The Effects of Inorganic Arsenic On Apoptosis and Autophagy in Human Hepatic Sellate Cells

doi:10.21203/rs.3.rs-1145060/v1

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The Effects of Inorganic Arsenic On Apoptosis and Autophagy in Human Hepatic Sellate Cells

https://doi.org/10.21203/rs.3.rs-1145060/v1

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Endemic arsenism is a major disease concern in China, with arsenic poisoning and induced potential lesions key issues on a global level. The liver is the main target organ where arsenic is metabolized; chronic exposure to arsenic-induced liver fibrosis is also closely related to autophagy, however, the exact mechanisms are remain unclear. In this study, we explored the effects of NaAsO₂ on apoptosis and autophagy in human hepatic stellate cells(HSC). We established a fibrosis model in the HSC line, LX-2 which was exposed to NaAsO₂ for 24h, 48h, and 72h. Cells were then transfected using an autophagy double-labeled RFP-GFP-LC3 adenoviral plasmid. Laser confocal microscopy indicated significant infection efficiencies and autophagy in LX-2. Flow cytometry was also used to investigate the effects of different NaAsO₂doses on apoptosis. NaAsO₂treatment upregulated the expression of autophagic markers, including microtubule-associated protein light chain A/B(LC3), ubiquitin binding protein(SQSTM-1/P62), autophagy related genes(ATGs), recombinant human autophagy effector protein (Beclin-1), and B cell lymphoma-2(BCL-2), but downregulated mammalian target of rapamycin(mTOR). Also, α-smooth muscle actin(α-SMA) expression was significantly upregulated in all NaAsO₂ groups. Furthermore, mTOR silencing via 3-methyladenine(3-MA) altered NaAsO₂induced autophagy, LC3, Beclin-1, and SQSTM-1/P62 expression were all upregulated in both NaAsO₂ and 3-MA-iAs groups. Altogether, NaAsO₂ induced HSC autophagy via apoptotic pathways. 3-MA inhibited LX-2 activity and reduced NaAsO₂-induced autophagy which may inhibit fibrosis progression caused by this toxin.

inorganic arsenic

liver fibrosis

autophagy

3-methyladenine

apoptosis pathways

Endemic arsenic disease is a serious environmental and geochemical issue endangering human health¹.According to 2016 statistics, nearly 150 million people in more than 70 global regions were variably threatened by high arsenic groundwater levels². Inorganic arsenic (iAs) is a ubiquitously distributed environmental and industrial toxicant, designated a class I carcinogen by the International Cancer Research Institute and the United States Environmental Protection Agency³. Arsenic has two oxidation states in the environment. iAs as a potential environmental carcinogen is not only associated with multiple cancers (skin⁴, lung⁵, liver⁶, kidney⁷, and bladder⁸), but is also detrimental to multiple organ systems, including the liver and kidneys, cardiovascular, respiratory, and reproductive systems⁹. The liver is the most important target organ of arsenic metabolism¹⁰. Epidemiological studies have linked chronic iAs exposure to an increased risk of liver disease, fibrosis, cirrhosis, and liver carcinogenesis^11–13. Arsenic carcinogenic mechanisms mainly relate to oxidative stress, DNA and chromosome damage, apoptosis, necrosis, and methylation. Importantly, in recent years, studies have also reported that autophagy plays a vital role in arsenic-related carcinogenic mechanisms¹⁴.

Autophagy is a conserved, tightly regulated process with key roles in protein and organelle quality control via intracellular molecule capture, degradation, and recycling¹⁵. The process is characterized by macroautophagy, microautophagy, and chaperone-mediated autophagy mechanisms¹⁶. Significantly, arsenic exposures affects cellular autophagy in a dose-dependent manner¹⁷. Multiple autophagy-related genes (ATGs) and signaling pathways co-regulate autophagy processes and biological functions¹⁸. Currently, 42 ATGs have been identified, with autophagy processes closely related to key signaling molecules, including, Beclin-1, VMP1, Atg5–Atg12, Atg4, and LC3, and also regulated by signal transduction pathways, including molecular target of rapamycin (mTOR), and phosphatidylinositol 3 kinase/protein serine threonine kinase (PI3K-Akt)^19,20. In this study, a comprehensive and reliable autophagy quantitative analytical method was required to investigate these complex mechanisms, therefore, we developed a double fluorescence RFP-GFP-LC3 system. The biggest advantage of the system is that no drug interventions are required, only a hunger-induced (serum-free medium) starvation mechanism is necessary to visually determine changes in autophagic activity and autophagic flow (autophagic flux). Thus using this strategy, autophagy was observed by fluorescence, indicating the successful construction of an RFP-GFP-LC3 lentivirus system in this study.

The effects of arsenic poisoning on liver fibrosis have been characterized but underlying mechanisms are unclear²¹. Autophagy as a fundamental degradation pathway in eukaryotic cells also induces liver fibrosis; hepatic stellate cell (HSC) activation contributes to liver fibrosis where autophagy processes provide energy for HSC activation by degrading lipid droplets, thereby promoting fibrosis²². In rat lungs, a high arsenic exposure (up to 10 µg/L) to drinking water triggered inflammation, oxidative damage, and inhibited autophagy²³. Moderate autophagy helped cell survival and protected cells from dysfunction²⁴, but excessive autophagy resulted in the gradual consumption of cell capacity, resulting in autophagic death²⁵.

3-Methyladenine (3-MA) is a phosphatidylinositol 3-kinases (PI3K) inhibitor. PI3K plays key roles in several biological processes, including controlling mTOR activation which is a key autophagy regulator. Also, 3-MA inhibits autophagy by blocking autophagosome formation by inhibiting class III PI3Ks²⁶. Similarly, 3-MA plays a dual role in autophagy; prolonged 3-MA treatment promotes autophagy under nutrient-rich conditions, whereas 3-MA inhibits starvation-induced autophagy²⁷. The inhibitor also prevents liver fibrosis by downregulating autophagy levels and inhibiting liver fibrosis progression caused by carbon tetrachloride (CCL4)²⁸. However, it is currently unclear if 3-MA inhibits arsenic-mediated fibrosis, therefore we investigated this phenomenon in this study.

In this study, we assessed HSC responses to different iAs³⁺ doses to characterize the full range of chronic arsenic exposure levels. Then, we investigated changes in autophagy protein levels during iAs³⁺-induced fibrosis. Finally, we silenced mTOR using 3-MA to alter iAs³⁺-induced autophagy. Our data may be used as a platform to explore changes in autophagy related-proteins during iAs-induced fibrosis in human HSCs, and importantly, lays a foundation for further liver fibrosis investigations.

2.1. Reagents and antibodies

The following reagents were used in this study. Sodium arsenite (iAs³⁺, analytical grade) (No.3 Chemical Reagent Factory, Beijing, China). RFP-GFP-LC3 lentivirus (Jikai Gene Biology Co. Shanghai China). The double fluorescence RFP-GFP-LC3 autophagy system was used in this research. The primary advantage of the system is the absence of any drug intervention to induce autophagy, instead, hunger (serum-free medium) induces changes in autophagy activity and autophagy flow (autophagic flux) and visualized under fluorescence microscopy. Once the system was authenticated, control and arsenic exposure groups were tested under normal and starvation conditions. Concomitant with changes in autophagy, we observed transcription and translation changes in autophagy-related genes, demonstrating that autophagy after arsenic exposure was independent of hunger, but that arsenic affected autophagy.

The real-time monitoring of RFP-GFP-LC3 adenovirus; RFP labeled and tracked LC3, whereas weakened GFP indicated lysosome and autophagosome fusion to form an autophagic lysosome. As GFP fluorescence was acid sensitive, GFP quenching occurred when the autophagosome and lysosome fused, therefore only red fluorescence was detected. This fluorescent protein expression system clearly indicated levels of autophagy flow and was an indispensable tool for our autophagy studies. Fetal bovine serum (FBS) and Dulbecco's modified eagle medium (DMEM) (American Hyclone Co.), Bicinchoninic acid assay (BCA) protein quantification kit (Thermo Scientific, USA). Electrotransfer and electrophoresis solution (Solarbio Co. Beijing, China). Annexin V-PE apoptosis detection kit I (Becton Dickinson) Annexin V-PE is a sensitive probe used to identify apoptotic cells. It binds to negatively charged phospholipid surfaces, with a higher specificity for phosphatidylserine (PS) than other phospholipids. Purified recombinant Annexin V was conjugated to phycoerythrin (PE). Annexin V-PE is routinely used to characterize primary cells or cell lines undergoing apoptosis. RevertAid First Strand cDNA Synthesis kit and QuantiNova SYBR Green PCR Kit (Thermo Scientific). Chromogenic agents (Invitrogen, USA). Chemiluminescence western blot detection kit (Thermo Scientific). Anti-GAPDH, anti-mTOR, anti-LC3, anti-Beclin 1, anti-BCL-2, anti-BCL-XL, anti-TMEM-49, anti-SQSTM-1/P62, and anti-α-SMA (Abcam Co, UK).

2.2. Cell culture

LX-2 cells (Punosai Life Technology Co. Wuhan China) were cultured at 37°C in 5% CO₂ and 95% air at 100% humidity in Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10% heat-inactivated FBS, 100 mg mL⁻¹ penicillin, and 100 mg mL⁻¹ streptomycin (Thermo Scientific). Cell media was changed once every two days. When cells reached 80–90% confluence, they were trypsinized in 0.25% trypsin and passaged into 6-well plates for iAs trioxide exposure.

2.3. Cell treatments and groups

LX-2 cells were seeded into 96-well plates at 5,000 cells per well. iAs³⁺ were added at different concentrations when cell confluence reached 60% (Table 1).

Table 1

Cell groups
Group	Arsenic concentration
high iAs³⁺group	5.00 µmol/L
middle iAs³⁺ group	0.50 µmol/L
low iAs³⁺ group	0.05 µmol/L
control group	0.00 µmol/L
infected only group	0.00 µmol/L

2.4. Cell infections

Before infection, LX-2 cells were resuspended at 1 × 10⁵/mL in 6-well plates for 16–24 h. The first well was the control (cells only). The virus was defrosted and diluted in cold PBS to 1×10⁷ TU/mL (MOI = 10) (TU/mL = number of bioactive virus particles per mL and MOI = multiplicity of infection). Virus was added to cells after 8–12 h, with growth in complete medium up to 72 h. Then, 2 µg/mL puromycin was added to wells and cultures maintained for 3–5 days, until all control cells died, At this point, live cells in remaining wells were assumed to be stably transfected with RFP-GFP-LC3. The medium was replaced with medium plus 0.5 µg/mL puromycin until cell fusion reached approximately 80%. At this point, cells were digested with pancreatic enzymes to ensure stable infection. At 14 days, cells were collected and assumed to be fully stable cell lines. RFP and GFP signals were observed using fluorescence microscopy (Borris Technologies, Co. Beijing, China). RFP was used to tag and track LC3, whereas weakened GFP indicated lysosome and autophagosome fusion to form autophagosomes/lysosomes. Fluorescence infection rates were detected by flow cytometry.

2.5. Apoptosis determination using flow cytometry

Flow cytometry (Bio-RAD,America)was used to analyze cell death processes. The Annexin V-fluorescein isothiocyanate (V-FITC) apoptosis detection kit (BioVision, USA) was used according to manufacturer's instructions. When the cell density reached 70–80%, the old medium was removed and different sodium arsenite concentrations in new medium added for 48 h. Cells were then harvested, washed in 1 × phosphate buffered saline (PBS; pH 7.4–7.5), and suspended in 500 µL 1 × Annexin V binding buffe. To this, 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) ( Trevigen ,America)were added and cells incubated for 5 min in the dark. Finally, cells were processed by flow cytometry.

2.6. Quantitative real-time PCR (qRT-PCR analysis)

Total RNA was isolated from LX-2 cells using the RNAiso kit (Takara, China) following manufacturer’s instructions. Total RNA was reverse-transcribed to cDNA using the Primer Script™ RT Master Mix kit (Takara Bio, Beijing, China). Then, qRT-PCR was performed using the SYBR ® Premix Ex Taq™II (2×) mix (Takara Bio) using a CFX 96-type RT fluorescence quantitative PCR instrument (Bio-Rad, USA). GAPDH was used as a reference gene. Cycle thresholds were determined for each sample and each gene amplification. Based on the 2^−ΔΔCt method, relative target gene expression was calculated. Primer sequences are shown (Table 2).

Table 2

Primers for quantitative real-time PCR
Gene	Forward (5’–3’)	Reverse (3’–5’)
LC3	AAGGCGCTTACAGCTCAATG	CTGGGAGGCATAGACCATGT
Beclin-1	CCATGCAGGTGAGCTTCGT	GAATCTGCGAGAGACACCATC
SQSTM-1/P62	GCACCCCAATGTGATCTGC	CGCTACACAAGTAGTCTGG
BCL-2	GGTGGGGTCATGTGTGTGG	CGGTTCAGGTACTCAGTCATCC
BCL-XL	GAGCTGGTTGACTTTCTC	TCCATCTCCGATTCAGTCCCT
ATG4	ACCAGAGTAAGGGCACCTCT	CCTCCTAATGCCCAAGACTG
ATG5	CGAGATGTGTGGTTTGGACGA	AATGCCATTTCAGTGGTGTGC
ATG8	GGCTCCCAAAGCTCGGATAG	TACAGCTGACCCATTGTGGC
GAPDH	CACCCACTCCTCCACCTTTG	CCACCACCCTGTTGCTGTAG
Note: LC3; microtubule-associated protein light chain A/B; Beclin-1; recombinant human autophagy effector protein; SQSTM-1/P62; ubiquitin binding protein; BCL-2; B cell lymphoma-2; BCL-XL; recombinant human B-cell leukemia/lymphoma-2 XL; ATG4; autophagy related 4; ATG5; autophagy related 5; ATG8; autophagy related 8; GAPDH; glyceraldehyde-3-phosphate dehydrogenase

2.7. Western blotting

After treatments, total protein was extracted using ice-cold RIPA lysis buffer (plus phenylmethylsulfonyl fluoride) and concentrations determined by bicinchoninic acid assay (BCA) protein quantification kit (Thermo Scientific). Protein samples (30 µg) were separated using 6%, 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with proteins transferred to 0.22-µm polyvinylidene fluoride membranes (Sangon Biotech, Shanghai, China). After blocking in 5% skimmed milk for 1 h at 37°C, blots were incubated with primary antibodies; LC3 antibody (dilution 1:1000), Beclin-1 (dilution 1:2000), SQSTM-1/P62 (dilution 1:1000), BCL-2 (dilution 1:500), and GAPDH (dilution 1:10000) overnight at 4°C. Then blots were rinsed three times with Tween (Sangon Biotech) plus PBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sangon Biotech) (1:8000) for 1 h. Immunoreactivity was visualized using an alkaline phosphatase color development kit (Sangon Biotech). Image analysis software ImageJ v1.51 (National Institutes of Health, USA) was used to calculate relative protein expression.

2.8. GFP and RFP fluorescence analysis

Cell lines infected for 14 days were harvested and cultured in confocal dishes (Sangon Biotech). Each dish was incubated with different NaAsO₂ and 3-MA concentrations for 48 h. 3-MA is an autophagy pathway inhibitor and was used to assess whether NaAsO₂ affected LX-2 autophagy. All subsequent arsenic cell assays were based on autophagy under fluorescence after transfection. A laser confocal microscope was used to capture GFP and RFP signals.

2.9. Statistical analysis

All experiments were performed at least 3 times. All data were represented as the mean ± standard error of the mean (SEM). Statistically significant differences between groups were analyzed using a single-factor analysis of variance (ANOVA) followed by two sided Student's t-test in the SPSS.22.0. P values < 0.05 were considered statistically significant.

3.1. The effects of NaAsO₂ on LX-2 morphology

LX-2 cells in the blank group grew well after 24 h; cells were fusiform or star-shaped, with clear boundaries (Figure 1a). In high dose iAs³⁺ groups after 24 h, cell numbers decreased with culture time and cell morphology had changed: cells were no longer stretched but were more circular in morphology (Figure 1b).LX-2 cells in In high dose iAs³⁺ groups after 72h, cell lost original morphology, and cell numbers decreased dramatically (Figure 1c).

3.2. Autophagy observations

RFP-GFP-LC3 punctae in RFP-GFP-LC3-LX-2 cells were detected using laser confocal microscopy: in the control group, RFP-GFP-LC3 punctae were uniformly distributed in the cytoplasm (Figure 2a, b). Autophagosome/lysosome fusion is shown (Figure 2c). After serum-free medium was added to induce starvation, red and green fluorescent punctae were increased, with red fluorescence more abundant than green. (Figure 2d, e), indicating autophagy. In Figure 2F, yellow fluorescence represented autophagy and red represented autophagic lysosomes, thus starvation conditions had induced autophagy. In the absence of drugs, hunger-induced autophagy was observed under fluorescence, indicating the successful construction of an autophagy model using lentiviral RFP-GFP-LC3.

3.3. The effects of NaAsO₂ on early apoptotic rates in LX-2 cells stably expressing RFP-GFP-LC3

When compared with the blank (apoptotic rate = 2.35% ± 0.07%) and infected group (apoptotic rate = 2.15% ± 0.07%), the apoptotic rate in the infected + low iAs³⁺ group was 8.35% ± 0.64%, infected + medium iAs³⁺group, 15.95% ± 1.48%, and 23.70% ± 0.85% in the infected + high iAs³⁺group. Thus, as arsenic concentrations increased, apoptosis rates increased in a dose-dependent manner. Apoptosis rates between groups were statistically significant (P < 0.05) (Table 3 and Figure 3).

Table 3

**The effects of** **NaAsO₂ on apoptotic rates in LX-2 cells after lentivirus infection** **(x̄±s %, n = 3).**
Group	Apoptosis rate (%)
Control group	2.35 ± 0.07
infected only group	2.15 ± 0.07
Infected + low iAs³⁺	8.35 ± 0.64^*
Infected + medium iAs³⁺	15.95 ± 1.48^*
Infected + high iAs³⁺	23.70 ± 0.85^*
Note: n = 3 represents the same cell, and the same treatment was repeated three times; * indicates statistically significant differences when compared with the blank group.

3.4. The effects of NaAsO₂ on LC3, Beclin-1, ATG4, ATG5, and ATG8 mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

To determine whether iAs³⁺ induced autophagy, we examined LC3, Beclin-1, ATG4, ATG5, and ATG8 expression levels in LX-2 cells after exposure to NaAsO₂.

The effects of iAs³⁺ on LC3, Beclin-1, ATG4, ATG5, and ATG8 mRNA expression in LX-2 cells after lentivirus infection are shown (Table 4). When we compared the blank and infection alone group, LC3 mRNA expression was significantly increased in the infection alone group. Thus, to better characterize autophagy, our transfection model may have also impacted other autophagy-related genes, thus after qRT-PCR, we used the transfection group as another control group. Similar to LC3, Beclin-1, ATG4, and ATG5 mRNA expression levels increased in infection groups. ATG8 mRNA was unaffected by transfection.

Table 4

The effects of NaAsO₂ on LC3, Beclin-1, ATG4, ATG5, and ATG8 mRNA expression in LX-2 cells after lentivirus infection (x̄±s n = 3).
Group	LC3	Beclin-1	ATG4	ATG5	ATG8
Blank group	1.25 ± 0.21	1.10 ± 0.53	1.13 ± 0.63	1.00 ± 0.04	1.01 ± 0.15
Infection group	20.09 ± 6.50^a	22.46 ± 0.66^a	2.38 ± 0.48^a	2.26 ± 0.25^a	1.43 ± 0.15
Infected + high As³⁺	36.57 ± 9.68^ab	13.38 ± 2.27^a	3.39 ± 0.17^a	1.65 ± 0.15^ab	1.75 ± 0.08^a
Infected + medium iAs³⁺	4.14 ± 0.00^bc	20.80 ± 6.95^a	4.67 ± 1.38^ab	2.62 ± 0.42^ac	2.89 ± 0.61^abc
Infected + low iAs³⁺	14.19 ± 6.17^ac	24.31 ± 7.09^ac	5.23 ± 0.64^abc	2.56 ± 0.21^ac	2.34 ± 0.55^ab
F	17.45	11.29	17.64	26.97	11.52
P	0.00	0.00	0.00	0.00	0.00
Note: compared with blank group, ^ap < 0.05; compared with infection group, ^bp < 0.05; compared with infected + high iAs³⁺, ^cp < 0.05.

In all transfection groups, LC3 mRNA expression levels tended to initially decrease, then increase with increasing As³⁺ doses (infected + low iAs³⁺: 14.19 ± 6.17; infected + medium iAs³⁺: 4.14 ± 0.00; infected + high As³⁺: 36.57 ± 9.68, p < 0.05) (Table 4).

Beclin-1 mRNA expression levels tended to decrease with increased iAs³⁺ dose (infected + low iAs³⁺: 24.31 ± 7.09; infected + medium iAs³⁺: 20.80 ± 6.95; infected + high As³⁺: 13.38 ± 2.27, p < 0.05). ATG4, ATG5, and ATG8 mRNA expression levels tended to initially increase, then decrease with increased iAs³⁺ dose (p < 0.05) (Table 4).

3.5. The effects of NaAsO₂ on BCL-2 and BCL-XL mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

To confirm iAs³⁺ induced apoptosis and to identify a relationship between autophagy and apoptosis, we investigated expression of the apoptosis-related genes, BCL-2 and BCL-XL in LX-2 cells after treatments. When compared with the blank and infection alone group (these groups exhibited no apoptosis-related gene expression differences when infected), BCL-2 mRNA levels in infected + high and medium iAs³⁺ groups were increased. BCL-XL mRNA levels were decreased in the infected + high iAs³⁺ group (p < 0.05) (Table 5).

Table 5

The effects of NaAsO₂ on BCL-2 and BCL-XL mRNA expression in LX-2 cells after lentivirus infection (x̄±s n = 3).
Group	BCL-2	BCL-XL
Blank group	0.75 ± 0.35	1.04 ± 0.24
Infection group	0.78 ± 0.22	1.12 ± 0.18
Infected + high iAs³⁺	6.93 ± 1.53^ab	0.52 ± 0.21^ab
Infected + medium iAs³⁺	2.81 ± 0.82^abc	0.71 ± 0.17^b
Infected+ low iAs³⁺	1.33 ± 0.34^c	0.81 ± 0.18
F	27.18	4.67
P	0.00	0.00
Note: compared with blank group, ^ap < 0.05; compared with the infection group, ^bp < 0.05; compared with infected + high iAs³⁺ group, ^cp < 0.05; compared with infected + medium iAs³⁺ group, ^dp < 0.05.

3.6. The effects of NaAsO₂ on LC3, Beclin-1, SQSTM-1/P62, TMEM-49, and mTOR protein expression in LX-2 cells

To further confirm autophagosome accumulation and apoptosis induced by arsenic, the translational levels of the autophagic markers, LC3, Beclin-1, SQSTM-1/P62, and TMEM-49, and the apoptosis marker, mTOR were examined in LX-2 cells by western blotting.

The effects of iAs³⁺ on LC3, Beclin-1, SQSTM-1/P62, TMEM-49, and mTOR protein expression in LX-2 cells after lentivirus infection were analyzed. When we compared the blank with the infection alone group, LC3 and Beclin-1 expression levels were both significantly increased. SQSTM-1/P62, TMEM-49, and mTOR levels were unaffected.

In all infection groups, when compared with the infection alone group, LC3 and mTOR expression levels tended to decrease in a dose-dependent manner. Beclin-1, SQSTM-1/P62, and TMEM-49 expression levels tended to increase first, then decrease with increased iAs³⁺ dose (p < 0.05) (Table 6, Figure 4).

Table 6

**The effect of NaAsO**₂ **on LC3, Beclin-1, SQSTM-1/P62, TMEM-49, and mTOR expression in LX-2 cells (x̄±s, n = 3).**
Group	LC3	Beclin-1	SQSTM-1/P62	TMEM-49	mTOR
Blank group	0.04 ± 0.01	0.25 ± 0.01	0.21 ± 0.06	0.37 ± 0.03	0.81 ± 0.02
Infection group	0.20 ± 0.06^a	0.53 ± 0.03	0.20 ± 0.06	0.37 ± 0.01	0.93 ± 0.02
Infected + high iAs³⁺	0.09 ± 0.01^b	0.44 ± 0.05	0.24 ± 0.07	0.42 ± 0.02	0.57 ± 0.11^ab
Infected + medium iAs³⁺	0.15 ± 0.00^a	0.83 ± 0.03^a	0.37 ± 0.04^ab	0.58 ± 0.03^abc	0.47 ± 0.07^ab
Infected+ low iAs³⁺	0.16 ± 0.0^ac	1.20 ± 0.02^abc	0.45 ± 0.01^abc	0.49 ± 0.00^abcd	0.50 ± 0.08^ab
F	11.08	11.75	8.31	30.40	17.18
P	0.00	0.00	0.00	0.00	0.00
Note: compared with blank group, ^ap < 0.05; compared with infection group, ^bp < 0.05; compared with infected + high iAs³⁺ group, ^cp < 0.05; compared with infected + medium iAs³⁺group, ^dp < 0.05.

3.7. The effects of NaAsO₂ on BCL-2 and BCL-XL protein expression in LX-2 cells

The effects of iAs³⁺ on BCL-2 and BCL-XL protein expression in LX-2 cells after lentivirus infection were also investigated. When we compared the blank with the infection alone group, BCL-XL levels were increased and BCL-2 levels unaffected. In all infection groups, when compared with the infection alone group, BCL-XL levels were first decreased, then increased with increasing iAs³⁺ dose (p < 0.05). BCL-2 levels were increased significantly in a dose dependent manner (p < 0.05) (Table 7, Figure 5).

Table 7

The effects of sodium arsenite on BCL-2 and BCL-XL expression in LX-2 cells after lentivirus infection (x̄±s, n = 3)
Group	BCL-2	BCL-XL
Blank group	0.30 ± 0.01	0.43 ± 0.01
Infection group	0.28 ± 0.07	0.56 ± 0.03^a
Infected + high iAs³⁺	1.30 ± 0.01^ab	0.84 ± 0.05^ab
Infected + medium iAs³⁺	0.90 ± 0.01^abc	0.53 ± 0.03^ac
Infected+ low iAs³⁺	0.82 ± 0.01^abc	0.38 ± 0.00^bcd
F	22.25	61.74
P	0.00	0.00
Note: compared with blank group, ^ap < 0.05; compared with infection group, ^bp < 0.05; compared with infected + high iAs³⁺ group, ^cp < 0.05; compared with infected + medium iAs³⁺group, ^dp < 0.05.

3.8. The effects of NaAsO₂ on α-SMA expression in LX-2 cells stably expressing RFP-GFP-LC3

To clarify if arsenic-induced autophagy was related to fibrosis, we assessed α-SMA expression in LX-2 cells after lentivirus infection. When we compared the blank with the infection alone group, α-SMA level were unaffected by infection. In all iAs³⁺ infection groups, when compared with the infection alone group, α-SMA levels were upregulated (p < 0.05) (Table 8, Figure 6).

Table 8

The effects of sodium arsenite on α-SMA expression in LX-2 cells after lentivirus infection (x̄±s, n = 3)
Group	α-SMA
Blank group	0.40 ± 0.07
Infection group	0.48 ± 0.04
Infected + high iAs³⁺	0.62 ± 0.01^a
Infected + medium iAs³⁺	0.78 ± 0.10^abc
Infected + low iAs³⁺	0.68 ± 0.06^ab
F	12.13
P	0.00
Note: compared with blank group, ^ap < 0.05; compared with the infection group, ^bp < 0.05; compared with infected + high iAs³⁺ group, ^cp < 0.05; compared with infected + medium iAs³⁺group, ^dp < 0.05.

3.9. The effects of the autophagy inhibitor, 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 mRNA levels in LX-2 cells stably expressing RFP-GFP-LC3

3-MA is an autophagy inhibitor and was used to evaluate arsenic-induced autophagy in LX-2 cells. Our data showed that except for the infected group, LC3 mRNA expression increased in all dose groups. Beclin-1 expression increased in the high dose trivalent arsenic, trivalent MA groups. SQSTM-1/P62 expression increased in trivalent arsenic high dose groups(p < 0.05). Beclin-1 expression levels increased in the trivalent arsenic high dose group and the 3-MA trivalent arsenic high dose group. SQSTM-1/P62 expression levels were upregulated in the trivalent high dose groups (p < 0.05). When compared to the 3-MA group, LC3 expression in the high dose 3-MA trivalent group was upregulated (Table 9).

Table 9

The effects of 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 mRNA expression levels in LX-2 cells after lentivirus infection (x̄±s, n = 3).
Group	LC3	Beclin-1	SQSTM-1/P62
Blank group	1.00±0.57	1.05±0.37	1.08±0.48
Infection group	3.59±0.30	1.68±0.96	2.12±0.04
3-MA group	8.49±0.48^ab	5.68±0.59^ab	4.93±0.31^a
Infected + high iAs³⁺	36.57±8.25^abc	8.29±1.20^abc	2.17±0.04^c
3-MA + high iAs³⁺	9.51±0.57^ab	11.20±0.44^abc	11.64±4.57^abc
F	102.98	265.08	28.13
P	0.00	0.00	0.00
Note: Compared with blank group, ^aP < 0.05; compared to the infection group, ^bp < 0.05; compared to the 3-MA group, ^cp < 0.05.

3.10. The effects of 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 expression in in LX-2 cells stably expressing RFP-GFP-LC3

When compared with the blank group, LC3 levels were increased in each dose group except the infected group. Higher Beclin-1 levels were observed in the high iAs³⁺, 3-MA + high iAs³⁺ groups. SQSTM-1/P62 levels were increased in in high iAs³⁺group (p < 0.05).

When compared to the 3-MA group, elevated LC3 levels were observed in 3-MA + high iAs³⁺ group. Beclin-1 levels were increased in high iAs³⁺ and 3-MA + high iAs³⁺ groups. SQSTM-1/P62 levels were also elevated in 3-MA + high iAs³⁺ (p < 0.05) (Table 10, Figure 7).

Table 10

The effects of 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 protein expression levels in LX-2 cells after lentivirus infection (x̄±s, n = 3).
Group	LC3	Beclin-1	SQSTM-1/P62
Blank group	0.29 ± 0.01	0.84 ± 0.07	0.75 ± 0.04
Infection group	0.38 ± 0.00	0.81 ± 0.03	0.83 ± 0.05
3-MA group	0.81 ± 0.06^ab	0.95 ± 0.04	0.74 ± 0.02^b
Infected + high iAs³⁺	1.01 ± 0.02^ab	1.25 ± 0.03^abc	0.96 ± 0.03^abc
3-MA + high iAs³⁺	1.49 ± 0.29^abc	1.41 ± 0.12^abc	0.83 ± 0.02^c
F	27.80	29.59	14.69
P	0.00	0.00	0.01
Note: Compared with blank group, ^aP < 0.05; compared to the infection group, ^bp < 0.05; compared to the 3-MA group, ^cp < 0.05.

4.1. LX-2 cell lines stably expressing RFP-GFP-LC3 lentivirus vectors contribute to autophagy observations

In recent years, autophagy has been associated with key roles in several human diseases²⁹. Increasingly, studies have confirmed autophagy maintains cellular and tissue homeostasis by regulating various physiological processes, including cytokine formation, pathogen clearance, antigen presentation, inflammatory responses, and innate and adaptive immune responses³⁰. Autophagy is a complex multi-step process, comprising initiation, nucleation, extension and closure, and lysosomal fusion³¹. Thus, the accurate dissection of autophagy is crucial for studying autophagic biological functions. In our study, we constructed an autophagic bi-functional lentiviral RFP-GFP-LC3 vector for stable transfection into cell lines. The molecular premise was that red and green punctae would represent autophagic mechanisms. Thus, characteristic autophagosomes could be fluorescently displayed at different stages in the autophagy pathway. The establishment of stable cell lines expressing RFP-GFP-LC3 provided a reliable cellular platform for detecting and assessing autophagic flux. Our approach was highly beneficial for studying the autophagic effects of arsenic-induced HSCs and lays the foundation for the identification of mechanisms underpinning arsenic induced liver fibrosis.

4.2. The effects of NaAsO₂ on autophagy levels in LX-2 cells

The liver is an important detoxification organ and is vulnerable when processing poisonous substances³². During hepatic fibrosis, HSCs invade target cells due to a variety of external factors, transforming them into myofibroblasts. α-SMA is present only in the liver and in large vascular smooth muscle. When HSCs are stationary, they do not express α-SMA, exhibit low proliferative activity, and low synthetic collagen capabilities. However, when the liver is damaged via inflammation or mechanical stimulation, HSCs activate and transform into myofibroblast-like cells, and express α-SMA³³ which is an important marker of HSC activation. Inorganic As causes liver injury, especially liver fibrosis³⁴. Tao et al. observed that chronic NaAsO₂ ingestion caused liver fibrosis and oxidative stress in Sprague-Dawley rats, along with collagen deposition, and HSC activation²¹. In our study, in LX-2 cell lines induced by iAs³⁺ for 48 h, α-SMA expression increased significantly, suggesting iAs³⁺ induced HSC activation in vitro, consistent with previous findings. Thus, an in vitro HSC induced iAs³⁺ model was successfully established.

Autophagy activity and biological functions are regulated by several ATGs and associated proteins, including LC3, SQSTM-1/P62, Beclin-1, ATG4, ATG5, ATG8, and ATG12³⁵. LC3 is involved in the formation of autophagy lysosomes and dissociates from lysosomal structures to digest damaged materials. P62 is a marker of autophagic flux which combines autophagy substrates with autophagy lysosomes for degradation³⁶. Therefore, LC3 and P62 are typical autophagy biomarkers. Wu et al. reported that arsenic induced autophagy in pancreatic islets was evidenced by elevated LC3-II levels and depressed P62 levels, both in vivo and in vitro³⁷. Based on our data, after trivalent and pentavalent arsenic treatment, LC3 mRNA expression levels were increased, and the highest increase was observed in the high trivalent arsenic dose group. In addition, the autophagy related gene, ATG5 strictly controls autophagy and plays a crucial role in autophagosome formation; previous studies have shown that ATG5 and ATG4 gene expression levels promote autophagy³⁸. In our study, ATG4, ATG5, and ATG8 gene levels were upregulated in each iAs³⁺ dose group. Moreover, Beclin-1 is a key regulator of autophagy; it promotes the transformation of LC3-I to LC3-II, thereby reflecting autophagy activation³⁹. Previous evidence has suggested mTOR is a regulatory autophagy inhibitor, promotes Beclin-1 release, and is an important regulator in anabolism and catabolism, cell growth, and proliferation⁹. Other studies reported that arsenite-treated cells depleted mTOR levels which in turn increased lysosomal activity and promoted autophagy via the formation of autophagy lysosomes²⁵. Our data showed that with increased iAs³⁺ doses, Beclin-1 and MEM-49 protein expression levels were upregulated and mTOR expression downregulated.

Common effector proteins associated with apoptosis and autophagy include BCL-2 and BCL-XL, and ATG5 respectively⁴⁰. BCL-2 and BCL-XL are well-characterized anti-apoptotic proteins and may be key factors in regulating autophagy; they inhibit Beclin-1 mediated autophagy by interacting with Beclin-1⁴¹. Studies have shown that the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin-1 (matrine is an alkaloid extracted from Sophora flavescens)⁴². Therefore, BCL-2 and BCL-XL have key roles in apoptosis and autophagy interactions. Beclin-1 also inhibits cell proliferation, as observed in several cancers, including, tongue squamous cell carcinoma⁴³, breast cancer⁴⁴, hepatocellular carcinoma⁴⁵, and colorectal cancer. Autophagy blocking also activated apoptotic pathways and eventually led to apoptosis and death. Li⁴⁶ reported that in Raji cells induced with As₂O₃, autophagy vacuoles were formed, increasing autophagy degradation via P62 substrates, accompanied by upregulated Beclin-1 and downregulated BCL-2 expression. Levine⁴⁷observed that Beclin-1 combined with BCL-2/BCL-XL to form a constitutive BCL-2/BCL-XL-Beclin-1 complex which reduced Beclin-1 monomer inhibition of autophagy. Our data showed that Beclin-1 levels were increased and BCL-2/BCL-XL levels inhibited in a dose-dependent trivalent arsenic manner, suggesting that with increased Beclin-1 monomers, apoptosis and autophagy were activated. These results were consistent with our studies, suggesting iAs³⁺ induced autophagy via apoptosis pathways.

4.3. The effects of 3-MA on autophagy induced by NaAsO₂ in LX-2 cells

3-MA is a commonly used inhibitor of the autophagy pathway⁴⁸. Reports have shown 3-MA inhibits phosphoinositide 3-kinase (PI3K) activity and blocks the formation of proautophagosomes, autophagosomes, and autophagic vacuoles²⁴. In our study, the formation of red and yellow fluorescent punctae in the 3-MA + trivalent arsenic high dose group was significantly reduced, while green fluorescent spots were increased, suggesting high-dose sodium arsenite inhibited autophagosome formation and promoted autophagy. Hence, 3-MA may further inhibit iAs³⁺-induced autophagy in LX-2 cells. Wang⁴⁹ reported that arsenic sulfide (AS₂S₂) induced autophagy in human osteosarcoma cells, with upregulated LC3 and Beclin-1 expression. Using 3-MA, the inhibitory effects of AS₂S₂ on cell viability may be enhanced. When Tris (1,3-dichloro-2-propyl) hydrochloride was used to induce SH-SY5Y cells, 3-MA reduced Beclin-1, LC3-II, and SQSTM1/P62 expression, and significantly promoted apoptosis.

In our study, Beclin-1 gene and protein expression in LX-2 cells were significantly upregulated in iAs³⁺ treatment and 3-MA + iAs³⁺ treatment groups, suggesting iAs³⁺ affected early-stage autophagy. Upregulated Beclin-1 was observed in the combined treatment group, further suggesting 3-MA exacerbated autophagic processes. LC3 mRNA and protein expression levels in trivalent group was increased and SQSTM-1/P62 protein expression levels increased suggesting autophagy turnover was inhibited. Therefore, arsenic affects autophagy formation. LC3 and SQSTM-1/P62 gene expression increased in the 3-MA combined iAs³⁺ group, suggesting this commination blocked downstream autophagic flow by inhibiting autophagosome degradation, consistent with our autophagosome distribution images.

This study successfully established an LX-2 cell line stably expressing RFP-GFP-LC3, thereby generating a robust cellular platform to perform autophagy studies. After different arsenite doses were used to induce LX-2 cells, apoptosis rates increased and the cell cycle was blocked in the G0–G1 phase thereby affecting cell replication and inducing apoptosis. Alpha-SMA levels were also significantly elevated suggesting HSC activation and the formation of hepatic fibrosis.Arsenic-induced autophagy occurrence in LX-2 cells may be mediated through the apoptotic pathway.Finally, 3-Methyladenine is able to reduce autophagage levels and may inhibit liver fibrosis induced by inorganic arsenic. However, further in vitro and in vivo research is required to explore how the mTOR pathway regulates arsenic-induced autophagy.

Funding

We acknowledge financial support from the National Natural Science Foundation of China, China 82160650 and Open topics of Microtrace Elements and Local Disease Key Laboratory of National Health Commission.

Acknowledgments

The authors are grateful to the National Natural Science Foundation of China (82160650) and Open topics of Microtrace Elements and Local Disease Key Laboratory of National Health Commission.

Data availability

Data from this study may be accessed upon reasonable request to the corresponding author.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Shunhua Wu designed the study. Yu Hui performed experiments. Fei Huang and Rishalaiti Tayier drafted the manuscript. Ang Li performed analyses and interpreted the data. All authors read and approved the final manuscript.

Chandio, T. A. et al. Fluoride and arsenic contamination in drinking water due to mining activities and its impact on local area population. Environ Sci Pollut Res Int 28, 2355–2368, doi:10.1007/s11356-020-10575-9 (2021).
Minatel, B. C. et al. Environmental arsenic exposure: From genetic susceptibility to pathogenesis. Environ Int 112, 183–197, doi:10.1016/j.envint.2017.12.017 (2018).
Loomis, D., Guha, N., Hall, A. L. & Straif, K. Identifying occupational carcinogens: an update from the IARC Monographs. Occup Environ Med 75, 593–603, doi:10.1136/oemed-2017-104944 (2018).
Mao, J. et al. Possible differences in the mechanism of malignant transformation of HaCaT cells by arsenite and its dimethyl metabolites, particularly dimethylthioarsenics. J Trace Elem Med Biol 61, 126544, doi:10.1016/j.jtemb.2020.126544 (2020).
Zhang, L., Huang, Y., Ling, J., Xiang, Y. & Zhuo, W. Screening of key genes and prediction of therapeutic agents in Arsenic-induced lung carcinoma. Cancer Biomark 25, 351–360, doi:10.3233/cbm-182333 (2019).
Liu, R., Tabuchi, T., Kitamura, T., Miyashiro, I. & Sobue, T. Long-term observational study on 6223 survivors of arsenic poisoning due to contaminated milk powder during infancy. Cancer Sci 111, 3873–3880, doi:10.1111/cas.14623 (2020).
Perry, A., Lynch, R. M., Rusyn, I. & Threadgill, D. W. Long-Term Combinatorial Exposure to Trichloroethylene and Inorganic Arsenic in Genetically Heterogeneous Mice Results in Renal Tubular Damage and Cancer-Associated Molecular Changes. G3 (Bethesda) 9, 1729-1737, doi:10.1534/g3.119.400161 (2019).
Song, Y., Jin, D., Chen, J., Liang, W. & Liu, X. Effects of Arsenic (+3 Oxidation State) Methyltransferase Gene Polymorphisms and Expression on Bladder Cancer: Evidence from a Systematic Review, Meta-analysis and TCGA Dataset. Toxicol Sci 177, 27–40, doi:10.1093/toxsci/kfaa087 (2020).
Chen, Q. Y. & Costa, M. PI3K/Akt/mTOR Signaling Pathway and the Biphasic Effect of Arsenic in Carcinogenesis. Mol Pharmacol 94, 784–792, doi:10.1124/mol.118.112268 (2018).
Xu, Y. et al. Assessing the potential value of Rosa Roxburghii Tratt in arsenic-induced liver damage based on elemental imbalance and oxidative damage. Environ Geochem Health 43, 1165–1175, doi:10.1007/s10653-020-00612-4 (2021).
Hu, Y., Xiao, T. & Zhang, A. Associations between and risks of trace elements related to skin and liver damage induced by arsenic from coal burning. Ecotoxicol Environ Saf 208, 111719, doi:10.1016/j.ecoenv.2020.111719 (2021).
Xue, J. et al. miR-21-regulated M2 polarization of macrophage is involved in arsenicosis-induced hepatic fibrosis through the activation of hepatic stellate cells. J Cell Physiol 236, 6025–6041, doi:10.1002/jcp.30288 (2021).
Fang, S. et al. Arsenic trioxide induces macrophage autophagy and atheroprotection by regulating ROS-dependent TFEB nuclear translocation and AKT/mTOR pathway. Cell Death Dis 12, 88, doi:10.1038/s41419-020-03357-1 (2021).
Wu, L. et al. Autophagy mediates bronchial cell malignant transformation induced by chronic arsenic exposure via MEK/ERK1/2 pathway. Toxicol Lett 332, 155–163, doi:10.1016/j.toxlet.2020.06.006 (2020).
Levine, B. & Kroemer, G. Biological Functions of Autophagy Genes: A Disease Perspective. Cell 176, 11–42, doi:10.1016/j.cell.2018.09.048 (2019).
Zhang, N. & Zhao, Y. Other Molecular Mechanisms Regulating Autophagy. Adv Exp Med Biol 1206, 261–271, doi:10.1007/978-981-15-0602-4_13 (2019).
Oshima, M., Seki, T., Kurauchi, Y., Hisatsune, A. & Katsuki, H. Reciprocal Regulation of Chaperone-Mediated Autophagy/Microautophagy and Exosome Release. Biol Pharm Bull 42, 1394–1401, doi:10.1248/bpb.b19-00316 (2019).
Markaki, M. & Tavernarakis, N. Autophagy mechanisms and roles: recent advances and implications. Febs j 287, 5024–5026, doi:10.1111/febs.15573 (2020).
Wang, M. et al. The expression characteristics and prognostic roles of autophagy-related genes in gastric cancer. PeerJ 9, e10814, doi:10.7717/peerj.10814 (2021).
Al-Bari, M. A. A. & Xu, P. Molecular regulation of autophagy machinery by mTOR-dependent and -independent pathways. Ann N Y Acad Sci 1467, 3–20, doi:10.1111/nyas.14305 (2020).
Tao, Y. et al. IRE1α/NOX4 signaling pathway mediates ROS-dependent activation of hepatic stellate cells in NaAsO(2) -induced liver fibrosis. J Cell Physiol 236, 1469–1480, doi:10.1002/jcp.29952 (2021).
Allaire, M., Rautou, P. E., Codogno, P. & Lotersztajn, S. Autophagy in liver diseases: Time for translation? J Hepatol 70, 985–998, doi:10.1016/j.jhep.2019.01.026 (2019).
Zhang, H. et al. More serious autophagy can be induced by ZnO nanoparticles than single-walled carbon nanotubes in rat tracheal epithelial cells. Environ Toxicol 36, 238–248, doi:10.1002/tox.23029 (2021).
Petiot, A., Ogier-Denis, E., Blommaart, E. F., Meijer, A. J. & Codogno, P. Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275, 992–998, doi:10.1074/jbc.275.2.992 (2000).
Wu, C. W., Lin, P. J., Tsai, J. S., Lin, C. Y. & Lin, L. Y. Arsenite-induced apoptosis can be attenuated via depletion of mTOR activity to restore autophagy. Toxicol Res (Camb) 8, 101–111, doi:10.1039/c8tx00238j (2019).
Chicote, J., Yuste, V. J., Boix, J. & Ribas, J. Cell Death Triggered by the Autophagy Inhibitory Drug 3-Methyladenine in Growing Conditions Proceeds With DNA Damage. Front Pharmacol 11, 580343, doi:10.3389/fphar.2020.580343 (2020).
Zhang, X. et al. Inhibition of autophagy by 3-methyladenine restricts murine cytomegalovirus replication. J Med Virol 93, 5001–5016, doi:10.1002/jmv.26787 (2021).
Peng, Y., Huang, K., Shen, L., Tao, Y. Y. & Liu, C. H. Cultured Mycelium Cordyceps sinensis allevi¬ates CCl4-induced liver inflammation and fibrosis in mice by activating hepatic natural killer cells. Acta Pharmacol Sin 37, 204–216, doi:10.1038/aps.2015.129 (2016).
Shang, D., Wang, L., Klionsky, D. J., Cheng, H. & Zhou, R. Sex differences in autophagy-mediated diseases: toward precision medicine. Autophagy 17, 1065–1076, doi:10.1080/15548627.2020.1752511 (2021).
Jiang, X., Chen, C., Liu, Y., Zhang, P. & Zhang, Z. Critical role of cellular glutathione homeostasis for trivalent inorganic arsenite-induced oxidative damage in human bronchial epithelial cells. Mutat Res Genet Toxicol Environ Mutagen 770, 35–45, doi:10.1016/j.mrgentox.2014.04.016 (2014).
Cheung, Y. W. S., Nam, S. E. & Yip, C. K. Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery. Int J Mol Sci 21, doi:10.3390/ijms21218051 (2020).
Mega, A., Marzi, L., Kob, M., Piccin, A. & Floreani, A. Food and Nutrition in the Pathogenesis of Liver Damage. Nutrients 13, doi:10.3390/nu13041326 (2021).
Zakaria, S. et al. Value of α-smooth muscle actin and glial fibrillary acidic protein in predicting early hepatic fibrosis in chronic hepatitis C virus infection. Arch Med Sci 6, 356–365, doi:10.5114/aoms.2010.14255 (2010).
Wang, Z. et al. Taurine protected As(2)O(3)-induced the activation of hepatic stellate cells through inhibiting PPARα-autophagy pathway. Chem Biol Interact 300, 123–130, doi:10.1016/j.cbi.2019.01.019 (2019).
Ganley, I. G., Wong, P. M., Gammoh, N. & Jiang, X. Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell 42, 731–743, doi:10.1016/j.molcel.2011.04.024 (2011).
Liu, X. et al. Regulation of FN1 degradation by the p62/SQSTM1-dependent autophagy-lysosome pathway in HNSCC. Int J Oral Sci 12, 34, doi:10.1038/s41368-020-00101-5 (2020).
Wu, W. et al. Pancreatic islet-autonomous effect of arsenic on insulin secretion through endoplasmic reticulum stress-autophagy pathway. Food Chem Toxicol 111, 19–26, doi:10.1016/j.fct.2017.10.043 (2018).
Kim, J. et al. Atg5-mediated autophagy controls apoptosis/anoikis via p53/Rb pathway in naked mole-rat fibroblasts. Biochem Biophys Res Commun 528, 146–153, doi:10.1016/j.bbrc.2020.05.083 (2020).
Hu, Y. J., Zhong, J. T., Gong, L., Zhang, S. C. & Zhou, S. H. Autophagy-Related Beclin 1 and Head and Neck Cancers. Onco Targets Ther 13, 6213–6227, doi:10.2147/ott.S256072 (2020).
Dalby, K. N., Tekedereli, I., Lopez-Berestein, G. & Ozpolat, B. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 6, 322–329, doi:10.4161/auto.6.3.11625 (2010).
Zhou, F., Yang, Y. & Xing, D. Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis. Febs j 278, 403–413, doi:10.1111/j.1742-4658.2010.07965.x (2011).
Yang, J. & Yao, S. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1. Int J Mol Sci 16, 25744–25758, doi:10.3390/ijms161025744 (2015).
Hu, Z. et al. Decreased expression of Beclin–1 is significantly associated with a poor prognosis in oral tongue squamous cell carcinoma. Mol Med Rep 14, 1567–1573, doi:10.3892/mmr.2016.5437 (2016).
Wang, N. & Feng, Y. Elaborating the role of natural products-induced autophagy in cancer treatment: achievements and artifacts in the state of the art. Biomed Res Int 2015, 934207, doi:10.1155/2015/934207 (2015).
Qiu, D. M. et al. The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance. BMC Cancer 14, 327, doi:10.1186/1471-2407-14-327 (2014).
Li, C. L. et al. Arsenic trioxide induces autophagy and antitumor effects in Burkitt's lymphoma Raji cells. Oncol Rep 32, 1557–1563, doi:10.3892/or.2014.3369 (2014).
Levine, B., Sinha, S. & Kroemer, G. Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy 4, 600–606 (2008).
Zhang, J. et al. 3-MA Enhanced Chemosensitivity in Cisplatin Resistant Hypopharyngeal Squamous Carcinoma Cells via Inhibiting Beclin -1 Mediated Autophagy. Curr Pharm Des 27, 996–1005, doi:10.2174/1381612826666201221150431 (2021).
Wang, G. et al. Arsenic sulfide induces apoptosis and autophagy through the activation of ROS/JNK and suppression of Akt/mTOR signaling pathways in osteosarcoma. Free Radic Biol Med 106, 24–37, doi:10.1016/j.freeradbiomed.2017.02.015 (2017).

No competing interests reported.

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The Effects of Inorganic Arsenic On Apoptosis and Autophagy in Human Hepatic Sellate Cells

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Reagents and antibodies

2.2. Cell culture

2.3. Cell treatments and groups

2.4. Cell infections

2.5. Apoptosis determination using flow cytometry

2.6. Quantitative real-time PCR (qRT-PCR analysis)

2.7. Western blotting

2.8. GFP and RFP fluorescence analysis

2.9. Statistical analysis

3. Results

3.1. The effects of NaAsO₂ on LX-2 morphology

3.2. Autophagy observations

3.3. The effects of NaAsO₂ on early apoptotic rates in LX-2 cells stably expressing RFP-GFP-LC3

3.4. The effects of NaAsO₂ on LC3, Beclin-1, ATG4, ATG5, and ATG8 mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.5. The effects of NaAsO₂ on BCL-2 and BCL-XL mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.6. The effects of NaAsO₂ on LC3, Beclin-1, SQSTM-1/P62, TMEM-49, and mTOR protein expression in LX-2 cells

3.7. The effects of NaAsO₂ on BCL-2 and BCL-XL protein expression in LX-2 cells

3.8. The effects of NaAsO₂ on α-SMA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.9. The effects of the autophagy inhibitor, 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 mRNA levels in LX-2 cells stably expressing RFP-GFP-LC3

3.10. The effects of 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 expression in in LX-2 cells stably expressing RFP-GFP-LC3

4. Discussion

4.1. LX-2 cell lines stably expressing RFP-GFP-LC3 lentivirus vectors contribute to autophagy observations

4.2. The effects of NaAsO₂ on autophagy levels in LX-2 cells

4.3. The effects of 3-MA on autophagy induced by NaAsO₂ in LX-2 cells

Declarations

References

Additional Declarations

Status:

Version 1

The Effects of Inorganic Arsenic On Apoptosis and Autophagy in Human Hepatic Sellate Cells

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Reagents and antibodies

2.2. Cell culture

2.3. Cell treatments and groups

2.4. Cell infections

2.5. Apoptosis determination using flow cytometry

2.6. Quantitative real-time PCR (qRT-PCR analysis)

2.7. Western blotting

2.8. GFP and RFP fluorescence analysis

2.9. Statistical analysis

3. Results

3.1. The effects of NaAsO2 on LX-2 morphology

3.2. Autophagy observations

3.3. The effects of NaAsO2 on early apoptotic rates in LX-2 cells stably expressing RFP-GFP-LC3

3.4. The effects of NaAsO2 on LC3, Beclin-1, ATG4, ATG5, and ATG8 mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.5. The effects of NaAsO2 on BCL-2 and BCL-XL mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.6. The effects of NaAsO2 on LC3, Beclin-1, SQSTM-1/P62, TMEM-49, and mTOR protein expression in LX-2 cells

3.7. The effects of NaAsO2 on BCL-2 and BCL-XL protein expression in LX-2 cells

3.8. The effects of NaAsO2 on α-SMA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.9. The effects of the autophagy inhibitor, 3-MA combined with NaAsO2 on LC3, Beclin-1, and SQSTM-1/P62 mRNA levels in LX-2 cells stably expressing RFP-GFP-LC3

3.10. The effects of 3-MA combined with NaAsO2 on LC3, Beclin-1, and SQSTM-1/P62 expression in in LX-2 cells stably expressing RFP-GFP-LC3

4. Discussion

4.1. LX-2 cell lines stably expressing RFP-GFP-LC3 lentivirus vectors contribute to autophagy observations

4.2. The effects of NaAsO2 on autophagy levels in LX-2 cells

4.3. The effects of 3-MA on autophagy induced by NaAsO2 in LX-2 cells

Declarations

References

Additional Declarations

Status:

Version 1

3.1. The effects of NaAsO₂ on LX-2 morphology

3.3. The effects of NaAsO₂ on early apoptotic rates in LX-2 cells stably expressing RFP-GFP-LC3

3.4. The effects of NaAsO₂ on LC3, Beclin-1, ATG4, ATG5, and ATG8 mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.5. The effects of NaAsO₂ on BCL-2 and BCL-XL mRNA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.6. The effects of NaAsO₂ on LC3, Beclin-1, SQSTM-1/P62, TMEM-49, and mTOR protein expression in LX-2 cells

3.7. The effects of NaAsO₂ on BCL-2 and BCL-XL protein expression in LX-2 cells

3.8. The effects of NaAsO₂ on α-SMA expression in LX-2 cells stably expressing RFP-GFP-LC3

3.9. The effects of the autophagy inhibitor, 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 mRNA levels in LX-2 cells stably expressing RFP-GFP-LC3

3.10. The effects of 3-MA combined with NaAsO₂ on LC3, Beclin-1, and SQSTM-1/P62 expression in in LX-2 cells stably expressing RFP-GFP-LC3

4.2. The effects of NaAsO₂ on autophagy levels in LX-2 cells

4.3. The effects of 3-MA on autophagy induced by NaAsO₂ in LX-2 cells