Eriocitrin attenuates ischemia reperfusion-induced oxidative stress and inflammation in rats with acute kidney injury by regulating the dual-specificity phosphatase 14 (DUSP14)-mediated Nrf2 and nuclear factor-κB (NF-κB) pathways

Abstract

Background: Ischemia reperfusion (IR)-induced acute kidney injury (AKI) is accompanied by increased inflammatory response and oxidative stress. Eriocitrin is a flavonoid that is mainly derived from lemon or citrate juice. It exhibits various pharmacological effects and is known to have antioxidant and anti-steatotic benefits. However, research on the effect of eriocitrin against IR-induced oxidative stress and inflammation in AKI is limited.

Methods: In this study, an OGD/R of HK-2 cell in vitro and rat model of AKI in vivo were constructed. Then the cell or rats were treated with eriocitrin at different doses (60, 30, 10 mg/kg). The levels of apoptotic were detected by flow cytometry. Inflammatory and oxidative stress factors in supernatant in vitro and tissue in vivo. Meanwhile, Western blot was used to detect the change of dual-specificity phosphatase 14 (DUSP14), Nrf2 and nuclear factor-κB (NF-κB).

Results: Eriocitrin attenuated apoptosis of the human renal tubular epithelial cell line HK-2 mediated by oxygen glucose deprivation/reperfusion via the repression of inflammation and oxidative stress in a dose-dependent manner. Eriocitrin also enhanced the levels of dual-specificity phosphatase 14 (DUSP14) and Nrf2, and decreased NF-κB phosphorylation. Furthermore, the in vivo experiments indicated that eriocitrin dose-dependently alleviated IR-induced AKI and apoptosis in rats. By elevating DUSP14, eriocitrin promoted the expression of Nrf2 and inactivated NF-κB, thereby downregulating inflammation and oxidative stress. Moreover, inhibiting DUSP14 expression with protein tyrosine phosphatase (PTP) inhibitor IV reversed the kidney-protective effects of Eriocitrin.

Conclusions: Eriocitrin protected IR-induced AKI by attenuating oxidative stress and inflammation via elevating DUSP14, thereby providing a theoretical basis for the treatment of IR-induced AKI.

Keywords: Acute kidney injury (AKI); dual-specificity phosphatase 14 (DUSP14); eriocitrin; inflammation; oxidative stress.

Figure 1

Eriocitrin reduced OGD/R-induced apoptosis in HK-2 cells. HK-2 cells were subjected to OGD/R and treated with different doses of eriocitrin (1–4 µM). (A) CCK-8 assay was employed to detect HK-2 cells viability; (B,C) flow cytometry was performed to determine HK-2 cell apoptosis. (D) Apoptosis-related proteins including caspase-3, Bax, and Bcl2 were examined by western blot. **P<0.01, ***P<0.001 vs. Con group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. OGD/R group. N=3. OGD/R, oxygen glucose deprivation/reperfusion; IR, ischemia reperfusion.

Figure 2

Eriocitrin notably attenuated OGD/R-induced inflammatory response and oxidative stress in HK-2 cells. HK-2 cells were subjected to OGD/R and treated with different doses of eriocitrin (1–4 µM). (A,B) RT-PCR (A) and ELISA (B) were performed to determine the expression levels of IL-1β, TNF-α, and IL-6. (C) ELISA was employed to detect the expression of MDA, SOD, and GSH-PX in each group. ***P<0.001 vs. Con group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. OGD/R group. N=3. OGD/R, oxygen glucose deprivation/reperfusion; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin-6; MDA, malondialdehyde; SOD, superoxide dismutase; RT-PCR, real-time polymerase chain reaction.

Figure 3

Eriocitrin modulated the Nrf2 and NF-κB pathway and upregulated DUSP14. HK-2 cells were subjected to OGD/R and treated with different doses of eriocitrin (1–4 µM). (A) RT-PCR was carried out to test DUSP14 expression at the mRNA level; (B) Cellular immunofluorescence was conducted to detect DUSP14 (marked by red color) in HK-2 cells. (C) Western blot was performed to examine the protein levels of DUSP14 Nrf2 and NF-κB. ***P<0.001 vs. Con group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. N=3. OGD/R group. DUSP14, dual-specificity phosphatase 14; OGD/R, oxygen glucose deprivation/reperfusion; RT-PCR, real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; NF-κB, nuclear factor-κB.

Figure 4

Inhibiting DUSP14 reversed the protective effect of eriocitrin on HK-2 cells. HK-2 cells were subjected to OGD/R and treated with different doses of eriocitrin (4 µM) and/or PTP inhibitor IV (50 Μm). (A,B). CCK-8 and flow cytometry were used to test the viability (A) and apoptosis (B) of HK-2 cells. (C,D) RT-PCR (C) and ELISA (D) were performed to test the effect of PTP inhibitor IV on the expression of IL-1β, TNF-α, and IL-6. D ELISA was carried out to examine changes in the levels of MDA, SOD, and GSH-PX. (E,F) Changes in DUSP14, Nrf2, and NF-κB were compared using western blot. (*P<0.05, **P<0.01, ***P<0.001 vs. OGD/R + eriocitrin 4 µm/mL). N=3. OGD/R, oxygen glucose deprivation/reperfusion; DUSP14, dual-specificity phosphatase 14; IR, ischemia reperfusion; PTP, protein tyrosine phosphatase; RT-PCR, real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 5

Eriocitrin reduced IR-induced apoptosis and kidney injury. An IR-induced kidney injury model was established using male SD rats. The rats were treated with eriocitrin at a high dose (60 mg/kg), medium dose (30 mg/kg), or low dose (15 mg/kg). (A) ELISA was used to detect the levels of BUN and creatinine in the serum. (B) HE staining was employed to detect glomerular and tubule damage in the different groups. (C) Immunohistochemical staining was performed to determine cell apoptosis (marked by caspase-3) in each group, the scale bar was 50 µM. (D) Apoptosis-related proteins including caspase-3, Bax, and Bcl2 were detected by western blot. *P<0.05, **P<0.01, ***P<0.001 vs. sham group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. N=10. IR group. IR, ischemia reperfusion; SD, Sprague-Dawley; ELISA, enzyme-linked immunosorbent assay; RT-PCR, real-time polymerase chain reaction.

Figure 6

Eriocitrin attenuated inflammation and oxidative stress IR-mediated renal tissue. An IR-induced kidney injury model was established using male SD rats. The rats were treated with eriocitrin at a high dose (60 mg/kg), medium dose (30 mg/kg), or low dose (15 mg/kg). (A,B) RT-PCR (A) and ELISA (B) were employed to detect the expressive changes of IL-1β, TNF-α, and IL-6 in rat kidney tissues. (C) ELISA was carried out to compare the expression levels of MDA, SOD, and GSH-PX in each group. ***P<0.001 vs. sham group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. IR group. N=10. IR, ischemia reperfusion; RT-PCR, real-time polymerase chain reaction; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin-6; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 7

Eriocitrin modulated the Nrf2 and NF-κB pathway by upregulating DUSP14. An IR-induced kidney injury model was established using male SD rats. The rats were treated with eriocitrin at a high dose (60 mg/kg), medium dose (30 mg/kg), or low dose (15 mg/kg). (A) RT-PCR was used to test the DUSP14 mRNA levels in the kidney tissues. (B) IHC was performed to detect DUSP14 expression, the scale bar was 25 µM. (C) Western blot was carried out to examine the expressions of DUSP14, Nrf2, and NF-κB in the kidney tissues. **P<0.01, ***P<0.001 vs. sham group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 vs. IR group. N=10. DUSP14, dual-specificity phosphatase 14; IR, ischemia reperfusion; NF-κB, nuclear factor-κB; RT-PCR, real-time polymerase chain reaction.

Figure 8

Inhibiting DUSP14 reversed the renal protective effects of eriocitrin. An IR-induced kidney injury model was established using male SD rats. The rats were treated with eriocitrin (60 mg/kg) and/or PTP inhibitor IV (5 mg/kg). (A) ELISA was employed to detect the levels of BUN and creatinine in rat serum. (B,C) RT-PCR (B) and ELISA (C) were employed to examine IL-1β, TNF-α and IL-6 expression. (D) ELISA was carried out to detect the levels of MDA, SOD and GSH-PX in serum. (E) IHC was performed to detect DUSP14 expression, the scale bar was 25 µM. (F) Western blot was used to explore changes in DUSP14, Nrf2, and NF-κB expression. *P<0.05, **P<0.01, ***P<0.001 vs. I/R + eriocitrin 60 mg/kg group. N=10. DUSP14, dual-specificity phosphatase 14; IR, ischemia reperfusion; SD, Sprague-Dawley; PTP, protein tyrosine phosphatase; ELISA, enzyme-linked immunosorbent assay; RT-PCR, real-time polymerase chain reaction; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; MDA, malondialdehyde; SOD, superoxide dismutase; NF-κB, nuclear factor-κB.