How the p62/Nrf2/Keap1 Pathway May Be Involved in Brain Problems Caused by Lead Exposure

Four‐week‐old male Sprague–Dawley (SD) rats, weighing 98.31 ± 8.58 g, were obtained from the Experimental Animal Center of Guangxi Medical University [SCXKG2020‐0003]. They were housed in a specific pathogen‐free (SPF) environment with a 12‐h light–dark cycle and ad libitum access to food and water. The experimental procedures adhered to the guidelines established by the Animal Protection and Experiment Committee of Guangxi Medical University and received approval from the Animal Protection and Use Committee of Guangxi Medical University.

Rats were randomly divided into two groups (Control and Pb‐exposed groups), each consisting of 10 individuals. Rats in the Pb group received intraperitoneal injections (i.p.) of 2 mg/kg Pb(C₂H₃O₂)₂ once daily for 5 days/week over 12 weeks to induce sub‐chronic Pb exposure. The control group received an equal volume of saline via i.p. After 12 weeks of Pb exposure, the Morris water maze test was conducted to evaluate learning and spatial memory performance in rats. The dose of Pb(C₂H₃O₂)₂ was chosen based on our previous study [23].

SH‐SY5Y cell line was obtained from the China Center for Type Culture Collection (CCTCC, Wuhan University, China), and cultured in a medium containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, MEM, and DMEM/F12 (1:1). Cells were maintained under humidified conditions at 37°C with 5% CO₂.

The MWM test was conducted to assess the learning and memory abilities of rats [24]. Rats were released into the maze from four different directions to find a hidden platform. Swimming distance and escape latency were recorded. Rats failing to locate the platform within 90 s during training were guided toward it. After 5 days of training, the platform was removed, and spatial memory ability was evaluated by recording how many times rats crossed the probe within 120 s.

Hematoxylin and eosin (HE) staining, as well as Nissl staining, were conducted in accordance with previously established protocols [25]. Samples were fixed, dehydrated, embedded in wax blocks, and sectioned into 5 μm‐thin slices. HE or Nissl staining was performed to observe morphological alterations.

The MARS6 Classic Microwave Digestion System was used to digest 50 mg of hippocampus and 300 μL of whole blood with 5 mL of nitric acid. Subsequently, the samples were evaporated on a graphite heating plate at 150°C until the liquid volume was reduced to approximately 0.5 mL. Samples were then cooled to room temperature and diluted with deionized water to reach a final volume of 10 mL. Pb levels were analyzed using Inductively Coupled Plasma Mass Spectrometry (ICP‐MS, Nexion 300D, USA).

The cytotoxicity test was performed following previously described protocols [26]. SH‐SY5Y cells were seeded at a density of 8000 cells per well in a 96‐well plate and incubated for 24 h under controlled humidified conditions with 5% CO₂ at 37°C. Subsequently, cells were exposed to media containing Pb concentrations of 0, 1, 5, 25, 50, 100, and 200 μmol/L for an additional 24 h. Each well was treated with a solution of Cell Counting Kit‐8 (CCK‐8, C0005, Targetmol, China), followed by incubation for two more hours. Absorbance at 450 nm was measured using an enzyme‐linked immunosorbent assay reader.

F‐actin staining was conducted following previously described protocols [27]. Briefly, the prepared cells were fixed with a 4% formaldehyde solution at room temperature for 10 min. They were then washed three times with PBS containing 0.1% Triton X‐100, each time for 5 min. Actin‐Tracker Red (1:100, C2205S, Beyotime, China) was diluted in PBS containing 5% BSA and 0.1% Triton X‐100, followed by incubation at room temperature in the dark for 40 min. Afterward, the cells were washed twice with PBS for 5 min and directly observed under a fluorescence microscope.

The measurement of the ROS procedure was conducted according to the previous study [24]. When Pb was exposed to Pb for 23.5 h, the positive control wells were treated with 4‐butyl hydrogen peroxide (Rosup) for 30 min. After that, SH‐SY5Y cells were treated with 10 μmol/L 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA, S0033s, Beyotime, China) for 1 h at 37°C in the dark. The detection was performed strictly following the manufacturer's instructions and previous studies. Fluorescence visualization was conducted using the EVOS fluorescence microscopy imaging system (Thermo, USA).

The detection of GPx activity procedure was conducted according to the previous study [28]. Cells were scraped and collected, followed by one wash with PBS. Cell lysis buffer (P0013, Beyotime, China) was used to lyse the cells, followed by centrifugation at 4°C and 12,000 g for 10 min. The supernatant was used for enzyme activity determination. Protein concentration was measured using the BCA assay kit (P0012, Beyotime, China). In a 96‐well plate, 50 μL of the test sample was added (with an additional 50 μL of Cell lysis buffer in the blank control well), followed by the addition of 40 μL of GPx detection working solution (S0056, Beyotime, China). Subsequently, each well received 10 μL of a 30 mM hydrogen peroxide reagent solution and was thoroughly mixed. Absorbance at 340 nm was measured using an enzyme‐linked immunosorbent assay reader. Results were standardized against protein concentration.

Cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Subsequently, permeabilization was performed by incubating the cells with PBS containing 0.1% Triton X‐100 at room temperature for 10 min. Following this, the cells were blocked with 5% BSA at room temperature for 30 min. Primary antibodies, including anti‐Nrf2 (1:500, Cell Signaling Technolog (CST), #12721) and anti‐LC3 (1:100, CST, #12741) were incubated overnight with the samples at 4°C. After three washes with PBS for 5 min each time, appropriate dilutions of anti‐rabbit IgG (1:1000, Alexa Fluor 488 Conjugate, CST, #4412S) or anti‐rabbit IgG (1:1000, Alexa Fluor 594 Conjugate, CST, #8889) in PBS were added to the samples and incubated for 1 h in a dark environment at room temperature. The samples were then stained with DAPI for 5 min after being rinsed three times in PBS for 5 min each time. Finally, the samples were observed under a Ti2‐U inverted fluorescence microscope (Nikon Eclipse, Japan).

The procedures of animal brain tissue immunofluorescence were conducted according to the previous study [29]. Brain specimens were collected, fixed, dehydrated, embedded in wax blocks, and sliced into 5 μm‐thick sections. After deparaffinization and antigen retrieval procedures, the slices underwent a blocking process with 5% BSA for half an hour before being subjected to overnight incubation at 4°C using Rabbit anti‐Nrf2 (1:500, CST, #12721). After three rinses in PBS for 5 min each time, the sections were exposed to anti‐rabbit IgG (HRP) at room temperature for 1 h, followed by FITC‐Tyramide (TSA) (Servicebio, G1222) treatment in darkness for 10 min. After the tyramide reaction, slides were washed and treated with 3% H₂O₂ for 15 min to quench HRP activity. To further ensure specificity, a mild stripping buffer (pH 2.0 glycine‐HCl) was used between sequential rounds to remove previous antibodies. Subsequently, the sections underwent antigen retrieval once again and were then incubated with Rabbit anti‐Keap1 (1:200, CST, #8047) or Rabbit anti‐P62 (1:100, CST, #39786). After three rounds of PBS rinses, the sections were subjected to incubation with anti‐rabbit IgG (HRP) at room temperature for 1 h. CY3‐TSA (Servicebio, G1223) or iF647‐TSA (Servicebio, G1232) was then applied in darkness for 10 min. After three rounds of PBS rinses, the sections were incubated with DAPI for 5 min. The sections were examined using the microscope slide scanner (Pannoramic DESK, Hungary). The procedures were conducted according to the previous study.

The western blot procedures were conducted according to the previous study [29]. The antibodies used in this study included anti‐β‐actin (1:1000, CST, #4967), anti‐GAPDH (1:1000, CST, #2118), anti‐Nrf2 (1:1000, CST, #12721), anti‐Keap1 (1:1000, CST, #8047), anti‐HO‐1 (1:1000, CST, #43966), anti‐LC3 (1:1000, CST, #12741), anti‐P62 (1:1000, CST, #39786), and anti‐Tau (1:1000, CST, #4019). Additionally, the secondary antibodies of anti‐rabbit IgG (1:3000, CST, #7074) and anti‐mouse IgG (1:3000, CST, #7076) were used. The protein bands' optical density was measured using an iBright Western Blot imaging system (iBright Fl1000, Thermo).

The data analysis was performed using GraphPad Prism, version 9.5. All experiments were repeated three or four times independently. All data were subject to tests for normality. Satisfy the normal distribution, and use student's t‐test or one‐way ANOVA analysis. We employed Dunnett's multiple comparisons test for pairwise comparisons. Repeated measures analysis of variance was used for MWM Test data. Non‐normal data used corresponding non‐parametric tests. Results are presented as means with standard deviations, and statistical significance was determined at p < 0.05.

After Pb exposure for 12 weeks, both blood and hippocampal Pb levels in rats were significantly increased (Figure 1A), indicating the successful establishment of the Pb exposure model. Compared to the control group, rats exposed to Pb showed a significant increase in escape latency on the fourth and fifth days, as well as a decrease in platform crossing times (Figure 1B). HE staining revealed that the hippocampal neurons in the control group were arranged regularly, with dense cell nuclei and no obvious cell loss. In contrast, neurons in the CA3 and DG regions of Pb‐exposed rats exhibited a loose tissue pattern, increased nuclear condensation, and less distinct cell nuclei (Figure 1C). The nissl bodies exhibit a blue hue in Nissl staining; however, under pathological conditions, their quantity diminishes, resulting in a lighter or even complete absence of the blue color. Our results showed that nissl staining indicated lighter staining and fewer nissl bodies in neurons stained with Pb compared to the control group, suggesting impaired neural function due to long‐term Pb exposure (Figure 1D).

To elucidate the relationship between Pb‐induced neurotoxicity and oxidative stress, we employed SH‐SY5Y cells and a rat model of Pb exposure. Treatment of SH‐SY5Y cells with varying concentrations of Pb for 24 h resulted in a significant decrease in cell viability at 50 μM, with a more pronounced decrease as the concentration increased (Figure 2A). Subsequent toxicological experiments were conducted at concentrations of 25 μM (no significant changes in cell viability), 50 μM (initial damage observed), and 100 μM (obvious damage occurred). F‐actin staining results showed that there were noticeable phenotypic changes in the cells, such as cell shrinkage and nuclear condensation at 50 μM, which became more apparent at 100 μM (Figure 2B). DCFH‐DA probe detection revealed that ROS levels were increased after Pb exposure in a dose‐dependent manner (Figure 2C). Furthermore, the activity of the GPx antioxidant enzyme decreased with increasing concentration of Pb (Figure 2D). Additionally, immunofluorescence results demonstrated reduced levels of nuclear and cytoplasmic Nrf2 protein expression (Figure 3A). Western blot analysis revealed decreased expression of Nrf2 and its downstream target HO‐1, accompanied by increased Keap1 expression (Figure 3B). Consistent findings were also observed in hippocampal protein expression levels in Pb‐exposed rats (Figure 3C), indicating that neuronal oxidative damage is induced by Pb exposure while disrupting the Nrf2/Keap1 pathway.

To further investigate neuronal autophagy following oxidative damage caused by Pb exposure, both in vitro and in vivo studies were conducted. The results showed no significant increase in the ratio of LC3II/LC3I, a marker protein for autophagy, in SH‐SY5Y cells after Pb exposure (Figure 4A). However, the protein expression of P62 increased with escalating concentrations of Pb (Figure 4A). Similarly, immunofluorescence analysis demonstrated no notable elevation in the expression of LC3 protein (Figure 4B), consistent with in vivo findings (Figure 4D). Importantly, there was a substantial upregulation observed in the protein expression of Tau, an AD‐related protein, following Pb exposure (Figure 4C,E), suggesting that Pb exposure disrupts autophagy and mediates neurodegeneration.

To further elucidate the relationship between Nrf2/Keap1 and the autophagy protein P62, immunofluorescence detection was performed on the hippocampus of Pb‐exposed rats. The results indicated that there was co‐localization of Nrf2, Keap1, and P62 proteins in the Pb‐exposed group. The Nrf2 protein expression decreased, and the P62 and Keap1 protein expressions increased in the Pb‐exposed group (Figure 5), consistent with the results of Western blot. This suggests that the Keap1 protein binds to P62, inhibiting its localization to autolysosomes and thus suppressing autophagy.

To further investigate the role of oxidative reactions, disruption of the Nrf2/Keap1 pathway, and inhibition of autophagy caused by Pb exposure, SH‐SY5Y cells were treated with the antioxidant N‐acetyl cysteine (NAC). The results demonstrated that treatment with NAC effectively reduced ROS levels in Pb‐exposed SH‐SY5Y cells (Figure 6A). Moreover, NAC ameliorated the downregulation of protein expressions of Nrf2 and HO‐1 induced by Pb while concurrently decreasing Keap1 expression (Figure 6B). Furthermore, NAC decreased P62 protein expression without significantly altering the LC3II/LC3I ratio (Figure 6C), suggesting that autophagy may not be necessary in cells after antioxidant treatment, possibly due to the absence of oxidative damage.

To activate Nrf2, SH‐SY5Y cells exposed to Pb were pretreated with the Nrf2 activator Artemisitene (ATT) (MedChemExpress, MCE, HY‐122550) [30]. The results demonstrated that ATT pretreatment significantly increased Nrf2 expression and heme oxygenase‐1 (HO‐1) levels, while decreasing Keap1 expression compared to the Pb‐exposed group (Figure 7A). Additionally, phosphorylated Tau (P‐Tau) expression was notably reduced. These findings suggest that enhancing Nrf2 activity potentiates the antioxidant capacity of SH‐SY5Y cells, partially alleviating lead‐induced neurodegenerative pathology. Furthermore, ATT pretreatment led to a slight reduction in P62 expression and a modest increase in the LC3II/LC3I ratio compared to the lead‐exposed group, although the difference did not reach statistical significance (Figure 7B). This observation implies that Nrf2 activation may mitigate lead‐triggered autophagy impairment.

To enhance cellular autophagy, SH‐SY5Y cells exposed to Pb were pretreated with the autophagy activator Rapamycin (RAPA) (MCE, HY‐10219) [31]. Compared to the Pb‐exposed group, RAPA pretreatment resulted in a reduction in P62 expression (albeit statistically insignificant) and an elevated LC3II/LC3I ratio, suggesting that RAPA promotes autophagic flux in lead‐exposed SH‐SY5Y cells (Figure 8B). Furthermore, RAPA pretreatment downregulated Keap1 expression and modestly increased Nrf2 and heme oxygenase‐1 (HO‐1) levels in lead‐exposed cells, though these differences were not pronounced (Figure 8A). Concurrently, phosphorylated Tau (P‐Tau) expression was reduced (Figure 8B), indicating that autophagy induction alleviates lead‐induced neurotoxicity.

Notably, both Nrf2 activation by ATT and RAPA‐mediated P62 reduction were associated with decreased Keap1 protein levels. These findings collectively highlight the critical role of the P62/Nrf2/Keap1 signaling pathway in mitigating lead‐induced neurodegenerative dysfunction.