Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (3): 549-558.doi: 10.3864/j.issn.0578-1752.2023.03.012

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Inhibitory Effect of N-acetylcysteine on Bisphenol A-Induced Apoptosis and Inflammatory Response in Porcine Kidney Cells

TAO WenJing(), ZHANG ZiTing(), LIU Yuan, SONG Dan(), LI XiangChen   

  1. College of Animal Science and Technology/College of Veterinary Medicine, Zhejiang A&F University/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Hangzhou 311300
  • Received:2021-11-11 Accepted:2022-05-12 Online:2023-02-01 Published:2023-02-14
  • Contact: SONG Dan E-mail:2822780147@qq.com;3106286165@qq.com;songdan2020@zafu.edu.cn

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Abstract:

【Background】 Bisphenol A (BPA) is widely used in the industrial manufacturing of plastic materials, which seeps out from plastic products and exposes to various environmental media such as food, water, soil, and air, resulting in long-term exposure of animals. It is passed to offspring through the placenta and breast milk, interfering with animal growth and development and adversely affecting animal growth performance and production efficiency. N-acetylcysteine (NAC), as a recognized potent antioxidant, can regulate various pathophysiological processes, such as oxidative stress, apoptosis, and inflammation. However, the regulatory role of NAC on BPA-induced porcine kidney cell injury remains unclear. 【Objective】This study aimed to explore the potential role of the antioxidant NAC on BPA-induced apoptosis and inflammatory responses in PK15 cells.【Method】 PK15 cells were selected as experimental materials, and the activities of catalase (CAT), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were measured by the corresponding antioxidant detection kits. PK15 cells were treated with different concentrations of NAC (0, 2, 5, 10 mmol·L-1) and then co-treated with BPA, and then the cell viability was detected by CCK-8 to select the optimal concentration of NAC. The expression of apoptosis-related genes (BAX, BCL-2 and Caspase3) and inflammatory genes (IL-8, IL-6, IL-1β and TNF-α) as well as protein expression were detected by real-time quantitative PCR (qRT-PCR) and western blotting. The number of apoptotic cells and nuclear factor kappa B (NF-κB) nuclear translocation were detected by immunofluorescence staining. 【Result】 The results showed that BPA significantly reduced the activities of CAT, T-SOD and GSH-Px in PK15 cells, compared with the control group (P<0.05). CCK-8 results showed that BPA significantly decreased PK15 cell viability in contrast to control group, whereas the different concentrations of NAC significantly promoted cell viability, and 5, 10 mmol·L-1 NAC pretreatment significantly promoted cell viability when compared with BPA alone. qRT-PCR and western blotting showed that BPA treatment significantly increased BAX and Caspase3 mRNA and protein expression, and decreased the BCL-2 mRNA and protein expression, whereas NAC pretreatment could reduce the increased BAX and Caspase3 expression and increase the decreased BCL-2 expression induced by BPA. Hoechst33258 fluorescence staining indicated that the cells treated with BPA showed strong blue fluorescence staining and obvious nuclear shrinkage, whereas NAC pretreated cell showed weak blue fluorescence. BPA treatment significantly increased the relative mRNA expression of inflammation-related factors (IL-8 and IL-6) (P<0.05), whereas NAC pretreatment inhibited BPA-induced increased inflammation-related factors (IL-8, IL-6 and IL-1β mRNA) relative expression. Immunofluorescence analysis of NF-κB nuclear translocation showed that NF-κB was mainly distributed in cytoplasm in the control group, whereas NF-κB was mainly distributed in the nucleus after BPA treatment and NAC pretreatment reduced nuclear NF-κB expression. 【Conclusion】 NAC significantly increased PK15 cell viability and inhibited PK15 cell apoptosis and inflammatory response induced by BPA.

Key words: Bisphenol A, N-acetylcysteine, apoptosis, inflammatory response, PK15 cell, oxidative stress

Table 1

Real-time quantitative PCR primer"

引物名称 Primer name 引物序列
Primer sequence (5′-3′)		 产物大小
Size (bp)
BAX F: TTTGCTTCAGGGTTTCATCC
R: GACACTCGCTCAACTTCTTGG		 113
BCL-2 F: GCGACTTTGCCGAGATGT
R: CACAATCCTCCCCCAGTTC		 116
Caspase3 F: TTGGACTGTGGGATTGAGAC
R: TTCGCCAGGAATAGTAACCAG		 121
IL-6 F: CCCTGAGGCAAAAGGGAAAGA
R: AGGAAATCCTCAAGGCTGCG		 148
IL-8 F: AGAGTGGACCCCACTGTGAA
R: TTGTTGTTGCTTCTCAGTTCTCT		 137
IL-1β F: GGAAGTGATGGCTAACTACGG
R: CTGGATGCTCCCATTTCTCA		 124
TNF-α F: GCCCAAGGACTCAGATCATCG
R: ATTGGCATACCCACTCTGCC		 104
GAPDH F: ACCCAGAAGACTGTGGATGG
R: TTGAGCTCAGGGATGACCTT		 207

Fig. 1

Effects of BPA on the activity of antioxidant enzymes in PK15 cells"

Fig. 2

Cell viability was detected by CCK-8 assay A: Effects of different concentrations of NAC on the viability of PK15 cells; B: Effects of NAC on PK15 cells cell viability induced by BPA"

Fig. 3

Effects of NAC on BPA-induced apoptosis gene expression in PK15 cells"

Fig. 4

Effects of NAC on BPA-induced apoptosis protein expression in PK15 cells A: Western blotting results of BAX, BCL-2 and cleaved-Caspase3 proteins; B: Statistical results of BAX, BCL-2 and cleaved-Caspase 3 protein expression"

Fig. 5

Cell apoptosis detection by using Hoechst33258 fluorescent staining(40×)"

Fig. 6

Effects of NAC on BPA-induced inflammation related gene expression in PK15 cells"

Fig. 7

Effects of NAC on BPA-induced nuclear translocation of NF-κB in PK15 cells (60×)"

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