Tea Polyphenols Mitigate TBBPA-Induced Renal Injury Through Modulation of ROS-PI3K/AKT-NF-κB Signalling in Carp (Cyprinus carpio)

Abstract

Tetrabromobisphenol A (TBBPA), a widely utilised brominated flame retardant, demonstrates toxicological effects in aquatic organisms. Tea polyphenols (TPs), natural compounds found in tea leaves, exhibit both antioxidant and anti-inflammatory activities. The kidney is one of the major metabolic organs in common carp and serves as a target organ for toxic substances. This study evaluated the therapeutic potential of TPs in mitigating TBBPA-induced nephrotoxicity in common carp. Common carp were exposed to 0.5 mg/L TBBPA in water and/or fed a diet supplemented with 1 g/kg TPs for 14 days. In vitro, primary renal cells were treated with 60 μM TBBPA and/or 2.5 μg/L TPs for 24 h. Methods included histopathology, TUNEL assay for apoptosis, ROS detection, and molecular analyses. Antioxidant enzymes (SOD, CAT) and inflammatory cytokines (IL-1β, IL-6, TNF-α) were quantified using ELISA kits. Results showed that TBBPA induced oxidative stress, and activated the ROS-PI3K/AKT-NF-κB pathway, thereby resulting in inflammatory responses. TBBPA upregulated apoptosis-related genes (Caspase-3, Bax, and Bcl-2) and induced apoptosis. TBBPA upregulated the expression of RIPK3/MLKL, thereby exacerbating necroptosis. TPs intervention significantly mitigated these effects by reducing ROS, suppressing NF-κB activation, and restoring antioxidant enzyme activities (SOD, CAT). Moreover, TPs attenuated apoptosis and necrosis in the carp kidney, thereby enhancing the survival ability and immunity of common carp.

Keywords: aquaculture feed additives; inflammatory injury; oxidative stress; tea polyphenols.

Figure 1

Effects of TBBPA exposure and TPs intervention on the morphology of carp kidney. (A) HE staining of kidney tissue in the Con group. (B) HE staining of kidney tissue in the TBBPA group. (C) HE staining of kidney tissue in the TBBPA + TPs group. (D) HE staining of kidney tissue in the TPs group. The black arrows in the figure indicate congestion of the renal tissue, the yellow arrows indicate inflammatory cell infiltration, and the green arrows indicate oedema of the renal tubules. The left figure is magnified 20×, and the right figure is magnified 40×. Each group included three independent biological replicates (n = 3). The data are expressed as mean ± SEM and analysed using one-way analysis of variance (ANOVA).

Figure 2

Effects of TBBPA exposure and TPs intervention on renal oxidative stress of common carp. (A) Changes in ROS levels in the kidney. (B) MDA content was detected in kidney tissue. (C) SOD content was detected in the kidney. (D) CAT content was detected in the kidney. (E–H) Changes in the content of ROS in cells, magnification 10×. (I) Detection of MDA content in primary renal cells. (J) Detection of SOD content in primary renal cells. (K) Detection of CAT content in primary renal cells. Each group consisted of three independent biological replicates (n = 3). Data are expressed as mean ± SEM. Analysis was performed using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3

Effects of TBBPA exposure and TPs intervention on the levels of inflammatory factors in renal tissue. (A–C) The mRNA levels of inflammatory factors in renal tissue. (D–F) Protein levels of inflammatory factors in renal tissue. (G–I) Expression levels of mRNA of inflammatory factors in primary renal cells. (J–L) Expression levels of the protein of inflammatory factors in primary renal cells. Each group included three independent biological replicates (n = 3). The data are expressed as mean ± SEM. Analysis was performed using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4

Effects of TBBPA exposure and TPs intervention on the expression of the PI3K/AKT-NF-κB pathway. (A) The mRNA levels of key proteins in the PI3K/AKT/NF-κB pathway in renal tissue. (B,C) Expression levels of key proteins in the PI3K/AKT/NF-κB pathway in renal tissue. (D) The mRNA levels of key proteins in the PI3K/AKT/NF-κB pathway in primary renal cells. (E,F) Levels of key proteins in the PI3K/AKT/NF-κB pathway in primary renal cells. Each group included three independent biological replicates (n = 3). Data are expressed as mean ± SEM. Analysis was performed using one-way analysis of variance (ANOVA). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5

Effects of TBBPA exposure and TPs intervention on renal apoptosis of common carp. (A) TUNEL results of kidney tissue; green represents apoptosis. (B) Tunnel fluorescence ratio. (C) The mRNA expression of Caspase-3, BAX, and BCL-2 genes in the kidney. (D,E) Protein expression levels of Caspase-3, BAX, and BCL-2 in the kidney. (F) The mRNA expression of Caspase-3, BAX, and BCL-2 in primary renal cells. (G,H) Protein expression of the Caspase-3 gene and BAX and BCL-2 in primary renal cells. Each group included three independent biological replicates (n = 3). Data are expressed as mean ± SEM. Analysis was performed using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 6

Effects of TBBPA exposure and TPs intervention on renal necrosis of carp. (A) The mRNA expression of RIPK1, RIPK3, and MLKL genes in the kidney. (B,C) Expression levels of RIPK1, RIPK3, and MLKL proteins in the kidney. (D) AO/EB results. Orange represents apoptotic cells, and red represents necrotic cells. Magnification 10×. (E) The mRNA expression of RIPK1, RIPK3, and MLKL genes in renal primary cells. (F,G) Protein expression levels of RIPK1, RIPK3, and MLKL in primary renal cells. Each group included three independent biological replicates (n = 3). Data are expressed as mean ± SEM. Analysis was performed using one-way analysis of variance (ANOVA). **, p < 0.01; ****, p < 0.0001.