in males, potentiating effects of co-exposure to neurotoxicants and testosterone, and neuroprotective impacts of female hormones (progesterone and estrogen), especially in the reduction of inflammation and oxidative stress [46].
According to the results, maternal aerobic training and maternal Cur supplementation, especially in males, increased muscle strength and muscle relaxant activity compared to the maternal Pb group.The most protective effect against maternal Pb(NO 3 ) 2 exposure was observed in the group treated with aerobic training and Cur supplementation.
A previous study demonstrated that maternal exercise during gestation could decrease metabolic impairments in the mother and fetus.This can enrich the skeletal muscle transcriptional profiles in offspring with genes regulating inflammation and immune responses [47,48].Besides, it is reported that maternal exercise before and during pregnancy can directly promote physical activity in offspring, It has been revealed that the upper and lower body of children with mothers doing sports during pregnancy is significantly more strong [45].Besides, a strong correlation was reported between maternal circulating testosterone levels during pregnancy and children's strength and endurance [46].On the other hand, a positive correlation was found between circulating testosterone levels in pregnant mothers and fetal testosterone levels [47][48][49].Thus, an increased strength observed in children with mothers who exercised during pregnancy can be justified to some extent.
Furthermore, maternal exercise was shown to activate AMP-activated protein kinase (AMPK) and DNA demethylation in the Ppargc1a gene promoter, which increases Ppargc1a expression consistently in offspring muscle associated with increased oxidative capacity and endurance.Hence, DNA demethylation in the Ppargc1a gene promoter is a critical control point for enhancing the offspring muscle function during fetal muscle development.It has been indicated that the level of apelin increases in the circulation of the fetus by mothers' exercises.Exercises stimulate the apelin-AMPK axis, thus increasing the cytosolic Ca2 + level [50][51][52].A relationship was shown between calcium concentration and force production in muscle [53].Thus, in the present study, the improvement of the apelin-AMPK pathway and the state of cytosolic calcium was stated as another mechanism involved in improving the strength in the training groups.
According to previous studies, moderate-intensity exercise training can enhance brain functions at both cellular and behavioral levels in rats with Alzheimer's disease [30], as well as in infants with mothers who exercised during pregnancy [31].The speed of running on the treadmill was considered 12 m/min (75% VO 2 max).
Due to mitochondrial disorders, Pb can influence the function of the brain neurons and functionality [54].It has been reported that mothers running on a treadmill for 40 min have better performance in Y-Maze behavioral tests, increased BDNF, and mitochondrial agents of brain neurons compared to mothers running on a treadmill for 20 and 30 min [31].Also, Pb can increase cellular oxidation and decrease antioxidant performance [55,56].On the other hand, long-term exercise can cause the neutralization of reactive oxygen species (ROS) and strengthen the antioxidant system (increased expression of superoxide dismutase [SOD]) [31,57].In the present work, the time for running on a treadmill was 40 min in the last week before pregnancy and during pregnancy.However, it is suggested to investigate the effects of different training variables, including the intensity, volume, and frequency of the exercise, in future studies.
Of note, there is concern about treadmill exercise as possible stress to maternal mice.Although acute exercise elicits stress temporally, both exercised and sedative maternal mice underwent the same protocol in this study, avoiding a possible interference of stress.On the other hand, regular treadmill exercise at moderate intensity, which was used in the current study, actually has protective effects on stressrelated symptoms [51][52][53].
In female offspring, this effect was first evident around sexual maturity and persisted into later adulthood.Increased physical activity was also observed in male offspring, but not later in adulthood [49].A human study showed that the mother's exercise program promoted the development of motor, social [54,55], mental, and cognitive skills [56,57] in children.Some studies have claimed that there is a positive effect of maternal exercise on neurobehavior in newborns [55].This correlation was likely due to genetic inheritance [50].This phenomenon resulted in better muscle strength and muscle relaxant activity, which can compensate for the adverse effects of maternal Pb exposure.However, the main mechanisms underlying these observations remain unknown and need future studies.
Similar to our study, the previous studies have shown that Cur treatment can reverse alterations (caused by maternal exposure to Pb) in locomotor and neuronal network development in offspring [58,59].Thus, this treatment allows normal locomotor behavior in offspring.Besides, it was demonstrated that Cur has anti-inflammatory properties targeting cellular signaling molecules, enzymes, and transcription factors, including nuclear factor-κB (NF-κB), activator protein 1 (AP-1), and cyclooxygenase-2 (COX-2) [60].Therefore, it is assumed that these properties of Cur not only may compensate for the side effects of Pb on offspring's muscle actions but also improve the development of muscle growth and metabolism by controlling inflammation.To confirm this hypothesis, some studies have suggested that Cur exerts anti-catabolic and protective effects on skeletal muscle [61,62].

Conclusion
Generally, this study showed that maternal exposure to Pb(NO 3 ) 2 significantly decreases muscle strength and muscle relaxant activity, especially in males.Maternal aerobic training and Cur supplementation compensate for the adverse effects of Pb exposure and have synergistic protective effects.These results can help build a foundation for determining the efficacy of maternal aerobic training and Cur supplementation for the protection of offspring's motor functions against heavy metal poisoning.

Fig. 1 Fig. 2
Fig. 1 Diagram of the animal study

Fig. 3 Fig. 4
Fig. 3 Linear wire hang test.a Control, b Pb +2 , c Pb +2 and MCur, and d pb+2 and exercise.f Pb +2 and MCur and exercise; latency to fall in male and female offspring in the linear wire hang test after maternal exposure to Pb(NO 3 ) 2 ; treatment with Micro-Cur and aerobic training.The significant difference between the groups was shown by "letters" (mean ± SD)