Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis attenuates cadmium￾induced apoptosis in duck renal tubular epithelial cells
Zejing Wei, Gaohui Nie, Fan Yang, Shaoxing Pi, Chang Wang, Huabin Cao,
Xiaoquan Guo, Ping Liu, Guyue Li, Guoliang Hu, Caiying Zhang
PII: S0269-7491(20)36608-2
DOI: https://doi.org/10.1016/j.envpol.2020.115919
Reference: ENPO 115919
To appear in: Environmental Pollution
Received Date: 1 July 2020
Revised Date: 17 October 2020
Accepted Date: 22 October 2020
Please cite this article as: Wei, Z., Nie, G., Yang, F., Pi, S., Wang, C., Cao, H., Guo, X., Liu, P., Li, G.,
Hu, G., Zhang, C., Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis attenuates cadmium￾induced apoptosis in duck renal tubular epithelial cells, Environmental Pollution, https://doi.org/10.1016/
j.envpol.2020.115919.
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Author statement
Zejing Wei: Conceptualization, Software, Formal analysis, Writing-Original Draft,
Data Curation, Visualization, Writing-Review & Editing, Validation.
Gaohui Nie: Methodology, Visualization, Resources.
Fan Yang: Validation, Formal analysis. Funding acquisition.
Shaoxing Pi: Data Curation, Validation, Formal analysis.
Chang Wang: Data Curation, Validation, Formal analysis.
Huabin Cao: Methodology, Visualization.
Xiaoquan Guo: Methodology, Formal analysis
Guyue Li: Visualization, Formal analysis.
Ping Liu: Visualization, Formal analysis.
Guoliang Hu: Resources, Validation.
Caiying Zhang: Conceptualization, Project administration, Writing-Review & Editing,
Funding acquisition. Journal Pre-proof
renal tubular Cd
epithelial cells kidney
ROS
NLRP3 NEK7
ASC
NAC
Caspase-1 GSDMD
Z-YVAD-FMK Pyroptosis
Caspase-9 Caspase-3
Bak-1
Bcl-2 MMP Cyt C release
Mito Apoptosis
Activation
Inhibition
IL-1β 、 IL-18 、
LDH 、 NO 、
Relative conductivity
p53
Bax
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1 Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis attenuates
2 cadmium-induced apoptosis in duck renal tubular epithelial cells
Zejing Weia1, Gaohui Nieb1, Fan Yanga
, Shaoxing Pia
, Chang Wanga
, Huabin Caoa
Xiaoquan Guoa
, Ping Liua
, Guyue Lia
, Guoliang Hua
, Caiying Zhanga* 4
5 1 These two are the equal first authors.
a
6 Jiangxi Provincial Key Laboratory for Animal Health, Institute of Animal
7 Population Health, College of Animal Science and Technology, Jiangxi Agricultural
8 University, No. 1101 Zhimin Avenue, Economic and Technological Development
9 District, Nanchang 330045, Jiangxi, P. R. China
b
10 School of Information Technology, Jiangxi University of Finance and
11 Economics, No. 665 Yuping West street, Economic and Technological Development
12 District, Nanchang 330032, Jiangxi, P. R. China
13 All authors have read the manuscript and agreed to submit it in its current form
14 for consideration for publication in the Journal
15 * Corresponding author
16 Address: College of Animal Science and Technology, Jiangxi Agricultural
17 University, No. 1101 Zhimin Avenue, Economic and Technological Development
18 District, Nanchang 330045, Jiangxi, P. R. China
19 Tel: +86-15870002893 (Caiying Zhang)
20 E-mail: [email protected]
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23 Abstract
24 Cadmium (Cd) is an occupational and environmental pollutant, which mainly
25 causes nephrotoxicity by damaging renal proximal tubular cells. To evaluate the
26 effects of Cd on pyroptosis and the relationship between pyroptosis and apoptosis in
27 duck renal tubular epithelial cells, the cells were cultured with 3CdSO4·8H2O (0, 2.5,
28 5.0, or 10.0 μM Cd), N-acetyl-L-cysteine (NAC) (100.0 μM), Z-YVAD-FMK (10.0
29 μM) or the combination of Cd and NAC or Z-YVAD-FMK for 12 h, and then
30 cytotoxicity was assessed. The results evidenced that Cd significantly increased the
31 releases of interleukin-18 (IL-18) and interleukin-1β (IL-1β), lactate dehydrogenase
32 (LDH) and nitric oxide (NO), relative conductivity and cellular reactive oxygen
33 species (ROS) level. Simultaneously, Cd also markedly upregulated NLRP3,
34 Caspase-1, ASC, NEK7, IL-1β and IL-18 mRNA levels and NLRP3, Caspase-1 p20,
35 GSDMD and ASC protein levels. Additionally, NAC notably improved the changes
36 of above indicators induced by Cd. Combined treatment with Cd and Z-YVAD-FMK
37 remarkably elevated Bcl-2 mRNA and protein levels, inhibited p53, Bax, Bak-1, Cyt
38 C, Caspase-9 and Caspase-3 mRNA levels and p53, Bax, Bak-1, Caspase-9/cleaved
39 Caspase-9 and Caspase-3/cleaved Caspase-3 protein levels, increased mitochondrial
40 membrane potential (MMP), decreased apoptosis ratio and cell damage compared
41 treatment with Cd alone. Taken together, Cd exposure induces duck renal tubular
42 epithelial cell pyroptosis through ROS/NLRP3/Caspase-1 signaling pathway, and
43 inhibiting Caspase-1 dependent pyroptosis attenuates Cd-induced apoptosis.
44 Keywords: Cadmium; Pyroptosis; Apoptosis; Renal tubular epithelial cell; Duck
45
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46 1. Introduction
47 Cadmium (Cd) is an environmental and occupational pollutant, which poses a
48 great threat to the health of animals and human beings (Levengood, 2003; Bandara et
49 al., 2011; Kiran et al., 2016; Hu et al., 2020). Various studies indicated that
50 environmental Cd pollution could not be ignored (Audry et al., 2004; Bandara, et al.,
51 2011; Gustin et al., 2020). Cd contents of rivers and soil were 0.04-0.18 μM and
52 800.37-1236.90 μM in certain areas, respectively (Bandara, et al., 2011; Zhang et al.,
53 2015). Moreover, Cd concentrations in kidneys of wood duck and humans living in
54 contaminated areas for a long time were as high as 5.0-27.6 μg/g and 36.6-58.3 µg/g
55 (wet weight), respectively, which might lead to chronic renal failure (Levengood,
56 2003; Uetani et al., 2006; Satarug, 2018). The main sources of Cd pollution include
57 non-ferrous metal production, nickel-Cd batteries, petroleum and coal combustion,
58 and industrial waste (Yang et al., 2020). With the rapid development of
59 industrialization process, the Cd burden is gradually increasing in the environment
60 (Audry, et al., 2004). At present, Cd pollution has become a global problem. Animals
61 are poisoned by ingestion of Cd pollutant through respiratory tract, skin, and digestive
62 tract (Setia et al., 2020). The main organs affected by Cd toxicity are kidney, liver,
63 testis, bone and so on (Wang et al., 2017; Rosales-Cruz et al., 2018). And kidney is
64 the most important target organ of Cd accumulation (Brzoska et al., 2003; Hagar and
65 Al, 2014; Zhuang et al., 2019). Cd induces nephrotoxicity mainly through damaging
66 renal proximal tubular cells (Wang et al., 2020). Numerous studies showed that Cd is
67 capable of inducing excessive reactive oxygen species (ROS) generation in kidney,
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68 which lead to oxidative stress, pyroptosis (Chen et al., 2016; Li et al., 2017) and
69 apoptosis (Li, et al., 2017; Zeeshan et al., 2017; Kang et al., 2019).
70 Pyroptosis and apoptosis regulate cell death, and are implicated in the
71 pathogenesis of many diseases (Kofahi et al., 2016). The pro-inflammatory cytokines
72 interleukin-18 (IL-18) and interleukin-1β (IL-1β) controlled by inflammatory bodies
73 mature and secrete, which induces inflammatory cell death known as pyroptosis
74 (Taabazuing et al., 2017). The main features of pyroptosis are Caspase-1 dependence,
75 swelling of cells and organelles, rupture of membrane, and intracellular substances
76 releases (Bergsbaken et al., 2009). The mechanism of pyroptosis activation is
77 considered to be Nod-like receptor protein 3 (NLRP3) inflammasome, which can be
78 activated by ROS (Tang et al., 2019; Wu et al., 2020). Once NLRP3 activates
79 Caspase-1, pyroptosis will inevitably occur (Strowig et al., 2012; Shi et al., 2017).
80 Therefore, the NLRP3/Caspase-1 pathway is very critical in regulating pyroptosis (Li
81 et al., 2018). Apoptosis is a genetically modulated mode of cell death, which is mainly
82 characterized by shrinking cell volume, disappearing mitochondrial membrane
83 potential (MMP), and releasing cytochrome C (Cyt C) into cytoplasm. It is regulated
84 by anti-apoptotic and pro-apoptotic families, such as Caspase family and Bcl-2 family
85 proteins (Castro et al., 2008; Siddiqui et al., 2015; Birkinshaw and Czabotar, 2017).
86 And p53 interacts with pro-apoptotic Bcl-2 family proteins, resulting in the release of
87 pro-apoptotic factor Bax/Bak, which induces the release of Cyt C, and then activating
88 Caspase-9 and downstream Caspase-3, eventually triggering apoptosis (Siddiqui, et
89 2015; Pistritto et al., 2016; Birkinshaw and Czabotar, 2017).
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90 Various studies showed that excessive ROS could induce oxidative stress which
91 activated NLRP3/Caspase-1 pathway pyroptosis and intrinsic apoptosis (Hagar and Al,
92 2014; Wu et al., 2018). Previous researches have revealed that pyroptosis and
93 apoptosis can interact with each other (Bergsbaken, et al., 2009; Rogers et al., 2017;
94 Taabazuing, et al., 2017; Wang et al., 2017). Our previous study found that apoptosis
95 was the predominant mechanism responsible for Cd-induced cellular death in duck
96 renal tubular epithelial cells (Zhuang, et al., 2019). However, the mechanisms of duck
97 renal tubular epithelial cell pyroptosis induced by Cd remain poorly understood, the
98 relationship between pyroptosis and apoptosis is still unclear. Thus, the purpose of
99 this experiment was to reveal whether Cd could induce pyroptosis via
100 ROS/NLRP3/Caspase-1 pathway and provide an interesting model for exploring the
101 crosstalk between pyroptosis and apoptosis in cytotoxicity induced by Cd in duck
102 renal tubular epithelial cells.
103 2. Materials and Methods
104 2.1 Cell isolation, culture and treatment
105 All of the experimental procedures involving animals were approved by the
106 Ethics Committee of Jiangxi Agricultural University. Cell isolation and culture
107 method were established in line with our previous methods (Zhuang, et al., 2019).
108 Cadmium sulfate (3CdSO4·8H2O) was applied as source of Cd. The duck renal
109 tubular epithelial cells were treated with a series of Cd concentrations (0, 2.5, 5.0,
110 10.0, 20.0, 40.0 or 80.0 μM) for 12 h, respectively, and then the cell viability was
111 calculated and was presented relative to the viability of the duck renal tubular
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112 epithelial cells that were not exposed to Cd in the form of a line chart. The half
113 maximal inhibitory concentration (IC50) of Cd on duck tubular epithelial cells for 12
114 exposure is 23.27 μM (Table S1, S2, Fig. S1). Hence, three concentrations (2.5, 5.0,
115 10.0 μM Cd) were selected in the study based on IC50, and experimental groups as
116 following: 0 μM Cd (Control group), 2.5 μM Cd group, 5.0 μM Cd group and 10.0
117 μM Cd group. Then, the cells were cultured with a series of N-acetyl-L-cysteine
118 (NAC) (Sigma-Aldrich USA) (0, 50.0, 100.0, 150.0, 200.0 or 250.0 μM) or
119 Z-YVAD-fluoromethylketone (Z-YVAD-FMK) (Abmole Houston, USA) (0, 2.5, 5.0,
120 10.0, 20.0 or 40.0 μM), respectively, and cell viability was detected by CCK-8 after
121 12 h treatment, the most suitable protective concentrations of NAC (100.0 μM ) and
122 Z-YVAD-FMK (10.0 μM) were selected (Table S3, S4), respectively. The
123 experiments were grouped as follows: 0 μM Cd (Control group), 10.0 μM Cd (Cd
124 group), 100.0 μM NAC (NAC group), 10.0 μM Z-YVAD-FMK (YVAD group), 100.0
125 μM NAC and 10.0 μM Cd (NAC+Cd group), 10.0 μM Z-YVAD-FMK and 10.0 μM
126 Cd (YVAD+Cd group), respectively. Since IC50 and the most suitable protective
127 concentrations of NAC and Z-YVAD-FMK were based on the results of 12 h
128 exposure, events of cells exposed to Cd or/and NAC (Z-YVAD-FMK) for 12 h were
129 chosen to assess the toxic effects in the study.
130 2.2 Determination of intracellular and extracellular Cd contents
131 Determination of Cd contents according to the method of Yang et al. (Yang et al.,
132 2018). After treatment, cells and culture medium were collected, and cells were
133 washed twice. Then cells and culture medium were digested with 65 % HNO3. The
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134 content of Cd in the sample was determined by flame atomic absorption
135 spectrophotometers (FAAS) (A3AFG, Beijing, China). The FAAS was adjusted to
136 flame atomic absorption mode, and the parameters were set to wavelength: 228.8 nm,
137 current: 2 mA. The correlation coefficient of the atomic absorption standard curve of
138 the measured sample must be greater than 0.999. Then Cd content was calculated
139 using the standard curve.
140 2.3 Assessment of LDH, NO releases and relative conductivity
141 The culture supernatant was collected after cells were treated for 12 h. The
142 release of LDH was assessed according to the kit instructions (Nanjing Jiancheng
143 Bioengineering Institute, China), the cell supernatant was collected and shifted to
144 96-well plates. The absorbance of each sample was measured at 450 nm by
145 Microplate Reader instrument. The cell supernatant was hatched with the equal
volume Griess reagent at 37 o
146 C for 15 min. Then, the absorbance of each sample was
147 measured (wavelength: 540 nm) through the Microplate Reader instrument (Thermo
148 Fisher, USA). The NO content was counted by the standard curve of sodium nitrite.
149 According to the instruction of conductivity meter (DDSJ-308A Shanghai precision
150 scientific instrument co., Ltd, China), the electrode and measurement conditions and
151 the conductivity meter were adjusted, and then the measurement was performed.
152 2.4 The assay of enzyme-linked immunosorbent
153 The IL-18 and IL-1β contents in cell supernatant were assayed by ELISA kits
154 (Mlbio Shanghai, China). 50 μL standard and sample was added to corresponding
155 wells, respectively. Then, 100 μL enzyme conjugates was added to the corresponding
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wells, sealed and incubated at 37 o
156 C for 1 h. After washing, 50 μL substrates A and B
were added to each well and reacted at 37 o
157 C for 15 min. Next, after adding 50 μL
158 stop solution, the optical density (OD) was read at 450 nm by a microtiter plate reader
159 in 15 min. The content of the sample could be calculated according to the standard
160 curve.
161 2.5 The detection of MMP and ROS level
162 After treatment, the cells were resuspended by trypsin and collected by
163 centrifugation. The MMP and ROS level were tested using the MMP kit (Keygen
164 Biotech Nanjing, China) and ROS kit (Beyotime Biotechnology Shanghai, China) in
165 line with the directions, respectively. Then the flow cytometer (C6 Plus flow
166 cytometer, BD, USA) or the inverted fluorescence microscope (Olympus Optical Co.,
167 Ltd., Tokyo, Japan) was used for cell measurement.
168 2.6 AO/EB staining assessment
169 Cells were treated with Cd or/and Z-YVAD-FMK for 12 h, then collected,
170 centrifuged and washed twice with PBS (Solarbio Biotechnology Beijing, China) and
adjusted cell content to 1×107
171 cells/mL with PBS. According to kit instructions, the
172 cells were fluorescently, and then observed under a fluorescence microscope
173 (Beyotime, Nanjing, China).
174 2.7 Real-time quantitative PCR (RT-qPCR)
175 The extraction method of total RNA was the same as that of our previous study
176 (Wang, et al., 2020), reverse transcription of 1 μL total RNA into cDNA using reverse
177 transcription kit (Takara, Japan), and then RT-qPCR was used to quantitatively detect
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178 mRNA relative level on ABI Quant Studio7 Flex PCR instrument. As described in
179 Table 1, each qPCR primer pair was adopted only when its amplification efficiency
180 fell between 95 % and 105%. The gene sequences of duck NLRP3, Caspase-1, ASC,
182 and β-actin were obtained from NCBI GenBank. The results were analyzed by SPSS
183 analysis software.
184 2.8 Western blot
185 After the cells were lysed completely, the protein concentration was assayed
186 according to the instructions of the BCA protein kit (Solarbio Biotechnology Beijing,
187 China). Separated the extracted protein by gel electrophoresis (TransGen Biotech
188 Beijing, China) and blotted onto PVDF membranes (0.45 µm) (Beyotime
189 Biotechnology Shanghai, China). The PVDF membrane was sealed with 5 % non-fat
190 milk powder for 1 h, and the membrane was incubated with the primary antibodies
191 anti-NLRP3 (1:500), anti-Caspase-1 p20 (1:500), anti-ASC (1:500), anti-GSDMD
192 (1:500), anti-p53 (1:500), anti-Bax (1:500), anti-Bcl-2 (1:500), anti-Caspase-9 (1:500),
193 anti-cleaved Caspase-9 (1:500) (Wanleibio, China), anti-Caspase-3 (1:1500),
194 anti-cleaved Caspase-3 (1:1500), and anti-GAPDH (1:5000) (proteintech, china)
overnight at 4 o
195 C, incubated with corresponding HRP-conjugated(1:5000) secondary
196 antibody for 1 h at room temperature. The blot bands were examined by Molecular
197 Imager Chemidoc XRS System (UVP, Ltd., US).
198 2.9 Statistical analysis
199 Each experiment was repeated at least 3 times. All data were processed with
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200 SPSS 25.0 software (SPSS Inc., Chicago, IL, USA), expressed as the mean ± SD. And
201 analyzed by one-way analysis of variance (ANOVA). P values < 0.05 were
202 considered statistically significant. Finally, data were presented using GraphPad
203 Prism 8.3 software.
204 3. Results
205 3.1 Cd exposure increases intracellular and extracellular Cd contents
206 The intracellular and extracellular Cd contents were analyzed by FAAS.
207 Compared to the control group, the intracellular and extracellular Cd contents were
208 significantly increased (P < 0.001) in all Cd treated groups and indicated a dose-effect
209 (Fig. 1A-B).
210 3.2 Cd induces duck renal tubular epithelial cell pyroptosis
211 Compared to the control group, the NLRP3, Caspase-1, ASC, NEK7, IL-18 and
212 IL-1β mRNA levels were up-regulated (P < 0.05, P < 0.01 or P < 0.001) in all Cd
213 treatment groups, especially in 5.0 μM Cd group and 10.0 μM Cd group (Fig. 2A-F).
214 Simultaneously, the results of western blotting showed that the Caspase-1 p20,
215 NLRP3, ASC and GSDMD protein levels were markedly elevated (P < 0.05, P < 0.01
216 or P < 0.001) in all Cd treatment groups compared to the control group, and the
217 changes of 10.0 μM Cd group were the most apparent (Fig. 2G-K).
218 3.3 NAC improves Cd-induced changes of pyroptosis-related indexes
219 The morphologically characteristics of pyroptosis are cell swelling and the
220 formation of membrane pores, which changes cell membrane permeability. Therefore,
221 the releases of IL-18, IL-1β, LDH and NO and relative conductivity in cell
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222 supernatant were detected in the experiment. As shown in Fig. 3A-E, the above
223 indicators in all Cd-treated groups were higher than those in the control group,
224 especially in 10.0 μM Cd group (P < 0.001). NAC could significantly reduce (P <
225 0.01 or P < 0.001) the changes of above indicators (Fig. 3F-J).
226 3.4 NAC improves Cd-induced changes of intracellular ROS level
227 The ROS fluorescence intensity was markedly elevated (P < 0.001) in all Cd
228 groups compared to the control group (Fig. 3K-N). In addition, NAC could
229 significantly reduce (P < 0.001) the changes of above indicators (Fig. 3O-R).
230 3.5 NAC reduces Cd-induced pyroptosis-related factors expression levels
231 The mRNA levels of Caspase-1, NLRP3, ASC, NEK7, IL-18 and IL-1β notably
232 elevated (P < 0.01 or P < 0.001) in the Cd group compared to the control group, but
233 mRNA levels of above the genes markedly decreased (P < 0.01 or P < 0.001) in the
234 NAC+Cd group compared with the Cd group (Fig. 4A-F). In addition, Fig. 4G-K
235 showed that Caspase-1 p20, NLRP3, ASC and GSDMD protein levels in the Cd group
236 markedly elevated (P < 0.001) compared to the control group, and the levels of above
237 these proteins in the NAC+Cd group remarkably down-regulated (P < 0.05 or P <
238 0.01) compared to the Cd group.
239 3.6 Z-YVAD-FMK improves Cd-induced changes of morphology and
240 pyroptosis-related indicators
241 In the control group and YVAD group, the normal morphology was observed,
242 cell vacuolation and density, and decreased cell size were found in the Cd group. In
243 addition, the degree of cell damage in the YVAD+Cd group was less than that in the
244 Cd group (Fig. 5A). As shown in Fig. 5B-F, compared to the control group, IL-18,
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245 IL-1β, LDH and NO releases, and relative conductivity in the Cd and YVAD+Cd
246 groups were notably elevated (P < 0.05 or P < 0.001), but they were dramatically
247 decreased (P < 0.01 or P < 0.001) in the YVAD+Cd group compared with the Cd
248 group. As shown in Fig. 5G-K, compared to the control group, Caspase-1 mRNA and
249 protein levels in the YVAD group were remarkedly reduced (P < 0.01), but the levels
250 of NLRP3 mRNA and protein were not statistically different (P > 0.05) in the YVAD
251 group, the Caspase-1 and NLRP3 mRNA and protein levels in the Cd group were
252 markedly increased (P < 0.01 or P < 0.001), and NLRP3 and Caspase-1 mRNA and
253 protein levels of YVAD+Cd group were dramatically lower (P < 0.01 or P < 0.001)
254 than those of Cd group.
255 3.7 Z-YVAD-FMK elevates Cd-induced changes of MMP
256 Changes in MMP were presented in Fig. 6A-B. Compared to the control group,
257 the MMP was remarkedly reduced (P < 0.001) in the Cd group, but the MMP of
258 YVAD+Cd group was markedly elevated (P < 0.001) compared with the Cd group.
259 3.8 Z-YVAD-FMK reduces Cd-induced apoptosis
260 Compared to the control group, p53, Bak-1, Bax, Cyt C, Caspase-9 and
261 Caspase-3 mRNA levels of Cd group were dramatically elevated (P < 0.001), and
262 Bcl-2 mRNA level was markedly decreased (P < 0.001). But Z-YVAD-FMK addition
263 could notably ameliorate (P < 0.05 or P < 0.001) this situation (Fig. 6C-I). The data
264 of western blot revealed that p53, Bax, Caspase-9/cleaved-Caspase-9 and Caspase-3/
265 cleaved Caspase-3 protein levels were markedly elevated (P < 0.05 or P < 0.001) in
266 the Cd group compared to the control group, but they were markedly downregulated
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267 (P < 0.05, P < 0.01 or P < 0.01) in the YVAD+Cd group compared to the Cd group.
268 But the result of Bcl-2 protein level was opposite (Fig. 6J-P). The results of AO/EB
269 staining were presented in Fig. 6R-S. Apoptosis ratio was remarkedly increased (P <
270 0.001) in the Cd and YVAD+Cd groups compared to the control group, but apoptosis
271 ratio of Cd group was markedly elevated (P < 0.001) compared with the YVAD+Cd
272 group.
273 4. Discussion
274 Cd is the most important heavy metal pollutant and kidney is the vital target
275 organ of Cd toxicity (Hagar and Al, 2014). The proximal tubule of the kidney is the
276 main site for Cd accumulation (Johri et al., 2010). Pyroptosis is a Caspase-1-
277 dependent programmed cell death, which is involved in certain pathological
278 inflammatory processes (Jang et al., 2015). In current study, the mechanism of
279 pyroptosis induced by Cd and the relationship between pyroptosis and apoptosis were
280 investigated based on duck renal tubular epithelial cells model. Primarily, events Cd
281 exposure for 12 h were selected to assess the toxic effects. Our results demonstrated
282 that Cd induced duck renal tubular epithelial cell pyroptosis via
283 ROS/NLRP3/Caspase-1 pathway, and inhibiting Caspase-1 dependent pyroptosis
284 might attenuate Cd-induced apoptosis.
285 Accumulating evidence have shown that Caspase-1 activation by NLRP3
286 inflammasome causes a massive inflammatory cell death form known as pyroptosis
287 (Tang, et al., 2019; Wu, et al., 2020). The NLRP3 inflammasome, composing of three
288 substances, including NLRP3, ASC and pro-Caspase-1, serves as a platform to
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289 activate Caspase-1 and pro-inflammatory cytokines IL-1β and IL-18 will be cleaved
290 by activated Caspase-1 (Strowig, et al., 2012). ASC is composed of both a Caspase
291 recruitment domain (CARD) and a pyrin domain (PYD) (Dick et al., 2016). The PYD
292 of NLRP3 interacts with ASC protein, after that ASC protein recruits pro-Caspase-1,
293 which causes NLRP3 inflammasome activation (Lamkanfi and Dixit, 2014).
294 Therefore, downstream of NLRP3, ASC plays a vital role in the assembly of
295 inflammasome and the recruitment of Caspase-1 (Shi, et al., 2017). As a
296 NLRP3-binding protein, NEK7 can regulate NLRP3 activation, acts as a key mediator
297 of NLRP3/Caspase-1 signaling pathway activation (He et al., 2016; Kim et al., 2019).
298 Caspase-1 exists as an inactive precursor pro-Caspase-1 in cell, which can be
299 activated by NLRP3 inflammasome (Bergsbaken, et al., 2009). Caspase-1 activation
300 results in the release of IL-18 and IL-1β as well as pyroptosis (Man and Kanneganti,
301 2016). Activated and secreted IL-18 and IL-1β can bind to and activate their receptors,
302 then leading to the activation of multiple cytokines involved in inflammation cascade
303 (Haldar et al., 2015). Gasdermin D (GSDMD) is an endogenous pore-forming protein,
304 and its N-terminal domain has been shown to form membrane-embedded pores (Shi et
305 al., 2015). Multiple studies revealed that GSDMD was important for IL-18 and IL-1β
306 releases and pyroptosis of inflammatory Caspase-dependence (Kayagaki et al., 2015;
307 Shi, et al., 2015). Moreover, the releases of LDH and NO and the increase of relative
308 conductivity in cell supernatant are the predominant characteristics of pyroptosis
309 (Fink et al., 2008). Recent studies have shown that heavy metals can activate NLRP3
310 inflammasome, up-regulate NLRP3, Caspase-1, IL-18 and IL-1β mRNA levels, and
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311 Caspase-1 p20, NLRP3 and ASC protein levels, promote IL-18 and IL-1β releases in
312 cell supernatant, and induce pyroptosis (Ahn et al., 2018; Liao et al., 2019).
313 Furthermore, various studies revealed that pyroptosis was accompanied by the
314 increase of NEK7 mRNA level and GSDMD protein level (Chen et al., 2019; Ruan,
315 2019; Sharif et al., 2019; Tang, et al., 2019) as well as LDH and NO releases, the
316 increase of relative conductivity in cell supernatant (Yuan et al., 2017). In the study,
317 data evidenced that Cd could upregulate Caspase-1, NLRP3, NEK7, ASC, IL-18 and
318 IL-1β mRNA levels and Caspase-1 p20, NLRP3, ASC and GSDMD protein levels in
319 duck renal tubular epithelial cells, Besides, Cd treatment increased IL-18, IL-1β, LDH
320 and NO releases, and relative conductivity in cell supernatant, which is consistent
321 with the above results. These results suggest that Cd can induce duck renal tubular
322 epithelial cell pyroptosis. As an inhibitor of pyroptosis, Z-YVAD-FMK can inhibit
323 Caspase-1 activation, reduce IL-18 and IL-1β releases, and inhibit the occurrence of
324 pyroptosis in cells (Wu et al., 2010; Zhang et al., 2017). In our study, as expected,
325 Caspase-1 mRNA and protein levels were significantly decreased after treatment with
326 Z-YVAD-FMK, further confirming that Z-YVAD-FMK inhibited duck renal tubular
327 epithelial cell pyroptosis by preventing the activation of Caspase-1. Moreover, the
328 difference in Caspase-1, NLRP3 mRNA and protein levels, IL-18, IL-1β, LDH and
329 NO releases as well as relative conductivity between Cd treatment and basal condition
330 in the presence of Z-YVAD-FMK revealed that Cd could cause
331 NLRP3-Caspase-1-dependent pyroptosis.
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332 Overwhelming evidence suggested that Cd induced ROS accumulation,
333 ultimately leading to oxidative stress in kidney (Jung et al., 2015; Xia et al., 2015; Liu
334 et al., 2019; Zhuang, et al., 2019; Wang, et al., 2020). It is well established that ROS
335 plays an essential role in NLRP3-Caspase-1-dependent pyroptosis (Kong et al., 2019).
336 The activation of ROS is the major pathway of NLRP3 inflammasome activation.
337 Excessive ROS induces oxidative stress, which activated the NLRP3/Caspase-1
338 pathway and caused pyroptosis (Wu, et al., 2020). And, ROS inhibitor suppressed
339 NLRP3 inflammasome activation (Zheng et al., 2014). In the present study, Cd
340 induced the increase of intracellular ROS level (Fig. 3K-N). However, the
341 relationship between ROS and pyroptosis induced by Cd in duck renal tubular
342 epithelial cells is still unknown. NAC is an important active oxygen radical scavenger
343 for mitochondria, which is mainly used to identify and inhibit ROS generation (Halasi
344 et al., 2013; Wu, et al., 2018). As a common and important antioxidant, NAC can
345 stimulate the synthesis of reduced glutathione (GSH) in cells, react directly with
346 oxygen free radicals, and enhance the activity of glutathione-s-transferase based
347 stimulation (Lavrentiadou et al., 2001). Hence, in this study, NAC was applied as a
348 ROS scavenger to explore the relationship between ROS and pyroptosis induce by Cd.
349 Our results manifested that comparing to the Cd group, NAC treatment notably
350 decreased the mRNA levels of Caspase-1, NLRP3, ASC, NEK7, IL-18, IL-1β, and the
351 protein levels of Caspase-1 p20, NLRP3, ASC, GSDMD. Moreover, NAC also
352 markedly relieved Cd-induced the changes of IL-18, IL-1β, LDH and NO releases,
353 relative conductivity as well as ROS level. Hence, we speculated that NLRP3
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354 inflammasome could be activated by Cd-induced excessive ROS, and then resulting in
355 ROS/NLRP3/Caspase-1 pathway pyroptosis in duck tubular epithelial cells.
356 Multiple studies have evidenced that pyroptosis and apoptosis are involved in the
357 toxicity mechanism of heavy metals (Pulido and Parrish, 2003; Ahn, et al., 2018; Liao,
358 et al., 2019). Pyroptosis is an inflammatory cell death of Caspase-1-dependence (Lee
359 et al., 2018). But apoptosis is a non-inflammatory cell death of Caspase-3-dependence
360 (Creagh, 2014). previous study showed that apoptosis occurred more slowly than
361 pyroptosis (Shi, et al., 2017). Kim et al. found that Z-YVAD-FMK could inhibit
362 Caspase-1 activation and reduce apoptosis ratio in mouse renal tubular epithelial cells
363 (Kim et al., 2018). Liao et al. reported that Cu caused chicken hepatocyte pyroptosis
364 and Z-YVAD-FMK reduced apoptosis induced by Cu (Liao, et al., 2019). Our
365 previous studies manifested that Cd could cause duck renal tubular epithelial cell
366 apoptosis (Zhuang, et al., 2019; Wang, et al., 2020). However, the relationship
367 between apoptosis and pyroptosis induced by Cd is still unclear. In our study,
368 Z-YVAD-FMK was applied as a Caspase-1 inhibitor to evaluate the relationship
369 between Cd-induced pyroptosis and apoptosis by detecting p53, Bcl-2, Bak-1, Bax,
370 Cyt C, Caspase-9 and Caspase-3 mRNA levels and protein levels, MMP and
371 apoptosis ratio. Pro-apoptotic p53 protein is normally known as tumor suppressor
372 protein, which can activate apoptotic signaling pathways and control apoptotic genes
373 such as those in the Bcl-2 family (Schramm et al., 2017). Bcl-2 belongs to
374 anti-apoptotic gene, mainly locates on the outer membrane of the mitochondria, and
375 regulates the apoptosis of the mitochondrial pathway (Birkinshaw and Czabotar,
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376 2017). Bax and Bak-1 are pro-apoptotic genes (Shi et al., 2010). Excessive Bax/Bak
377 dimer can changing mitochondrial membrane potential, then causing the release of
378 mitochondrial Cyt C into the cytoplasm, and eventually activating Caspase-3 (Cao et
379 al., 2016; Hu et al., 2019). Cyt C is an important part of the mitochondrial electron
380 transport chain and is a key step in initiating apoptosis (Phaneuf and Leeuwenburgh,
381 2002). Caspase-9 plays a role in the initial stage of apoptosis and participates in
382 apoptosis mediated by the mitochondrial pathway (Cao, et al., 2016; Hu, et al., 2019).
383 Caspase-3 is the executor caspase of apoptosis. Once the Caspase-3 apoptotic protease
384 activated the pro-Caspase-3, and inactive Caspase-3 will be lysed and activated as
385 cleave Caspase-3 to execute apoptosis (Larsen et al., 2010). MMP is a vital marker of
386 intrinsic apoptosis (Kalaivani et al., 2014; Pavon et al., 2019). Our results showed that
387 p53, Bax, Cyt C, Bak-1, Caspase-9, Caspase-3 mRNA levels and p53, Bax,
388 Caspase-9/cleaved Caspase-9, Caspase-3/cleaved Caspase-3 protein levels in the 10
389 μM Cd group were dramatically higher than those in the control group. But Bcl-2
390 mRNA and protein levels were the opposite. Bcl-2 expression downregulation can
391 destroy the integrity of mitochondrial outer membrane and enhances the release of
392 Cyt C from mitochondria, leading to the activation of mitochondrial pathway
393 apoptosis. And Cd exposure could decrease MMP and increase cell apoptosis ratio. In
394 the study, among the three doses of 2.5, 5, and 10 μM Cd, we chose the 10 μM Cd
395 dose that induced pyroptosis most significantly to further explore the relationship
396 between pyroptosis and apoptosis. The 10 μM Cd is lower than half of IC50 of Cd.
397 The results of RT-qPCR showed that Caspase-9 and Caspase-3 mRNA levels of 10
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398 μM Cd group were significantly higher than those of the control group, which is
399 consistent with the results of western blot. Western blot is a method to detect the
400 amount of target protein in all cells in the experiment. We tested Caspase-9 and
401 Caspase-3 levels, which were the overall level of all cells in the treatment groups.
402 Moreover, our results showed that the apoptosis ratio of 10 μM Cd group was
403 32.20 % ± 2.12, and the ratio of MMP positive was 30.50 % ± 1.05, which is the
404 upstream of Caspase-9/3, thus the signal of cleaved Caspase-9 and Caspase-3 detected
405 by western blot was not strong, which is expected and consistent with previous results
406 (Jeon et al., 2004; Petit et al., 2004; Lee et al., 2017; Bihaqi et al., 2018). Moreover,
407 Z-YVAD-FMK could dramatically improve the changes of the above apoptosis
408 related indicators induced by Cd. These results suggest that Z-YVAD-FMK can
409 decrease duck renal tubular epithelial cell apoptosis induced by Cd. Our results
410 implied that activation of Caspase-1 is involved in apoptosis induced by Cd. Hence,
411 we conclude that apoptosis is associated with pyroptosis in Cd-induced nephrotoxicity,
412 and inhibiting Caspase-1 dependent pyroptosis might attenuate Cd-induced apoptosis
413 in duck renal tubular epithelial cells
414 Conclusion
415 In brief, Cd exposure could induce duck renal tubular epithelial cell pyroptosis
416 through ROS/NLRP3/Caspase-1 signaling pathway, and inhibiting Caspase-1
417 dependent pyroptosis might attenuate Cd-induced apoptosis.
418 Conflict of Interest Statement
419 The authors declare that there are no conflicts of interest.
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420 Acknowledgments
421 This study was supported by the National Natural Science Foundation of China
422 (No. 31960722, Beijing, P.R. China). All authors thank all members of clinical
423 veterinary medicine laboratory in the College of Animal Science and Technology,
424 Jiangxi Agricultural University, for help in the experimental process.
425
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Highlights

Cadmium (Cd) toxicity was evaluated in duck renal tubular epithelial cells.

Cd could induce pyroptosis via ROS/NLRP3/Caspase-1 pathway.

N-acetyl-L-cysteine (NAC) could inhibit pyroptosis induced by Cd.

Inhibiting Caspase-1-dependent pyroptosis might weaken Cd induced-apoptosis.
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Conflict of Interest Statement
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
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