Cathepsin B mediates the lysosomal-mitochondrial apoptosis pathway in arsenic-induced microglial cell injury

Abstract

Arsenic is a prevalent environmental pollutant that targets the nervous system of living beings. Recent studies indicated that microglial injury could contribute to neuroinflammation and is associated with neuronal damage. Nevertheless, the neurotoxic mechanism underlying the arsenic-induced microglial injury requires additional research. This study explores whether cathepsin B promotes microglia cell damage caused by NaAsO₂. Through CCK-8 assay and Annexin V-FITC and PI staining, we discovered that NaAsO₂ induced apoptosis in BV2 cells (a microglia cell line). NaAsO₂ was verified to increase mitochondrial membrane permeabilization (MMP) and promote the generation of reactive oxygen species (ROS) through JC-1 staining and DCFDA assay, respectively. Mechanically, NaAsO₂ was indicated to increase the expression of cathepsin B, which could stimulate pro-apoptotic molecule Bid into the activated form, tBid, and increase lysosomal membrane permeabilization by Immunofluorescence and Western blot assessment. Subsequently, apoptotic signaling downstream of increased mitochondrial membrane permeabilization was activated, promoting caspase activation and microglial apoptosis. Cathepsin B inhibitor CA074-Me could mitigate the damage of microglial. In general, we found that NaAsO₂ induced microglia apoptosis and depended on the role of the cathepsin B-mediated lysosomal-mitochondrial apoptosis pathway. Our findings provided new insight into NaAsO₂-induced neurological damage.

Introduction

Over 200 million people are exposed to arsenic and its toxicity, and its concentration in groundwater exceeds the limit level for drinking water (10 μg/L) set by the WHO as well as the Environmental Protection Agency of the United States in more than 35 countries across the globe. ¹ Though recently more and more arsenic derivatives and its compounds are being synthesized and discovered, and some of them are used in the treatment of cancer (especially leukemia),^2,3 as a result of long-term exposure, arsenic can enter and accumulate in the body via the respiratory tract, digestive tract, and skin contact, causing arsenic poisoning. Moreover, prolonged exposure to arsenic can cause severe medical conditions, including diabetes, high blood pressure, cardiovascular disease, neurological disease, and cancer.

The toxicity of arsenic to the nervous system has received significant research attention. Previous studies have reported that arsenic exposure could potentially causes neurodevelopmental abnormalities and behavioral disorders among children and adults. ⁴ Various forms of arsenic, such as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and NaAsO₂, can breach the blood-brain barrier, accumulating in different parts of the brain, resulting in neurological diseases. ⁵ Several studies have confirmed that arsenic exposure could trigger neuronal apoptosis, interfere with neuronal cytoskeletal proteins ⁶ and increase the expression of inflammatory cytokines in astrocytes. ⁷ Notably, microglia regulate the development, homeostasis, and pathogen defense of the central nervous system, and inorganic arsenic (iAs) always shows more significant toxicity to microglia.^8,9 So increasing attention is directed toward the role of microglia in the damage to the nervous system caused by iAs. It is generally accepted that excessive activation of microglia caused by arsenic is attributed to the release of pro-inflammatory cytokines (such as IL-1β, IL-6, TNF- α, NO), resulting in neurotoxicity or a series of uncontrollable inflammatory reactions. ¹⁰ Recent experiments suggested that apoptosis of microglia through oxidative stress, Nrf2/HO-1 pathways caused by spinal cord injury and stroke could further increase the damage to the nervous system.^11,12 Nevertheless, few studies have reported whether arsenic induces apoptosis of microglia and causes neurological damage.

Mitochondria and lysosomes play essential roles in substance metabolism and signal transmission. Arsenic is believed to cause mitochondrial oxidative stress and change a series of signaling pathways and functions, leading to cell damage and apoptosis. ¹³ There have been numerous attempts to confirm that the death of cells exposed to arsenic could be induced by lysosomal destruction. ¹⁴ Lysosome dysfunction contributes to many diseases, including lysosomal storage diseases (LSD), neurodegeneration, autoimmune diseases, and cancer.^15,16 Cathepsin is the most critical proteolytic enzyme in lysosomes. Additional studies have indicated that overexpression of cathepsin could increase lysosomal membrane permeabilization (LMP) and selective release of cathepsin, and then initiate a cascade of cell signaling events, resulting in cell apoptosis.^17,18 As an essential member of cathepsins, cathepsin B regulates various cell functions, including cytokine exocytosis, lysosome protein cleavage, and cell death induction. ¹⁹ The released cathepsin B can cleave Bid to generate active form tBid at either Arg65 or Arg71, as evidenced by tumor cell studies. ²⁰ After cleavage by cathepsin B, Bcl-2 members are transferred to mitochondria to activate and bind Bax/Bak1, ²¹ causing mitochondrial membrane permeabilization (MMP), which induces the mitochondrial apoptotic pathway. ²² Notably, the cathepsin B-mediated lysosome-mitochondrial apoptosis pathway plays a crucial role in various diseases, ²³ and Wang et al. ¹⁵ have discovered that the destruction of lysosomes and mitochondria could cause the death of cells exposed to arsenic. However, lysosome-mitochondrial-mediated microglial apoptosis is unclear in arsenic-induced microglial injury. Therefore, we aimed to explore the effects of arsenic on microglia and the regulatory pathways by investigating the mitochondrial function and lysosomal integrity and whether cathepsin B increases under NaAsO₂ exposure and could activate Bid, which could possibly increase MMP and promote mitochondrial apoptosis.

Materials and methods

Cell culture

Kunming Cell Bank of the Traditional Culture Preservation Committee provided the BV2 mouse microglial cell line. BV2 was cultured in 5% CO₂ at 37°C in Roswell Park Memorial Institute (RPMI) 1640 (30–2003, Gibco, USA) medium supplemented with 20% fetal bovine serum (30–2020, Gibco, USA) and 1% penicillin/streptomycin (15,140,122, ThermoFisher, USA) in an incubator (ThermoFisher, USA). The medium was changed daily and subcultured once every 2–3 days. During the experiment, cells in the logarithmic phase of growth were used. BV2 cells were exposed to 0, 2, 4, 8 and 16 μmol/L NaAsO₂ (7784-46-5, Sigma, US) for 24 h. To further analyze the impact of cathepsin B on BV2 cells, cells were pretreated with CA074-Me (HY-100,350, MedChemExpress, US) for 1.5 h before co-incubating with four or 8 μmol/L NaAsO₂ for 24 h.

CCK-8 assay

BV2 microglia with perfect growth conditions were inoculated into a 96-well plate (NEST, China), and the cell seeding density was adjusted to 5000/well. A blank control group (without any intervention treatment) and four experimental groups, each with six multiple replicates, were set up. After 24 h of culture, the culture medium containing 0, 2, 4, 8 and 16 μmol/L NaAsO₂ was changed and cultured for 24 h. Then the supernatant was discarded, and 100 μL of RPMI1640: CCK-8(C0038, Beyotime, China) = 9:1 working solution was added to each well. After 2 h of incubation, at 450 nm, the OD value of each well was measured using a Multiwell microplate reader (Bio-Rad, US).

Annexin and PI staining

Apoptotic cell death on BV2 cells was assessed using the Annexin V-FITC and PI staining detection kit (C1067S, Beyotime, China), followed by a bi-parametric FACS assessment. Cells (1 × 10⁴/cm²) within the logarithmically growing phase were plated into 12-well plates (NEST, China) with fresh culture medium for 24 h. After that, BV2 cells were rinsed and centrifuged with phosphate-buffered saline (PBS) at 1500 rpm for 5 min after being treated with NaAsO₂ (0, 2, 4, 8, and 16 μmol/L) for 24 h, and then we counted 100,000 cells into centrifuge tubes. Thereafter, Binding Buffer 200 μL, Annexin V-FITC 10 μL, and PI 5 μL were sequentially added. Cells were incubated in an optimum temperature condition for 15 min, and their apoptosis rate was determined within 0.5 h using a flow cytometer (BD Biosciences, US).

Measurement of mitochondrial transmembrane potential

The mitochondrial transmembrane potential ΔΨm was measured using the fluorescent probe JC-1 (C2003S, Beyotime, China). BV2 cells were selected in the logarithmic growth phase and seeded in 6-well plates (NEST, China) for 24 h. After the corresponding treatment, the cells in each group were washed with PBS once, and then 1 mL of cell culture medium was added. For each well, we then added 1 mL of JC-1 buffer: JC-1as 200:1 staining solution and re-incubated it in the incubator for 20 min. Once the cells were stained, we discarded the supernatant and washed them twice with 1 × JC-1 buffer. To conclude, 2 mL of medium was added, observed under a fluorescent microscope (×200, Nikon, Japan), and photographed. Image J software (NIH, USA) was used to determine the mean fluorescence intensity.

Determination of intracellular reactive oxygen species generation

A reactive oxygen species detection kit (S0033S, Beyotime, China) was used to measure intracellular ROS levels. DCFH-DA was diluted in serum-free medium to a final concentration of 10 μmol/L. In a 96-well plate, BV2 cells in the logarithmic growth phase were cultured with NaAsO₂ for 24 h, and the cell density was adjusted to 10,000 cells/well. There were six replicates in each set. After incubating with DCFH-DA for 20 min at 37°C, the cells were washed with a serum-free medium. The excitation wavelength of ROS was adjusted at 488 nm, and the emission wavelength at 525 nm. The fluorescence intensity at 488 nm was measured with a fluorescence spectrophotometer (Bio Tek, US). Meanwhile, Fluorescence images of intracellular ROS were obtained with a fluorescence microscope (×100, Nikon, Japan). After the corresponding treatment in 6-well plates, we added DCFH-DA working solution and incubated for 20 min. Then cells were washed three times with medium, and Fluorescence images of intracellular ROS were obtained with a fluorescence microscope (×100, Nikon, Japan). Image J was used to assess the mean fluorescence intensity.

Measurement of lysosomal membrane integrity

The stability of lysosomal membrane integrity was determined by Lys-Tracker Green (C1047S, Beyotime, China). A small amount of Lys-Tracker Green was added to the cell culture medium based on the 1: 13,333-1: 20,000 ratios and the working solution was prepared at 37°C. After adding trypsin to digest the cells, we counted 10,000 cells into centrifuge tubes. Then the working solution was added, and the cells were set for 15 min at 37°C. After incubation, the cells were centrifuged for 5 min at 1200 rpm/min before discarding the supernatant. After that, PBS was added for washing before centrifuging at 1200 r/min for 5 min to wash the cells. Lastly, PBS was added and thoroughly mixed with the remaining cells in the tube before detection via flow cytometry (BD Biosciences, US).

Immunofluorescence assay

An immunofluorescence assay was performed using the polyclonal antibody against Bid (1:200, YT0488, ImmunoWay) to measure Bid release. A 6-well plate was inoculated with BV2 cells. Upon reaching 60% confluence, NaAsO₂ was added and treated for 24 h. Exactly 4% cell fixed solution was added at room temperature for 30 min, followed by two washes using PBS for 5 min each. Further, 0.5% Triton X-100 (85,111, Thermofisher, US) was incubated at room temperature for 20 min before washing with PBS 3 times, 5 min per time. The cells were probed with Bid polyclonal antibody for overnight incubation at 4°C. After washing three times with PBS for 5 min, Alexa Fluor 488 goat anti-rat IgG polyclonal antibody was added (1: 200, RS3208, ImmunoWay) and incubated at 37°C for 1 h. The cells were eventually stained by DAPI (C1002, Beyotime, China) for 15 min and analyzed on an immunofluorescence microscope. The mouse anti-cathepsin B antibody was used to detect cathepsin B release via Immunofluorescence (1:100, Proeintech, China). Average fluorescence intensity was quantified by Image J software (NIH, USA).

Western blot

In order to isolate proteins, BV2 cells were seeded in a Petri plate measuring 90 mm in diameter. We removed the medium after 60% confluence and treated it with NaAsO₂ (0–8 μmol/L). Incubation was carried out for 24 h after exposure to a fresh medium. RIPA buffer (R0010, solarbio, China) and protease inhibitor cocktail (539,133, solarbio, China) were used to isolate the protein after incubation (RIPA: PMSF = 100:1). An assay kit for BCA protein concentration was used (PC0020-50T, solarbio China) in order to determine the protein concentration. Polyvinylidene difluoride (PVDF) membranes (88,518, Thermofisher, US) were used to transfer 20 g of protein resolved by 10% SDS-PAGE. The membranes were blocked with blocking buffer (3% BSA) before incubation with polyclonal antibodies, including β-actin (1:5000, YM3028, ImmunoWay), Bax (1:1500, 50,599-2-Ig, Protrintech), Bcl-2 (1:1000, 12,789-1-AP, Proteintech), caspase-3 (1:750, 19,677-1-AP, Proteintech), cytochrome c (1:1000, 10,993-1-AP, Proteintech), cathepsin B (1:800, 12,216-1-AP, Proteintech), Apaf-1 (1:1000, YT5378, ImmunoWay) and Bid (1:800, YT0488, ImmunoWay) for 4 h at ambient temperature. At intervals of 10 min, membranes were washed three times with TBST (10 mM Tris-HCL, 150 mM NaCl, and 0.05% Tween-20). The blots were incubated with secondary antibodies conjugated to horseradish peroxidase (1:10,000, RS0002 or RS0001, ImmunoWay) for at-least 2 h before washing in TBST three times. With the help of immobilon Western chemiluminescent HRP substrate, blots were observed (vazyme, US) at Amersham Biosciences, and the blot density was determined by Image J software (NIH, USA).

Statistical assessment

SPSS 26.0 & GraphPad Prism 8.4.3 software was used for statistical analyses, and the Shapiro-Wilk test was employed to test normality.A one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare group means ± standard errors (SE). A two-tailed (a) probability p less than 0.05 was considered statistically significant. At least three replications of the experiment were conducted. A statistically significant difference was defined as one with a p-value less than .05.

Results

NaAsO₂ induced microglia cells apoptosis

Previous studies have suggested that arsenic can cause damage and apoptosis of microglia. ⁵ To verify this conclusion, the viability of microglia cells was determined to investigate the effect of NaAsO₂ on microglia cells. CCK-8 test indicated a decreased cell viability in a concentration-dependent manner (0, 2, 4, 8, 16 μmol/L), with 48% reduction at 16 μmol/L (Figure 1(a)). The capacity of NaAsO₂ was then investigated to induce BV2 apoptosis by FITC-conjugated Annexin V (Ann V-FITC/PI) staining and flow cytometric analysis. It has been found that NaAsO₂ induced apoptosis in BV2 cells in a dose-dependent manner (0, 2, 4, 8 μmol/L) (Figures 1(b) and (c)), with 8 μmol/L showing the highest apoptotic effects (% apoptotic cells: 21.4%) in comparison to 4 μmol/L (% apoptotic cells: 16.86%.), 2 μmol/L (% apoptotic cells:13.9%.) and blank (% apoptotic cells: 7.86%.). These results showed that NaAsO₂ could cause injury to microglia.

Figure 1.

Effects of NaAsO₂ on cell viability of BV2 cells. (a) Impact of NaAsO₂ on BV2 cell viability. (b) The apoptotic rate of BV2 cells increased by NaASO₂ in a dose-dependent manner. (c) The bar diagram represents the % apoptosis rate in all cells. (*p < .05, **p < .01).

Mitochondrial depolarization and intracellular ROS generation by NaAsO₂

Considerable research efforts have focused on arsenic’s effects on mitochondrial dysfunction. ²⁴ To further explore whether NaAsO₂ also caused mitochondrial depolarization and ROS generation in microglia, JC-1 dye was used to measure mitochondrial membrane potential; in healthy mitochondria, it emits red fluorescence, whereas in damaged mitochondria, it emits green fluorescence. When BV2 cells were exposed to NaAsO₂ (0, 2, 4, 8 μmol/L), the fluorescence of the JC-1 dye changed from red to green, indicating the loss of mitochondrial membrane potential (Figure 2(a)). Since the loss of mitochondrial membrane potential often indicates the onset of apoptosis, ²⁵ we speculated that mitochondrial apoptotic pathway could be activated. Subsequently, the generation of intracellular ROS was established by the intensity of DCFH-DA fluorescence measured by a spectrofluorometer and images obtained from a fluorescence microscope. With increasing NaAsO₂ concentration, ROS generation substantially increased (Figure 2(b)–(d)). The results revealed that NaAsO₂ might damage the mitochondria, causing ROS generation.

Figure 2.

Effects of NaAsO₂ on the mitochondrial function in BV2 cells. (a) An analysis of the mitochondrial membrane potential by staining with JC-1 in NaAsO₂. A bar diagram illustrates the fluorescence of JC-1 in all cells (red/green). (b) Images of fluorescent microscopy of cells stained with DCFH-DA. (c) Bar diagrams represent ROS fluorescence intensity in all cells by a fluorescence microscope. (d) Intracellular ROS measurement by a spectrofluorometer. (*p < .05, **p < .01).

NaAsO₂ induced lysosomal membrane permeabilization and cathepsin B increase

Lysosomes are important targets in heavy metal damage, and cathepsins released from lysosomes can cause cell damage in various ways. ²⁶ CA074 has been demonstrated to specifically disrupt cathepsin B activity.^27,28 Because the methylated form of CA074 (CA074-Me) has high membrane permeability and can transform rapidly into its unmethylated form, it is suitable and reasonable to use CA074-Me for cathepsin B suppression. To further confirm whether NaAsO₂ promotes the damage of microglia induced by the increased cathepsin B and lysosomal destruction, the Lyso-Tracker Green dye was employed to confirm the NaAsO₂-induced increase in LMP and the protective impact of CA074-Me as a cathepsin B inhibitor on lysosomal membrane integrity. Consequently, after treating BV2 with different concentrations of NaAsO₂ for 24 h (0, 2, 4, 8 μmol/L), green fluorescence intensity was recorded as 2662, 2329, 2137, and 1548 respectively (Figure 3(a)) because there was a loss of integrity within the lysosomes. Meanwhile, CA074-Me (5.10 μmol/L) was added to BV2 cells for 1 h with NaAsO₂ (8 μmol/L) and primarily increased the green fluorescence intensity by 622 and 736 respectively (Figure 3(b)), suggesting that the NaAsO₂-mediated increase in LMP could be cathepsin B-dependent. Releasing cathepsin B from lysosome indicates apoptosis, and these results (Figure 3(c)–(e)) showed an increase in cathepsin B after NaAsO₂ exposure. As a comparison, there was nearly no expression of cathepsin B in the control group. Similarly, this effect was reduced by adding CA074-Me. These data indicated that increased LMP and cathepsin B are closely related to BV2 injury.

Figure 3.

Effects of NaAsO₂ on lysosome permeabilization and cathepsin B release in BV2 cells. (a, b) NaAsO₂ induces lysosomal destabilization, and CA074-Me can reverse this process. The bar diagram represents the fluorescence intensity of lysosomes in all cells. (c, d) The Western blot images show an increase in cathepsin B induced by NaAsO₂. However, CA074-Me can reverse this process. Bar diagrams represent the densitometric assessment of the relative protein expression of cathepsin B. (e) Immunofluorescence images show cathepsin B distribution. Bar diagrams represent the mean fluorescence intensity of Cathepsin B in all cells. (*p < .05, **p < .01).

NaAsO₂ increased bid expression, whereas cathepsin B inhibitor CA074-Me reduced the activation of bid

It was demonstrated in several studies that Bid, being activated by cathepsin B, plays an irreplaceable role in the lysosome-mitochondrial pathway. ²⁰ The level of Bid was assessed to explore whether cathepsin B-mediated activation of Bid acted as a bridge connecting the destruction of lysosomes to the mitochondrial apoptosis pathway. We found that treatment with NaAsO₂ (0, 2, 4, 8 μmol/L) induced the cleavage of the pro-apoptotic Bid protein to produce tBid, the truncated version (p15), and showed a parallel increase in Bid (p22) expression (Figures 4(a) and (c)) through Western blot. Immunofluorescence images also showed an increase in total Bid (Figure 4(e)). After treating with CA074-Me, tBid and Bid expression were downregulated in the BV2 cells (Figures 4(b) and (d)). These results indicated that NaAsO₂ upregulated the expression of Bid, and cathepsin B induced the activation of Bid, which could play a pivotal role in mitochondrial apoptosis.

Figure 4.

Effects of cathepsin B on the level of Bid under NaAsO₂ exposure. (a, b) The Western blot images show that the upregulated expression of Bid and tBid under NaAsO₂ and CA074-Me can inhibit this process. (c, d) Densitometric assessment of relative protein expression of Bid as well as tBid. (e) Immunofluorescence images show the distribution of Bid. Bar diagrams represent Bid fluorescence intensity in all cells. (*p < .05, **p < .01).

NaAsO₂ triggers the intrinsic pathway of apoptosis, whereas cathepsin B inhibitor CA074-Me relieves this process

Mitochondrial depolarization always implies the initiation of the intrinsic apoptotic pathway. ²⁹ To investigate whether the intrinsic apoptotic pathway was activated after the increase in MMP, Western blot was performed on proteins involved in the intrinsic apoptotic pathway. Figure 5 showed images of Western blot. Figures 5(b) and (c) illustrated the densitometric analysis normalized with β-actin of Bax highly expressed in NaAsO₂ treated cells, and Bcl-2 was expressed poorly. Figures 5(d) and (e) showed protein expression of cytc and Apaf-1 upregulated in NaAsO₂-treated cells, often representing the formation of the apoptotic bodies, and promoting cell apoptosis. Figure 5(f) showed the protein level of caspase-3 upregulated in NaAsO₂-treated cells, representing the caspase-dependent mitochondrial apoptosis. The cytc and caspase-3, two critical proteins involved in the intrinsic apoptotic pathway, were downregulated after treating BV2 cells with CA074-Me (Figure 5(g)–5(i)). These results showed that mitochondrial depolarization could initiate the intrinsic apoptotic pathway in BV2 cells, and CA074-Me could inhibit this process.

Figure 5.

Effects of NaAsO₂ on the mitochondria-mediated apoptosis in BV2 cells. The impact of NaAsO₂ upon protein level (a, g). Images of Western blot. (b) The upregulated Bax expression under NaAsO₂. (c) The downregulated expression Bcl-2 expression under NaAsO₂. (d). The upregulated Apaf-1 expression under NaAsO₂. (e) The upregulated cytc expression under NaAsO₂. (f) The increased expression of caspase-3 under NaAsO₂. (h) The inhibitory impact of CA074-Me on cytc expression. (i) The inhibitory impact of CA074-Me on the level of caspase-3. (*p < .05, **p < .01).

Discussion

Arsenic and its compounds are significant environmental pollutants threatening human health. ³⁰ Numerous studies have indicated that arsenic exposure potentially causes the occurrence and development of various neurodegenerative disorders.^31,32 The current types of research on arsenic toxicity to the central nervous system primarily focus on the damage of neurons and the increase of inflammatory factors in astrocytes and microglia. ³³ However, the apoptosis of microglia may further promote the production of inflammatory factors and aggravate the damage to the nervous system. ¹¹ In addition, mitochondria and lysosomes are the main targets of arsenic damage, and how mitochondria interact with lysosomes needs further exploration. Our study investigated the cathepsin B-mediated lysosomal-mitochondrial pathway to explore the specific mechanism of arsenic-induced microglial injury. To our knowledge, this is the first study to explore the cytotoxicity of arsenic to BV2.

Namgung U et al. revealed that arsenic could cause typical apoptotic manifestations of cultured rat neurons, including karyorrhexis and pyknosis. ³⁴ Meanwhile, several studies indicated that nematode infection, exogenous toxic substances including LPS ³⁵ and endogenous β-amyloid protein, ³⁶ and stroke ³⁷ could result in apoptosis of BV2 microglia. So, we hypothesized that arsenic would similarly affect BV2 cells. As expected, the viability of cells treated with NaAsO₂ significantly decreased, and the apoptosis rate of BV2 cells also revealed similar results.

Mitochondria are essential energy-regulating organelles in cells. Several existing studies have supported the role of mitochondrial dysfunction and oxidative damage in neurodegenerative disease and arsenic-induced apoptosis.^23,38 Our study demonstrated that MMP decreased in NaAsO₂-treated cells. The initiation of the intrinsic apoptosis pathway caused by increased MMP often induces mitochondrial fragmentation, triggering oxidative phosphorylation and uncoupling the electron transport chain.^39,40 Previous studies have found that NaAsO₂ could trigger intracellular ROS production in a dose-dependent and reduce the viability of cells.^5,41 DCFH-DA detection was also employed to show intracellular ROS production to validate these findings in BV2 cells.

Lysosomes play an essential role in iron metabolism and autophagy ¹³ and could also be one of the targets of arsenic-induced BV2 cell damage. Increased LMP causes cell death and neuroinflammation in neurological diseases, resulting in behavioral deficits and neurodegeneration. ⁴² Our study discovered that LMP of BV2 cells was also increased following arsenic exposure. Since the new theory of programmed cell death on the lysosome-mitochondrial pathway was proposed, more and more diseases have been proven to be linked to this pathway. Pan et al. ¹⁴ also illustrated that the destruction of lysosomes and mitochondria could kill INS-1 cells exposed to arsenic. As a vital lysosome enzyme, cathepsin B regulates cell damage through various signaling pathways. Tao et al. discovered that arsenic causes damage to hepatic stellate cells (HSCs) cell through the autophagic-CTSB-NLRP3 inflammasome pathway.^43,44 Yang et al. ²⁶ revealed that excess zinc increases the levels of cathepsin B/D in PK-15 cells. Herein, we confirmed that the increased cathepsin B could destroy lysosomes and connect the lysosome-mitochondrial pathway by observing LMP after using a cathepsin inhibitor (CA074-Me).

The Bid is a vital member of the Bcl-2 family, which functions in a truncated form and can activate mitochondria and promote apoptosis in the full-length form. ⁴⁵ Bid can be cleaved by cathepsin B and has been used universally to account for the connection of the lysosome-mitochondria pathway. ²⁰ Similarly, we also discovered that arsenic increased the level of Bid and its active form tBid. Once Bid is activated, Bax can be upregulated, and Bcl-2 can be downregulated, stimulating cytc release, APaf-1 activation, and promoting mitochondria-dependent caspase signaling pathway that causes apoptosis.^28,46 We also found coincident results to these regulations at the protein level. CA074-Me inhibited the cleavage of Bid and reduced the levels of cytc and caspase-3, which play a key role in the intrinsic apoptotic pathway, confirming that cathepsin B could activate mitochondrial apoptosis through Bid. Therefore, the lysosomal-mitochondrial apoptosis pathway could be connected.

Although our study provides some new insights into arsenic-induced neurological damage, there are some potential limitations of our study. Lysosome-mitochondria pathway is not only connected by cathepsin B. Cathepsins released from lysosomes after destruction, including cathepsin D, cathepsin C and cathepsin L can also cause damage to mitochondria and lead to apoptosis. Therefore, future research could focus on whether other cathepsins are also involved in the lysosome-mitochondrial pathway. Meanwhile, with the gradual deepening understanding of arsenic and its compounds, the damage of organic arsenic to microglia is different from that of organic arsenic due to the different chemical structures ⁴⁷ so organic arsenic on microglia warrants further investigation.

In conclusion, we found that NaAsO₂ could reduce the viability of BV2 cells and promote the apoptosis of BV2 cells in a dose-dependent manner. We also found that arsenic could increase MMP and generate a large amount of ROS. Meanwhile, CA074-Me could inhibit the arsenic-induced increase of cathepsin B and lysosomal damage. Furthermore, also as a critical protein connecting the mitochondria-lysosome pathway, Bid significantly increased in the arsenic-exposed group. Meanwhile, the protein levels of Bcl-2, Apaf-1, caspase-3, cytc increased, and the level of Bax decreased signally in the exposure of NaAsO₂. Additionally, in the CA074-Me + NaAsO₂ group, the level of cytc and caspase-3 was down-regulated compared to the NaAsO₂ group. Thus, our study showed that the increased cathepsin B could participate in destroying lysosomes, cleaving Bid, and promoting BV2 cell apoptosis via the lysosome-mitochondrial pathway. By clarifying the path and mechanism of arsenic-induced microglia apoptosis, this study is anticipated to provide a novel target for arsenic-induced central nervous cell damage.