The Effects of Mung Bean Peptide and Its’ Complexes on the Treatment of Lead Poisoning

Objective . To investigate the eﬀects of mung bean peptide and its’ complexes on promoting lead excretion and neuroprotection of zebraﬁsh. Methods. The lead poisoning models of zebraﬁsh were established by lead acetate solution; the models were treated with high and low concentrations (58.3 and 175 μ g/mL) of mung bean peptides, with high, medium, and low concentrations (27.8, 83.3, and 250 μ g/mL) of mung bean peptide complexes, separately. The eﬀects of the mung bean peptide complexes on the lead content, axonal ﬂuorescence intensity, and peripheral motor nerve length changes were identiﬁed in the zebraﬁsh model, and the eﬀects of mung bean peptide and its’ complexes on zebraﬁsh’s lead excretion, axonal protection rate, and peripheral movement promotion rate of nerve regeneration were calculated. Results . The eﬀects of high concentration of mung bean peptide (175 μ g/mL) in promoting lead excretion was 29% ( p < 0 . 05), and the eﬀect of high concentration of mung bean peptide complexes (250 μ g/mL) in promoting lead excretion was 30% ( p < 0 . 05). The other concentrations of mung bean peptide and its’ complex groups did not show a noticeable lead excretion eﬀect. The protective eﬀects of mung bean peptide at concentrations of 58.3 and 175 μ g/mL against zebraﬁsh axonal injury were 98% and 101% ( p < 0 . 01), and the peripheral nerve regeneration promotion eﬀects were 29% ( p > 0 . 05) and 42% ( p < 0 . 05), respectively. The protective eﬀects of mung bean peptide complexes at concentrations of 27.8, 83.3, and 250 μ g/mL against zebraﬁsh axonal injury were 85%, 78%, and 93% ( p < 0 . 01); peripheral nerve regeneration promotion rates were 46%, 50%, and 50% ( p < 0 . 05). Conclusion . The mung bean peptide and its’ complexes can eﬀectively promote the discharge of lead in the zebraﬁsh lead poisoning and have protective and regeneration eﬀects on zebraﬁsh nerves.


Introduction
Lead (Pb) is a common neurotoxic heavy metal pollutant, which is widely used in industrial activities, and enters the environment through different routes [1,2]. Acute lead exposure can cause neurotoxic effects in animals, such as abnormal behavior, learning disabilities, and hearing loss [3]. In addition to its apparent neurodevelopmental toxicity, lead can also destroy the redox state of the body by directly inducing the body to produce reactive oxygen species and consuming intracellular antioxidants, leading to oxidative damage to the body, which would cause injuries to tissues and organs, especially in the nervous system [4,5].
Lead can easily pass through the blood-brain barrier, inhibiting the development of the central nervous system [6]. e normal development of glial cells has an important influence on the migration of neurons and the establishment of synaptic connections. Lead can also lead to the premature differentiation of glial cells, and the interactions between glial cells and neurons cannot proceed normally [7]. Besides, lead can accumulate in endothelial cells, and it can also affect the formation of synapses through calcium-like effects and nerve cell adhesion molecules [8].
Mung beans are rich in protein, starch, minerals, vitamins, and essential amino acids, among which the protein content is 19.5% to 33.1% [9,10]. In the past 20 years, researchers have found that the peptides from food-derived proteins have a wide range of biologically active functions, such as antioxidation, lowering blood pressure, lowering cholesterol, and improving the body's immunity [11][12][13][14][15][16][17]. In recent years, more and more attention has been paid to the studies on the health mechanism of mung bean peptides (MBPs), especially the neuroprotective mechanism. However, there is no report on repairing nerve injury caused by lead poisoning with MBPs [16].
Zebrafish has the characteristics of small size, short reproduction cycle, rapid embryo development, transparent embryo body, and high homology with mammals [18]. e effects of complexes on the nervous system in live or intact fixed specimens through special staining can be quickly detected. Organic damage is a model activity widely used in developmental toxicology, genetic biology, drug development, and environmental monitoring [19][20][21][22]. In order to further explore the effects of mung bean peptides and mung bean peptide complexes in promoting lead excretion and protecting nerves, a zebrafish lead poisoning model through induction was established, and the effects of different concentrations of mung bean peptide and mung bean peptide complexes on zebrafish in promoting lead excretion and neuroprotection were observed.

Drugs and Main Reagents.
Mung bean peptide and mung bean peptide complexes were supplied from Zhongshiduqing (Shandong) Biological Co., Ltd. (Heze, China); calcium sodium edetate was purchased from Huazhong Haiwei Gene Technology Co., Ltd. (Beijing, China); lead acetate trihydrate and other reagents were provided by Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Animals.
e wild-type AB strain of zebrafish was reproduced by natural paired mating. A total of 2130 fishes with an age of 2 hpf were used to determine the maximum tolerated concentration (MTC) of mung bean peptide complexes, evaluate the lead excretion effects of mung bean peptide and mung bean peptide complexes, and evaluate the protective effects of injured axonal. Motor neuron fluorescent zebrafish (NBT strain) was reproduced by natural paired mating. A total of 240 fishes, with an age of 2 hpf, were used to evaluate the effect of mung bean peptide and mung bean peptide complexes on the promotion of peripheral motor nerve regeneration. e above zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to each 1L of reverse osmosis water, conductivity was 480∼510 μS/cm; pH is 6.9∼7.2; hardness was 53.7∼71.6 mg/L CaCO 3 ).

Determination of Mung Bean Peptide Complexes MTC.
2102 hpf wild-type AB strains of zebrafish were randomly selected in a six-well plate (30 fish per well, each well with a capacity of 3 mL), and the mung bean peptide complexes (50, 100, 250, 500, and 1000 μg/mL) were dissolved in water to obtain different concentrations. A normal control group (zebrafish treated with water for fish farming) and model control group were set up. e dead zebrafish were observed and removed every day. After the mung bean peptide complexes were treated to 72 hpf, the number of deaths of the zebrafish was counted, and the MTC of the zebrafish was determined according to the toxicity and death of the zebrafish.

Evaluation of the Effects of Mung Bean Peptide and Mung
Bean Peptide Complexes in Promoting Lead Excretion. A total of 240 wild-type AB strains of zebrafish were randomly chosen 2 hours (2 hpf ) after fertilization and placed in a sixwell plate. ere were 30 fish in each well with a capacity of 3 mL each, and a total of 6 parallel groups were treated. e models were treated with mung bean peptide (58.3 and 175 μg/mL) and mung bean peptide complexes (27.8, 83.3, and 250 μg/mL) in water solution separately, and the positive control drug was injected with calcium sodium edetate 125 ng per fish. Lead acetate was added to water to establish a lead poisoning model. After 70 hours, samples of zebrafish were collected by washing the liquid and sucking up the water for fish farming with EP tubes. Every 60 zebrafishes were set as a series of samples, and each of them was detected by ICP-MS. e lead content (C) of a zebrafish sample was converted to the ratio of total lead to the wet weight of the zebrafish, and C of the mung bean peptide and mung bean peptide complexes were used to evaluate the lead excretion effects of the complexes. e calculation formula for the lead excretion effect is as follows: 2.6. Protective Effect on Axonal Injury. 2402 hpf transgenic wild-type AB strains of zebrafish were randomly chosen in a six-well plate (30 fish in each well, and each with a volume of administration, the axonal-specific antibodies were stained, and the pictures were taken under a fluorescence microscope. Zebrafish head axonal fluorescence intensity (S) was collected to evaluate the axonal protection of the mung bean peptide and its' complexes. e formula for calculating the protective effect of axonal damage is as follows:

Promoting Regeneration of Peripheral Motor Neuron
Injury. After 70 hours, pictures were taken under a fluorescence microscope to collect the average length of peripheral motor nerves (L). e motor nerve length was used to evaluate the motor nerve regeneration effect of mung bean peptide and its' complexes. e formula for the promotion of regeneration of motor nerve injury is as follows:

Statistical
Analysis. e data were expressed as (x ± s), and the differences were tested among the groups by T-test.

Determination of the MTC of Mung Bean Peptide
Complexes.
e mung bean peptide complexes induced 100% death of zebrafish at the concentrations of 500 μg mL −1 and above, and no apparent toxic phenotype was found at the concentrations of 250 μg mL −1 and below. erefore, the MTC of mung bean peptide complexes is determined to be 250 μg mL −1 (Table 1). e concentration of mung bean peptide complexes was reduced to MTC, and 27.8 μg mL −1 (1/9 MTC), 83.3 μg mL −1 (1/3 MTC), and 250 μg mL −1 (MTC) were selected in the pharmacodynamic evaluation experiment. e mung bean peptide complexes contain 70% mung bean peptides. erefore, the concentrations of the mung bean peptides in the regeneration test of peripheral motor nerve were set at 58.3 μg mL −1 (1/3 MTC) and 175 μg mL −1 (MTC), respectively.

Lead Excretion Effect of Mung Bean Peptide and Its'
Complexes. After successfully establishing zebrafish lead poisoning models, they were treated with mung bean peptide and its' complexes, respectively. After 70 hours, the zebrafish samples were collected by washing the liquid and sucking up the water for fish farming with EP tubes. ree parallel samples were tested for each experimental group, and each 60 zebrafish was considered as one sample. e lead content of zebrafish in the lead poisoning model control group (9.01 μg g −1 ) was significantly higher than that of the normal control group (3.11 μg g −1 ), indicating that the model was successfully constructed (p < 0.01). e positive control group (calcium sodium edetate 125 ng per fish) contained 8.38 μg g −1 of lead, and the effect of promoting lead excretion was 7%, indicating that calcium sodium edetate has a certain tendency to promote lead excretion under the experimental dose conditions. Compared with the model control group, the lead content of the zebrafish in the mung bean peptide high concentration group (175 μg/mL) was significantly reduced (p < 0.05), and the lead excretion effect was 29%; compared with the model control group, the lead content of the zebrafish in the mung bean peptide complexes high concentration group (250 μg/mL) was significantly reduced (p < 0.05), and the lead excretion effect was 30%. ere was no noticeable lead excretion effect in the other concentrations of mung bean peptide and mung bean peptide complexes, which shows that the appropriate concentration of mung bean peptide and mung bean peptide complexes have significant effects of promoting lead excretion ( Table 2).

Mung Bean Peptide and Its' Complexes Protect the Injured
Axonal. Compared with model group, calcium sodium edetate had a protective rate of 85% against zebrafish axonal injury (p < 0.05), mung bean peptide with the concentrations of 58.3 and 175 μg/mL had a protective effect on zebrafish, and the protection rates of axonal injury were 98% (p < 0.01) and 101% (p < 0.01). Compared with the model group, the mung bean peptide complexes with the concentrations of 27.8, 83.3, and 250 μg/mL protected the injured axonal in zebrafish, and the rates were 85% (p < 0.01), 78% (p < 0.01) and 93% (p < 0.01). e results showed that the appropriate concentration of mung bean peptide and mung bean peptide complexes had noticeable protective effects on zebrafish injured axonal (Figure 1 and Table 3).

e Protection of Mung Bean Peptide and Its' Complexes Promoting the Regeneration of Injured Peripheral Motor
Neuron. Compared with the model group, calcium sodium edetate promoted the regeneration of zebrafish peripheral nerves by 46% (p < 0.05). Compared with the model group, the mung bean peptide with the concentrations of 58.3 and 175 μg/mL had effects on the regeneration of peripheral nerve in zebrafish, and the regeneration rates were 29% (p > 0.05) and 42% (p < 0.05). Compared with the model group, mung bean peptide complexes with the concentrations of 27.8, 83.3, and 250 μg/mL promoted the peripheral nerve regeneration, and the rates were 46% (p < 0.05), 50% (p < 0.05), and 50% (p < 0.05). e results indicated that the appropriate concentrations of mung bean peptide and mung bean peptide complexes could significantly promote the regeneration of peripheral nerves in zebrafish (Figure 2 and Table 4).    Note: compared with the model control group, * p < 0.05, * * p < 0.01, and * * * p < 0.001.

Conclusion
Lead is harmful to multiple systems and organs in the human body, including bone marrow, hematopoietic system, immune system, nervous system, digestive system, kidney, and heart [22,23]. With the development of industrialization and transportation in recent years, environmental lead pollution was increased, and the impact on human health has become increasingly severe [24]. Clinically, the treatment of lead poisoning mainly uses edetate calcium disodium and penicillamine, but these drugs have many side-effect reactions, such as kidney damage and allergic reactions [25]. Natural products have significant effects on the discharge of lead [26][27][28]. It has been reported that alliin in garlic can bind with lead to reduce the toxicity of lead-poisoned rats; konjac powder can specifically bind to lead and promote its discharge without affecting absorbing calcium, iron, and zinc, so the refined konjac powder can be used as a health food for preventing and controlling lead poisoning [29,30]. Mung bean is recognized as a safe and nontoxic health food, which has attracted wide attention from all over the world [31]. At present, most focuses of mung bean is the processing of mung bean starch and vermicelli, ignoring the development and utilization of its byproduct mung bean protein [32,33]. Under the investigated concentration range of this work, the mung bean peptide and its' complexes provided by Zhongshiduqing (Shandong) Biological Co., Ltd. have significant effects on the excretion of lead, have a significant regenerative effect on zebrafish peripheral motor nerve damage caused by lead poisoning, and have an apparent protective effect on axonal injury.
After nerve injury, the neuron cell bodies cannot be regenerated, and the leading related functions will also be constantly under attack [34][35][36][37]. At present, the so-called nerve regeneration refers to the regeneration of axons,   including axon growth, axon extension, rebuilding synaptic connections with target cells, realizing nerve reinnervation, and restoring function [38,39]. Under normal circumstances, after peripheral nerve injury, Wallerian degeneration occurs at the proximal end of some nerves and all distal ends, which leads to degeneration and necrosis of nerve fibers and myelin sheaths to make nerve function disorders [40,41]. In this work, it was found that high-concentration mung bean peptide complexes (250 μg/mL) have a protection rate of 93% against zebrafish axonal damage and a 50% promotion rate of peripheral neuron injury, indicating that mung bean peptide can protect zebrafish axonal and promote the regeneration of the peripheral motor nerve. As a kind of agricultural product, mung bean is easy to grow, and the output of mung bean is very high. It is expected to develop mung bean peptide complexes as a leadpromoting health functional food, which would provide an adequate, nontoxic, green lead-emission health food for patients with chronic lead poisoning.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.