The Preventive Effect of Lactobacillus Fermentum SCHY34 Isolated from Traditional Fermented Yak Yogurt on Lead Acetate-Induced Neurological Damage in SD Rats


 Background: Lead is a heavy metal that is widespread in nature and has extremely strong chemical toxicity in the human body especially harmful to the human nervous system. Lactic acid bacteria (LAB) can be used to maintain the balance of the intestinal microbiota, and recent reports have shown that LAB can remove metal ions through adsorption. In this experiment, Sprague-Dawley (SD) rats were treated with 200 mg/L of lead acetate solution daily to induce chronic lead poisoning, and oral LF-SCHY34 to study its mitigation effects and mechanisms on rat neurotoxicity.Results: Through electron microscopy and energy spectrum analysis, it was found that the surface of the lactic acid bacteria adsorbed a large amount of lead ions, and the O and N elements in the bacteria were significantly reduced. Animal experiments showed that LF-SCHY34 maintained the morphology of rat liver, kidney, and hippocampi, reduced the accumulation of lead in the blood, liver, kidney, and brain tissue, reduced the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN) in rat serum, and increased δ-aminolevulinic acid dehydratase (δ-ALAD) activity in serum. Further, LF-SCHY34 alleviated the lead-induced decline in spatial memory and response capacity of SD rats, and also regulated the secretion of neurotransmitters and related enzyme activities in the brain tissue of rats. LF-SCHY34 inhibited the secretion of glutamate and the activity of monoamine oxidase in brain tissue, promoted the synthesis of glutamine (Glu), norepinephrine (NE), and cyclic adenosine monophosphate (cAMP), and increased the activities of glutamine synthetase (GS), acetylcholinesterase (AchE), and adenylate cyclase (AC). In addition, the expression of genes related to cognitive capacity in rat brain tissues such as calmodulin (CaM), protein kinase A (PKA), brain-derived neurotrophic factor (BDNF), c-fos, c-jun was increased by LF-SCHY as was the expression of the antioxidation genes and the anti-apoptotic gene. Conclusions: Compared with the lead poisoning treatment drug EDTA, LF-SCHY34 not only had greater lead discharge capacity than EDTA, but also had a greater alleviating effect on organ damage and oxidative damage caused by lead. As a food-grade LAB, LF-SCHY34 has great potential and research value for removing heavy metals from food and alleviating the toxicity of heavy metals.


Introduction
Lead is a multiaffinity toxic heavy metal that can accumulate in the environment over time, pollute the environment, and directly or indirectly pollute food. Lead in the environment can also enter the human body through various channels such as the respiratory tract, digestive tract, skin, and mucous membranes. [1] The Joint FAO/WHO Expert Committee on Food Additives (JECFA) set the limit for daily lead intake to 1.3 μg/kg BW for adults and 0.6 μg/kg BW for children. Lead first enters the blood after being absorbed by the human body, 1% of which is combined with proteins in the plasma and remains there. The remaining 99% is combined with red blood cells in the form of soluble lead glycerophosphate or lead hydrogen phosphate and enters other organs with the blood circulation to be absorbed and accumulate in tissues. [2] The chemical properties of lead are relatively stable, and it does not easily decay or transfer. In the human body, lead has no physiological effects. Lead accumulates in the body and can damage the human nervous system, reproductive system, and circulatory system, and cause damage to the corresponding tissues and organs such as the brain, kidney, liver, and cardiovascular organs. [3] Children are more sensitive to the toxicity of lead than adults, and the nervous system is the most sensitive organ. Lead mainly affects the peripheral nervous system of adults and the central nervous system of children, especially the central nervous system of developing children. [4] Lead has a strong neural affinity. A child's blood-brain barrier is not well developed, the central nervous system is relatively fragile, and it has higher permeability to lead. In addition, excretion is not ideal, therefore, a child's nervous system has the weakest resistance to lead toxicity. [5] Current research shows that the direct mechanism of damage for lead to the nervous system is mainly the following: 1. Enter the brain tissue through the blood-brain barrier, combine with nerve cells in the brain tissue, change cell function and morphology, and obstruct the supply of nutrients and energy [6]; 2. Cause neurotoxicity by inhibiting the release and conduction of neurotransmitters [7]; 3.
Competitively inhibit Ca 2+ in the body, form a lead-calmodulin complex, affect the normal flow of Ca 2+ in brain tissue, interfere with the uptake and release of Ca 2+ by nerve cell membranes, disrupt intracellular Ca 2+ homeostasis and cause neurotoxicity [8]; 4. Interfere with the synthesis of brain-derived neurofactors, immediate-early genes, and synaptophysin (SYN), and affect the expression of proteins related to learning and memory, which in turn leads to impairment of learning and memory [9]; 5. Inhibit the growth and repair of synapses, affecting the normal function of synapses and reducing neuronal synaptic plasticity. [10] In addition to direct damage to neurons, lead can also cause the production of oxygen free radicals in the organism. [11] Free radicals cannot be excreted through metabolism and cause oxidative damage, and the organism cannot repair itself before oxidative damage occurs, leading to metabolic imbalance. [4] The body contains active antioxidant substances such as superoxide dismutase (SOD), glutathione superoxide dismutase (GSH-Px), and catalase (CAT), which can effectively antagonize the oxidative damage caused by the production of free radicals in the organism. Lead can reduce the activity of antioxidant enzymes in the cell, inhibit the activity of sulfhydryldependent enzymes, reduce the defense of the plasma membrane against ROS, increase the lipid peroxidation of neuronal cells, and reduce active of glutathione (GSH) and SOD. These effects induce oxidative damage to neuronal cells. [12] The traditional treatment for lead poisoning is based on the combination of metal chelating agents with vitamins. At present, the metal chelating agents commonly used in clinical treatment of lead poisoning are 2,3-dimercaptosuccinic acid (DMSA) and calcium sodium edetate (EDTA). [13] However, the efficacy of chelating agents in the treatment of lead poisoning varies greatly between individuals. Moreover, the byproducts produced by chelating agents after entering the body can cause serious gastrointestinal reactions, persistent loss of appetite, and metal odors in the oral cavity. [14] It can also cause the more serious "over complexing syndrome" which occurs when the metal complexing agent combines with toxic heavy metals, calcium, copper, and other essential trace elements to form stable complexes that are excreted from the body.
This results in a lack of trace elements in the body, causing symptoms such as nausea, vomiting, fatigue, and loss of appetite, which can affect treatment effects and the patient's health. Long-term use or one-time large-dose use of chelating agents can cause damage to liver and kidney function, and irreversible damage to the liver and kidneys.
[15] Therefore, to improve the quality of life of lead poisoning patients and reduce potential damage to groups at high risk for lead exposure, it is urgent to develop new methods for the prevention and treatment of lead poisoning.
Probiotics can colonize the host and exert probiotic effects. Several studies suggest that probiotics in humans and animals have an adsorption effect on toxic heavy metal ions. [16] The possible mechanism for the detoxification of heavy metals by probiotics is that they react with heavy metals through surface adsorption, intracellular adsorption, and extracellular adsorption to purify heavy metal pollution. In addition, probiotics can also change the valence of heavy metals and reduce toxicity through oxidation or reduction. [17] Lactic acid bacteria (LAB) in humans and animals are an edible probiotic that plays an important role in maintaining the microecological balance of the body and improve immune function. They have many sources, are easy to obtain, and are convenient to cultivate. In recent years, the removal of heavy metals in vivo and in vitro using LAB has become an important method for the biodegradation of heavy metals. [18] The plateau environment is characterized by low temperature, low pressure, high altitude, strong ultraviolet radiation, and hypoxia. [19] Microorganisms that have lived in this environment over a long period of time have unique physiological functions due to natural selection and genetic evolution. Yak fermented milk contains LAB with good genetic stability, strong stress resistance, and excellent fermentation performance.
Under the traditional fermentation mode of the plateau, it not only retains population abundance but also produces plateau characteristics of fermented yogurt with high nutritional value. [20] Traditionally fermented yak yogurt is a mixed fermented milk of LAB and yeast, with a complex microbial flora. Through sequencing analysis, studies have found that more than 100 species of microorganisms are involved in fermentation in traditionally fermented yak yogurt. The main strains include Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus delbrueckii (Bulgaria subspecies), Leuconostoc mesenteroides, Lactobacillus casei, and Streptococcus thermophilus. [21] The LF-SCHY34 used in this study was isolated from the yak yogurt of Sichuan Hongyuan. In vitro experiments found that LF-SHY34 exhibited effective resistance to artificial gastric acid, anti-bile salt capacity, and strong lead ion adsorption. Animal experiments verified the protective effect of LF-SCHY34 on the nerves, liver, and kidneys of lead-exposed SD rats and showed alleviation of lead-induced oxidative damage.
Materials and Methods:

Experimental Strain
Lactobacillus fermentum SCHY34 was isolated from yogurt in Hongyuan, Sichuan, China using de Man, Rogosa and Sharpe (MRS) medium. LF-SCHY34 was identified using the Basic local alignment search tool (BLAST) from the National Center of Biotechnology Information (NCBI). This strain is currently stored in the China General Microbiological Culture Collection Center (Beijing, China) and the preservation number is CGMCC No. 18795.

Determination of Survival Rate of LF-SCHY34 in Artificial Gastric Juice
Artificial gastric juice is a mixture of 0.2% NaCl and 0.35% pepsin. The pH was adjusted to 3.0 with 1 mol/L HCl and then filtered and sterilized with a 0.22 μm sterile filter. The LP-KFY04 was activated twice in 5 ml MRS liquid medium and centrifuged at 3,000 rpm for 10 min to collect the bacteria. The bacterial liquid was washed twice with sterile saline and resuspended in 5 ml saline 1:1 (v/v), mixed with the sterile artificial gastric juice, shaken, and placed in a constant temperature incubator at 37°C.
The number of viable bacteria was determined at 0 h and 3 h, and the survival rate of LF-SCHY34 in artificial gastric juice was calculated using the formula (1): Survival rate (%) = 3 h viable count (CFU/mL)/0 h viable count (CFU/mL) × 100.
3. Determination of the growth efficiency of LF-SCHY34 in bile salts Activated LP-KFY04 was inoculated twice at 2% (v/v) into sterilized MRS-THIO medium (0.2% sodium thioglycolate was added to MRS medium) containing 0.0% and 0.3% porcine bile salts. After culturing in a constant temperature shaker at 37°C for 24 h, control blank medium (uninoculated MRS-THIO medium) and the inoculated medium were added to a 96-well plate (200 μl/well) and the Optical Density (OD) was measured at a wavelength of 600 nm. Growth efficiency was calculated using the formula (2): Growth efficiency (%) = (OD600 of 0.3% bile salt medium − blank medium)/(OD600 of 0.0% bile salt medium − blank medium) × 100 4. In vitro lead ion adsorption capacity test LF-SCHY34 was cultured in MRS medium at 37°C for 18h, centrifuged at 8,000 × g at 4°C for 20 min and washed twice with ultrapure water. The final concentration of LF-SCHY34 was adjusted to 1 g/L and added 1:1 (v/v) into a 50 mg/L lead ion solution (AlCl3·6H2O). The mixture was co-cultivated at 37°C for 24 h, centrifuged at 4°C for 20 min at 8,000 × g and washed with ultrapure water twice. The supernatant was placed under an atomic absorption spectrophotometer to determine the initial lead ion concentration (Ci) of lead ions and the post-adsorption lead ion concentration (Cf).

LF-SCHY34 surface hydrophobicity test
The LF-SCHY34 MRS medium suspension was centrifuged at 1,500 × g for 10 min, washed twice with saline, and centrifuged to collect the bacteria. The cell concentration was adjusted with physiological saline until the OD value was 1.000 at a wavelength of 580 nm. The absorbance adjusted suspension (2 ml) was mixed with 2 ml of xylene, vortexed for 120 min, placed at room temperature for 30 min and 1 ml of the upper aqueous phase was absorbed. Normal saline was used as the blank control.
The absorbance value (A0) of the blank control group and the sample absorbance value (A1) was measured at 580 nm. The surface hydrophobicity of LAB was calculated using the formula (4): surface hydrophobicity (CSH%) = (A0-A1)/A0 × 100 6. Scanning Electron Microscope and Scanning Energy Spectrum Analysis of LF-

SCHY34 Bacteria Before and After Adsorption of Lead Ions
Bacteria without the lead ion solution and bacteria after absorption of lead ions using a 50 mg/L lead ion solution were centrifuged at 8,000 × g for 20 minutes, washed with sterilized ultrapure water three times, centrifuged under the same conditions as above, and then poured into glutaraldehyde to fix for 1.5 h. The solution was then washed THREE times with phosphate buffer solution (PBS), centrifuged at 6,000 ×g for 10 min, dehydrated once with ethanol of different concentrations (50%, 70%, 90%, 100%), and then centrifuged at 6,000 ×g for 10 min. The elute was divided with ethanol and tert-butanol mixture (v/v = 1/1) and pure tert-butanol once, centrifuged at 6,000 × g for 10 min, frozen at −20°C for 30 min, and put it into a freeze dryer for 4 h. Finally, an ion sputtering coating device was used to coat the sample with a layer of metal film at a thickness of 100-150 A. The coated sample was then put it into an observation room and the element composition was analyzed using an energy dispersive spectrometer. The rats in the EDTA group were injected with EDTA at a concentration of 50 mg/kg every day from the 8th week to the 12th week, and the LF-SCHY34 group was given 1 × 10 9 CFU/kg (b.w.) LF-SCHY34 daily from the 1st week to the 12th week ( Figure 1).
After 12 weeks, all rats were fasted for 12 h and then anesthetized with ether. Blood was taken from the orbital vein and the mice were sacrificed. The liver, kidney, and brain tissues of the rats were collected under liquid nitrogen and stored at −80°C for later use.

Morris water maze experiment
The water maze had a diameter of 150 cm and a height of 50 cm and was divided into four quadrants. A platform with a height of 38 cm was set at the intersection of the four quadrants, and the water level was approximately 2 cm above the platform. Before daily training, the rats were placed in the water maze room to adapt for 30 minutes; each rat was trained once a day. The rats were placed into the water facing the wall of the pool at a fixed position in one of the four quadrants of the maze. The rats swam until they found the platform and then they were allowed to stay on the platform for 20 s. The time from when the rat went into the water until it found the platform was recorded and defined as escape latency. After 20 s of rest on the platform, the entry point was changed to another quadrant and the experiment repeated. If the platform was not found within 120 s, the rat was placed on the platform and allowed to stay for 20 s; escape latency was recorded as 120 s. On the sixth day of training, the platform was withdrawn and the water entry point remained unchanged. The rats were placed in the water for 120 s and the escape latency, residence time in the target quadrant, number of shuttles to the target location, and swimming speed were measured.

Active avoidance experiment
Rats were put into any one of two chambers in the experimental box and allowed to adapt for 5 s. Then, a beeping sound lasting 20 s was initiated and a 50V electrical stimulation given for the next 10 s. After the rats were subjected to electrical stimulation, they would move to the other chamber which was devoid of electrical stimulation. After repeated conditioning, the rats would run to the other room after receiving the conditioned stimulation (the beeping sound). This training was done 30 times each day for 4 consecutive days. On the fifth day, the conditioned reflex latency and the number of conditioned reflexes were tested.
11. H&E and Nissl staining, immunohistochemical sectioning and histomorphological observation H&E staining: SD rat liver, kidney, and hippocampus tissues were fixed in 10% formalin (v/v) for 24 h. After the tissue was dehydrated, it was embedded in paraffin and then cut into 0.5 μm sections. The deparaffinized tissue was stained with hematoxylin and eosin. After dehydration, the slides were mounted with neutral gum and histological morphology was observed and photographed under an optical microscope (BX43; Olympus, Tokyo, Japan).
Nissl-staining: SD rats' hippocampus tissues were fixed in 10% formalin (v/v) solution, dehydrated, and embedded in paraffin. Sections were then deparaffinized, washed with distilled water, placed in tar purple staining solution at 37℃ for 10 min, and then washed with distilled water. Purple Nissl bodies were differentiated using 950 mL/L ethanol, dehydrated, cleared, and mounted. Staining was observed with a microscopic image analysis system. Immunohistochemical sectioning: The hippocampal tissue of SD rats fixed with 10% formalin (v/v) solution was dehydrated and embedded in paraffin. After sectioning, the tissue was deparaffinized and then repaired with citric acid antigen retrieval buffer at pH 6.0. A 3% hydrogen peroxide solution was used to block endogenous peroxidase, samples were blocked with serum, incubated with the primary antibody (GFAP, GB11096, Servicebio Biological Technology Co., Ltd., Wuhan, China) and then the secondary antibody (GB23303, Servicebio). Samples were then stained with diaminobenzidine (DAB, G1211, Servicebio) and counterstained with hematoxylin.
Stained samples were dehydrated, mounted on slides with neutral gum, and histological morphology was observed under an optical microscope. The collected blood (500 μL) or 50 mg of each tissue was placed in a tetrafluoroethylene digestion tank and 5 mL nitric acid was added for digestion. After cooling, 1 mL of a mixed solution containing 12.5% ammonium dihydrogen phosphate and 2.5% magnesium nitrate was added, using 2% nitric acid to make the volume to 2mL. To determine the absorbance, 20 μL of this solution was added in a Graphite furnace atomic spectrophotometer. The lead content in the blood was calculated from the standard curve. 16. Analysis of mRNA in SD rat brain tissue TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract RNA from the liver tissue and the concentration was adjusted to 1 μg/μL. A cDNA reverse transcription kit (ThermoFisher Scientific) was used to convert RNA to cDNA. The synthesized cDNA was then mixed with 10 µL SYBR Green PCR Master Mix (ThermoFisher Scientific), 2 µL primers (Table 1), and distilled water, and then put into a qPCR instrument for processing. Quantitative PCR was performed in an automatic thermocycler for 95°C for 60 s; 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 35 s; and a final step of 95°C for 30 s and 55°C for 35 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene, and the 2 -ΔΔCt formula was used to calculate the relative mRNA transcription level. 17. Western blot analysis of SD rat brain tissue Brain tissue (100 mg) was homogenized in 1 mL RIPA buffer (ThermoFisher Scientific, Waltham, MA, USA) and 10 μL PMSF (ThermoFisher Scientific) and centrifuged at 12,000 × g for 5 minutes at 4°C. Protein was quantified using the BCA protein assay kit (ThermoFisher Scientific). The protein sample was mixed with sample buffer (ThermoFisher Scientific) 4:1, and heated at 95°C for 5 minutes. The samples were then added into the wells of an SDS-PAGE gel and run at 100V. The bands were then transferred onto PVDF membranes, the membranes were blocked with 5% Duncan's multiple range test and One-way Analysis of Variance were used to evaluate differences between the average of each group. Differences with p < 0.05 were considered statistically significant. Results:

SD rat in vitro experimental results
The in vitro antiartificial gastric juice, antibile salt experiment, surface hydrophobicity experiments, and lead ion adsorption capacity tests showed that the survival rate of LF-SCHY34 in artificial gastric juice was 88.71% ± 0.23%, the growth efficiency in bile salts was 85.32% ± 0.41%, the surface hydrophobicity rate was 43.78% ± 0.75%, and the lead ion adsorption rate was 69.58% ± 0.56%.     Table 2 shows the morphology of the energy spectrum of the bacteria before (a) and after (c) the adsorption of lead ions, and the scanning energy spectrum before (b) and after (d) the adsorption of lead ions. Analysis showed that after treatment with lead, the surface of the LF-SCHY34 bacteria had adsorbed a large amount of lead ions, the weight of C and P elements increased by a small amount, the weight of Pb elements increased significantly, and the weight of O and N elements decreased. Table 3 shows the incubation period, the number of shuttles to the target location, and the swimming speed data recorded for the rats in the Morris water maze experiment. Figure 4 shows the swimming trajectory diagram of the SD rats in the water maze. Table 4 shows the conditioned reflex latency and the number of conditioned reflexes of the rats tested in the active avoidance experiment. Table 3 and Table 4 show that in the performance of the Morris water maze experiment, the rats in the normal group had the shortest incubation period and the most shuttle times, followed by the rats in the LF-SCHY34 group, and then the rats in the EDTA group. In the active avoidance experiment, the conditioned incubation period of rats in the normal group was the shortest and the number of conditioned reflexes was the highest. The data from the rats in the LF-SCHY34 group was second only to the normal group. The incubation period of conditioned reflexes in the Morris water maze test and active avoidance experiment in the lead-induced group was significantly higher than that of the rats in other groups, and the number of shuttles and conditioned reflexes were significantly lower than that of the rats in all other groups. However, there was no significant difference in the average swimming speed of rats in all groups.

Analysis of Behavioral Indices of SD Rats
4. Histopathologic analysis of liver and kidney H&E sections, hippocampal Nisslstained sections, and immunological sections Figure 5 shows that the liver lobules and hepatic cords in the normal group were structured and ordered, and the central vein and hepatic sinusoids were clear. In the lead-induced rats, the liver lobules were blurred, the hepatic cords disorderly, and the monocytes were scattered in different intercellular spaces after aggregation. The hepatocytes showed focal necrosis and the infiltration of large inflammatory cells, internuclear inclusions, and nuclei fragmentation. The liver cells of the rats treated with EDTA and LF-SCHY34 were more orderly, with less inflammatory cell infiltration, and less damage and necrosis of the liver cells compared with the lead-induced rats.
In the normal group, the structures of the glomeruli and renal tubules was normal, the cells were tightly arranged, and the number of cells was normal. In the kidneys of SD rats in the lead-induced group, glomeruli were ruptured and the number of cell nuclei increased significantly. Glomeruli had a large number of vacuoles, the renal tubular walls were dilated with symptoms of hyperemia and swelling, epithelial cell granular degeneration, and cellular breakage, infiltrating lymphocytes, and the renal capsule cavity layer had disappeared. Compared with the lead induction group, the kidney slices of the SD rats in the EDTA and LF-SCHY34 groups had better cell integrity with less inflammatory cell infiltration and no significant renal tubular expansion. Although the structure of the glomeruli was slightly damaged, it was more complete than in the lead-induced group ( Figure 6).
The neurons in the hippocampi of the normal group were arranged neatly and densely, the cell morphology was regular and complete, and the cytoplasm contained rich and dense Nissl bodies. The expression of astrocytes was strong, the number of cells was large, the cell body was large with dark brown-yellow staining, with thick and long protrusions, fewer branches, and a large proportion of cells. In the lead-induced rats, the CA1 area, CA3 area, and the hippocampal neurons of the dentate gyrus were scattered and loose, the neurons were missing, the shape was irregular, most were triangular or polygonal, the nucleoli were not obvious, and the Nissl bodies in the cytoplasm were reduced. The number and proportion of astrocytes in the hippocampus was significantly reduced, cell morphology was damaged, the radial neurites were shorter, and the expression level was lower. The neurons in the hippocampi of rats in the LF-SCHY34 and EDTA groups were normal in appearance and arranged neatly.
The expression and cell morphology of astrocytes were similar to those in the normal group. (Figure 7-8) The liver cell morphology, kidney cell morphology, hippocampal tissue morphology, and astrocyte morphology of SD rats treated with LF-SCHY34 were closer to those of the normal group, and the effect was better than in the EDTA group.

Analysis of Lead Content in Blood, Liver, Kidney, and Brain Tissues of SD Rats
The lead content in the blood and tissues of the rats in each group is shown in Table 5. Results showed that the lead content in the blood, liver, kidney, and brain tissues of the normal group was the lowest among all groups. The lead content in the blood, liver, kidney, and brain tissues of rats in the lead induction group was the highest among all groups. Among these tissues, the lead content in the blood and kidneys of SD rats was the highest, followed by liver and brain tissue. The lead content in blood and tissues of the lead-induced group was approximately15 times that of the normal group. The lead content in blood, liver, kidney, and brain tissues of rats in the LF-SCHY34 and EDTA groups decreased to varying degrees compared with rats in the lead induction group. The lead content in the blood and tissues of the LF-SCHY34 group was 5-8 times that of the normal group, and the lead content in the blood and tissues of the EDTA group was 7-12 times that of the normal group.

Analysis of Oxidation Levels in Serum, Liver, Kidney, and Brain Tissues of SD Rats
Through analysis of the data in Table 6, we found that the levels of CAT, T-SOD, and GSH in the blood, liver, kidney, and brain tissue of SD rats in the normal group was the highest among the four groups, and the values of MDA and ROS were the lowest. This trend was the opposite of the lead-induced rats. The blood, liver, kidney, and brain tissues of the lead-induced rats had the lowest levels of CAT, T-SOD, and GSH, while MDA and ROS were the highest among the four groups. The trend of the oxidation index results of rats in the LF-SCHY34 group was closer to that of the normal group than the rats in the EDTA group. Table 7 shows the activities of ALT, AST, δ-ALAD, and the BUN and CRE content in the serum of SD rats. Due to the effect of lead ions, the activity of ATL, AST, and the content of BUN and CRE were the highest, and the δ-ALAD enzyme activity was the lowest in the lead-induced group. Among the four groups of rats, the normal group had the lowest ATL and AST enzyme activities, the highest δ-ALAD enzyme activities, and the BUN and CRE content was the lowest. The ALT and AST enzyme activities and the content of BUN and CRE in the EDTA and LF-SCHY34 groups were significantly lower than in the lead induction group. In addition, the δ-ALAD enzyme activity was significantly higher than that of the lead induction group and the intervention effect of LF-SCHY34 was better than EDTA.

Analysis of serum ALT, AST, BUN, CRE, and δ-ALAD indices in SD rats
8. Analysis of neurosignaling substance levels in the brain tissue of SD rats The brain tissue of SD rats in the normal group had the lowest glutamate content and the lowest monoamine oxidase activity. The other neurotransmitters and enzymes had the highest content and the highest activity in all groups. After lead exposure, the expression trends of neurotransmitters and enzymes in the other 3 groups were different from those in the normal group. The lead-induced rats had the highest Glu content and the highest MAO activity. The content of Gln, NE, and cAMP was the lowest of the four groups, and the activities of GS, AchE, and AC were the lowest of the four groups.
Both LF-SCHY34 and EDTA effectively alleviated the changes of lead ions induced on neurotransmitters and enzymes in brain tissue; LF-SCHY34 had a better alleviating effect than EDTA. (Table 8) 9. Analysis of mRNA and protein expression in SD rat brains This experiment detected the mRNA and protein expression of related genes in rat brain tissue. Analysis of mRNA expression of related genes (Table 9) showed that in the brain tissues of normal SD rats, the expression of BDNF and the early genes c-fos and c-jun was the highest. In addition, the expression of oxidation related SOD1, SOD2, and NOS1 was the highest, the expression of Nrf2 and HO-1 was the lowest, the expression of apoptosis related Bax and Caspase-3 was the lowest, and the expression of Bcl-2 was the highest. The mRNA expression of the BDNF, c-fos, c-jun, SOD1, SOD2, NOS1, and Bcl-2 genes in the brain tissue of the lead-induced group was the lowest and Bax and Caspase-3 were the highest. In addition, the mRNA expression of Nrf2 and HO-1 was slightly higher than that of the normal group, but much lower than the mRNA expression in the brain tissue of rats in the LF-SCHY34 and EDTA groups.
Protein level analysis (Figures 9-12) showed that the protein expression of BDNF, c-fos, c-jun, SOD1, SOD2, NOS1, Nrf2, HO-1, Bax, Bcl-2, and Caspase-3 in the brain tissues of rats in all groups were the same as the mRNA expression trends. Several other related proteins (CaM, PKA, NMDAR1, NMDAR2, SYN, GSH, and p-CREB) had the strongest protein expression in the brain tissue of rats in the normal group, and their expression in the brain tissue of rats in the lead-induced group was the lowest. The LF-SCHY34 group had the second highest protein expression intensity, followed by the EDTA group.

Discussion:
Due to environmental pollution, pollution of food and daily necessities, household pollution, poor hygiene, and eating habits, hyperleademia and lead poisoning have become modern diseases in developed and developing countries. [22] Although lead does not participate in the growth, proliferation, signal transduction, or other related physiological activities of cells, and has no physiological functions in the human body, lead exerts strong neurotoxicity and is particularly harmful to brain development and the nervous system. [23] As an edible probiotic, lactic acid bacteria create a healthy intestinal environment for the host by regulating the balance of bacterial populations and secreting beneficial metabolites. [24] In recent years, studies have been carried out using lactic acid bacteria to ameliorate lead poisoning. After entering the body, the lactic acid bacteria first pass through the mouth, esophagus, and stomach, and then enter the intestine and begin to function. Therefore, tolerance to gastric juice and intestinal juice determines the number of lactic acid bacteria that pass through the oral cavity and enter the intestine. [25] The stronger the tolerance, the greater the number of viable bacteria that can survive. The hydrophobicity of the surface of lactic acid bacteria reflects the adhesion ability of lactic acid bacteria. The stronger the hydrophobicity, the more effectively the probiotics can interact with the intestinal epithelial cells. [26] The survival rate of LF-SCHY34 in artificial gastric juice was 88.71% ± 0.23%, the growth efficiency in bile salts was 85.32% ± 0.41%, and the surface hydrophobicity was 43.78% ± 0.75%, which is significantly higher than a report by Ilavenil Soundharrajan et al. [27] The survival rate of lactobacillus TC50 in gastric juice is 70%, the survival rate in intestinal juice is 83%, and the surface hydrophobicity rate is 28.94 ± 7.5%.
The adsorption of Pb2+ by bacterial strains is mainly due to functional groups such as -OH, -NH, and -COOH participating in the adsorption process. [24] The mechanism of adsorption includes mainly surface electrostatic interaction, complexation, ion exchange, and intracellular accumulation. In addition, macromolecular substances such as nucleic acids, phosphate esters, polysaccharides, S-layer proteins, and fatty acids also participate in the adsorption process. [28] After lead adsorption, LF-SCHY34 bacteria showed a large amount of aggregation under the electron microscope. The elements O, N decreased, and the elements C, P, Pb increased, indicating that the -NH and -COOH on the cell surface of LF-SCHY34 bacteria participated in the adsorption of lead-hydroxyapatite. LF-SCHY34 bacteria removed 69.58% ± 0.56% of the lead ions in the solution in vitro, which is much higher than the 25% lead ion removal capacity of lactobacillus reported by Marc A. Monachese et. al. [16] Lead in the human body is mainly excreted through the kidneys. When the maximum excretion of lead by the kidneys is reached, lead is deposited in the proximal tubule epithelial cells, affecting cell metabolism and damaging the structure and function of the kidneys. When cells are damaged or necrotic, renal tubular reabsorption function decreases, causing CRE and BUN to remain in the blood. [29] Therefore, the concentration of blood creatinine and blood urea nitrogen can reflect kidney function.
Lead also inhibits the activity of δ-ALAD, which increases the ALA in the blood. δ-ALA is excreted in the urine, resulting in a decrease in the δ-ALA content in the blood.
The liver is the most important detoxification organ. [30] Experiments have shown that lead can cause different degrees of liver disease, cause severe inflammation, affect the activity of liver-related enzymes, and ultimately cause liver damage. ALT and AST are distributed in liver cells. When liver cells are damaged, ALT and AST in the cytoplasm is released into the blood. Therefore, the concentration of ALT and AST in the blood can indicate the degree of liver cell damage. [31] Through the detection of serum δ-ALAD, ALT, AST, CRE, and BUN, as well as the pathological analysis of liver and kidney sections, we found that LF-SCHY34 bacteria protected the integrity of liver and kidney cells, and relieved the liver and kidney damage in SD rats induced by lead.
Rodents have a strong motivation to escape water. The process of learning to escape from a water environment reflects the learning ability of the animal. Spatial positioning according to the surrounding environment and purposefully swimming to a safe place in the water (platform) reflects the animal's spatial memory capacity. [32] The active avoidance experiment is performed to give the rat a repeated harmful stimulus and cause it to learn to actively avoid the harmful stimulus, which can reflect the reaction ability and memory capacity of the rat. [33] The hippocampus is an important part of the brain responsible for learning and memory. [34] In the hippocampus, the DG area plays a vital role in the separation of patterns, or distinguishing similar field patterns, similar events, or similar spatial locations. The CA3 area is involved in memory recovery or pattern completion, i.e., responding to incomplete stimuli by recalling previously stored information. The CA1 area plays an important role in short-term learning and spatial patterns of objects and events. The CA1 and CA3 areas of the hippocampus are rich in location cells. When the location cells move to a certain part of the environment, they have a discharge response, and the spatial map of this area can be coded according to the discharge pattern of the location cells. Therefore, the CA1 and CA3 areas also play an important role in spatial navigation. [35] Astrocytes perform many functions in the brain and are a bridge between the peripheral environment and the central nervous system. Astrocytes not only participate in the composition of the blood-brain barrier, but also maintain the stability of the internal environment of the nervous system. They also participate in the elimination of metabolites produced by neuronal activities, such as glutamate and potassium ions, and secrete cytokines to mediate the immune response of the nervous system. Astrocytes can also release neurotransmitters, participate in the transmission and integration of nerve signals, adjust neuron excitability and synaptic conduction efficiency, affect the formation of synapses and the regulation of synaptic plasticity, and play an important role in learning and memory. [36,37] GFAP-positive astrocytes can be arranged regularly in the form of an obvious lamellar structure in the hippocampus. This orderliness is conducive to establishing a fixed positional relationship and a stable functional relationship between neurons, to better regulate the functional activity of neurons. [38] The rat brain tissue slices showed that LF-SCHY34 bacteria maintained the morphology and number of nerve cells in the DG, CA1, and CA3 regions of the rat hippocampus, protected astrocytes from lead toxicity, and stabilized the hippocampal structure. Combining the Morris water maze and active avoidance experiments showed that the rats in the LF-SCHY34 group had a short incubation period, rapid active avoidance, and better memory. These results indicate that LF-SCHY34 bacteria ameliorated the damage caused by lead to the learning and memory capacity of the rats, and protected the normal function of rat brain tissue. it induces phosphorylation at specific sites of the TrkB receptor and transmits BDNF signals to the nucleus for neuroprotection. [45] The immediate-early genes c-fos and cjun belong to a class of proto-oncogenes which can be induced by second messengers to respond quickly to external stimuli such as neurotransmitters, hormones, and nerve impulses. These genes express their expression products as third messengers to participate in the regulation of the transduction of signals closely related to learning and memory in cells. After normal learning and memory activities or after learning and memory impairment, regular changes in their expression occur. [46] SYN is a membrane protein closely related to the structure and function of synapses. It forms synaptic vesicle-specific membrane channels, participates in the transport and discharge of vesicles, and can also be used as a presynaptic terminal specific marker.
[47] N-methyl-D-aspartate (NMDA) is an effector receptor of ionotropic glutamate, which plays an important role in synaptic excitatory conduction, synaptic plasticity, learning, and excitotoxicity. Glutamate binds to the NMDA receptor, causing the Ca 2+ channel to open. After Ca 2+ enters the cell, it activates CaM, which further activates NOS1 and AC. [48] NOS1 produces nitric oxide in the nervous tissues of the central nervous system and peripheral nervous system and assists in cell communication and association with native membranes. [49] When lead ions enter the brain tissue, it damages astrocytes, inhibits the activity of glutamine synthetase in the cerebral cortex, prevents glutamate from synthesizing glutamine, and causes excess glutamate to accumulate in the astrocytes of the cerebral cortex. [50] The excess glutamate counteracts the glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) distributed on the cell membrane to reduce the reuptake of glutamate, thereby causing the accumulation of glutamate in the contact gap, leading to a series of symptoms of central nervous system excitement, ultimately leading to nervous system damage. [51] In addition, lead can also activate the activity of monoamine oxidase, produce ROS, and cause oxidative damage in brain tissues. [52] In addition, lead inhibits the activities of GS and AchE, reduces the secretion of NE, and damages the normal activities of brain tissue. [53] Lead also competitively binds to related proteins to inhibit Ca 2+ influx and disrupt intracellular Ca 2+ balance, thereby inhibiting CaM activation and the secretion of nNOS. This leads to inhibition of AC and cAMP, affecting PKA activation and CREB phosphorylation, ultimately suppressing the expression of BDNF, C-fos, c-jun, and SYN ( Figure 13). LF-SCHY34 bacteria can alleviate the neurotoxicity of lead to the brain tissue of SD rats, maintain the normal secretion and activity of various neurotransmitters and related enzymes, and ensure that the Ca 2+ channel is unblocked, thereby ensuring the supply of brain neurotrophic factors and energy. LF-SCHY34 bacteria can also activate the Nrf2/HO-1 antioxidant pathway and increase the expression of downstream SOD1, SOD2, and GSH, thereby reducing oxidative damage of brain tissue caused by lead. In addition, LF-SCHY34 bacteria can inhibit the expression of the apoptosis-related genes Bax and Caspase-3, and increase the expression of the anti-apoptotic gene Bcl-2, promoting the survival of nerve cells.

Conclusions:
LF-SCHY34 is a probiotic strain derived from traditional fermented yogurt that exhibits high antiacid and antibile salt capacity and high hydrophobicity. It also shows significant lead ion adsorption ability in vitro. LF-SCHY34 can prevent lead ions from entering the blood-brain barrier and from binding to nerve cells, avoiding obstacles in the supply of nutrients and energy to the brain, and protecting the integrity of brain tissue cells and tissues. It can also regulate the release of neurotransmitters and related enzymes, and avoid the neurotoxicity caused by the competitive inhibition of lead ions and Ca 2+ , leading to the destruction of intracellular Ca 2+ homeostasis. It can promote the expression of cAMP and downstream related genes, activate the antioxidant pathway Nrf2/HO-1 and the expression of the anti-apoptotic gene Bcl-2, and avoid oxidative damage of brain tissue and apoptosis of brain cells. It can also maintain the normal function of synapses and the normal activities of brain tissue.
In summary, LF-SCHY34 is a strain of lactic acid bacteria that has a strong protective effect on the structure and functional activities of brain tissue exposed to lead ions. Further study is need to examine the use of LF-SCHY34 in the diet to alleviate the toxic effects of heavy metals on the human body. Overall, LF-SCHY34 has great potential and research value.    Table 7. ALT, AST, BUN, CRE, and δ-ALAD in the serum of SD rats  image of lead-adsorbed lactic acid bacteria cells.