Puerarin attenuates cisplatin-induced rat nephrotoxicity: The involvement of TLR4/NF-κB signaling pathway

Abstract

Puerarin was a major isoflavonoid derived from the Chinese medical herb radix puerariae (Gegen). In present study effect of puerarin on cisplatin nephrotoxicity was evaluated. Rat model of nephrotoxicity was established by a single intraperitoneal injection of cisplatin (7mg/kg). Puerarin was administrated through caudal vein injection once per day at the dose of 10mg/kg, 30mg/kg and 50mg/kg. Biochemical assays showed that after cisplatin treatment the serum urea and creatinine increased significantly compared with control (P<0.05). Cisplatin treatment significantly increased xanthine oxidase (XO) activity and malondialdehyde (MDA) formation, and significantly decreased the levels and /or activities of enzymatic and non-enzymatic antioxidants (GSH, GPx, GST, GR, SOD, CAT), in the kidney tissues. Renal levels of TNF-α and IL-6, two important inflammatory cytokines, were also upregulated by cisplatin. Histopathological examination indicated that cisplatin treatment resulted in severe necrosis and degeneration, hyaline casts in the tubules, intertubular hemorrhage, congestion and swelling in glomerulus and leukocytes infiltration in the kidney tissues. Western blot results demonstrated that cisplatin increased TLR4 and NF-κB protein expression in the kidney tissues. However, all these changes induced by cisplatin were significantly attenuated by puerarin treatment in dose-dependent manner, which indicated the renal protective effect of puerarin. Cell culture experiments illustrated that puerarin alone treatment concentration-dependently inhibited COLO205 and HeLa tumor cell growth and dose-dependently promoted the antitumor activity of cisplatin in COLO205 and HeLa tumor cells. The promotion effects might be attributed to suppression of cisplatin-increased NF-κB p65 expression by puerarin. Taken together, findings in this study suggested that puerarin exhibited renal protection against cisplatin nephrotoxicity via inhibiting TLR4/NF-κB signaling, with no inhibition but promotion effect on the antitumor activity of cisplatin. Puerarin might be a promising adjuvant agent for cisplatin chemotherapy.

Fig 1

Effects of puerarin on changes of (A) malondialdehyde (MDA) levels and (B) glutathione (GSH) content in the kidney tissues of cisplatin-treated rats. ***P<0.05 versus control group; ^###P<0.05 versus cisplatin alone treated group.

Fig 2. Effects of puerarin on changes of oxidative biomarkers in kidney tissues of cisplatin-treated rats.

(A) glutathione reductase (GR) activities; (B) glutathione peroxidase (GPx) activities; (C) glutathione-S-transferase (GST) activities; (D) superoxide dismutase (SOD)activities; (E) catalase (CAT) activities; (F) xanthine oxidase (XO) activities. ***P<0.05 versus control group; ^###P<0.05 versus cisplatin alone treated group.

Fig 3. Photomicrographs of rat kidney sections stained with hematoxylin and eosin (H.E) (200×).

(A) Kidney sections from control group showed normal morphological view; (B) Kidney tissues section from puerarin alone treated rats (50mg/kg) showed normal morphological view. (C) Kidney tissue section from cisplatin alone treated rats (7 mg/kg) showed severe tubular degeneration and necrosis, hyaline casts in the tubules, intertubular hemorrhage, congestion and swelling in glomerulus; (D-F) Sections from puerarin plus puerarin treated rats. The respective dose of puerarin was (D) 10mg/kg, (E) 30mg/kg and (F) 50 mg/kg. Kidney sections (F) from cisplatin plus 50mg/kg puerarin treated rats showed predominantly normal renal histology with occasional degenerative changes when compared with cisplatin alone treated rats. Black arrow head indicates necrosis and degeneration; Black arrow indicates intertubular hemorrhage; White arrow indicates leukocytes infiltration; Two asterisks indicate hyaline casts in the tubules. Three asterisks indicate congestion and swelling in glomerulus.

Fig 4. Effects of puerarin on inflammatory cytokines production in kidney tissues of cisplatin treated rats.

(A) Tumor necrosis factor-α (TNF-α); (B) Interleukin-6 (IL-6). ***P<0.05 versus control group; ^###P<0.05 versus cisplatin alone treated group.

Fig 5. Expression of TLR4 and NF-κB p65 proteins in kidney tissues in each groups.

(A) Representative Western blot picture, showing the expression levels of TLR4 and NF-κB p65 proteins. β-actin was used as an internal control. (B) Changes in the expression level of TLR4 protein. (C) Changes in the expression level of NF-κB p65 protein. Data are presented as mean ± SD (n = 4). ***P<0.05 versus control group; ^###P<0.05 versus cisplatin alone treated group.

Fig 6. Influence of puerarin on the inhibitory activity of cisplatin in human COLO205 and HeLa cancer cells.

***P<0.05.

Fig 7

Western blot analysis about the effects of puerarin on NF-κB signaling pathway in COLO205 colon cancer cells (A) and HeLa cervical cancer cells (B).