[Protective effect of tumor necrosis factor receptor-associated factor 6 inhibitor C25-140 on acute kidney injury induced by diquat poisoning in mice].

Abstract

Objective: To investigate the protective effect and mechanism of tumor necrosis factor receptor-associated factor 6 (TRAF6) inhibitor C25-140 on acute kidney injury (AKI) induced by acute diquat (DQ) poisoning in mice.

Methods: A total of 80 SPF grade healthy male C57BL/6 mice were randomly divided into the normal control group, DQ model group, C25-140 intervention group, and C25-140 control group, with 20 mice in each group. The DQ poisoning mouse model was established by using one-time intraperitoneal injection of 1 mL of 40 mg/kg DQ solution. The normal control group and C25-140 control group were injected with an equal amount of pure water into the peritoneal cavity. After 4 hours of model establishment, the C25-140 intervention group and C25-140 control group were given intraperitoneal injection of C25-140 5 mg/kg. The normal control group and DQ model group were given equal amounts of pure water, once a day for 7 consecutive days. After 7 days, the mice were anesthetized, eye blood was collected, and renal tissue was collected after sacrifice. The pathological changes of renal tissue were observed under a light microscope and renal tissue structure and mitochondrial changes were observed under transmission electron microscopy. The levels of serum creatinine (SCr) and blood urea nitrogen (BUN) were measured. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of serum interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α). Western blotting was used to detect the protein expression levels of TRAF6, myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) in renal tissue. Chemical method was used to determine the content of serum malondialdehyde (MDA) and superoxide dismutase (SOD).

Results: During the observation period, there were no abnormal behaviors in the normal control group mice. The DQ model group mice gradually showed symptoms such as mental fatigue, fluffy fur, reduced activity, and low food intake after being exposed to the toxin, and severe cases resulted in death. The above symptoms were alleviated in the C25-140 intervention group compared to the DQ model group. Under light microscopy, HE staining showed infiltration of inflammatory cells, glomerulosclerosis, proximal tubular dilation, and vacuolization in the DQ model group, while the inflammatory response was reduced in the C25-140 intervention group compared to the DQ model group. Under transmission electron microscopy, the DQ model group showed relatively high levels of mitochondrial damage, severe swelling, increased volume, matrix dissolution, ridge fracture and loss. The degree of mitochondrial damage in the C25-140 intervention group was reduced compared to the DQ model group. Compared with the normal control group, the levels of serum SCr, BUN, IL-6, IL-1β, TNF-α, and MDA in the DQ model group were significantly increased, while the serum SOD level was significantly decreased. Compared with the DQ model group, the levels of serum SCr, BUN, IL-6, IL-1β, TNF-α, and MDA in the C25-140 intervention group were significantly reduced [SCr (μmol/L): 59.07±13.11 vs. 83.61±20.13, BUN (mmol/L): 25.83±9.95 vs. 40.78±11.53, IL-6 (ng/L): 40.76±7.03 vs. 83.33±21.83, IL-1β (ng/L): 53.87±7.82 vs. 91.74±12.53, TNF-α (ng/L): 102.52±32.13 vs. 150.92±31.75, MDA (μmol/L): 3.57±1.06 vs. 5.75±1.83], and the serum SOD level was significantly increased (kU/g: 162.52±36.13 vs. 122.72±22.13), and the differences were statistically significant (all P < 0.01). Western blotting results showed that the protein expression levels of TRAF6, NF-κB, and MyD88 in the renal tissue of DQ model group mice were significantly higher than those in the normal control group. The expression levels of the above-mentioned proteins in the C25-140 intervention group of mice were significantly lower than those in the DQ model group (TRAF6/β-actin: 1.05±0.36 vs. 1.74±0.80, NF-κB/β-actin: 0.57±0.07 vs. 1.03±0.75, MyD88/β-actin: 0.58±0.07 vs. 1.03±0.33, all P < 0.05).

Conclusions: TRAF6 inhibitor C25-140 can alleviate AKI induced by DQ poisoning in mice by regulating the Toll-like receptor 4 (TLR4)/TRAF6/NF-κB signaling pathway and downregulating the levels of inflammatory cytokines IL-1β, IL-6, and TNF-α.