TLR4/Inflammasomes Cross-Talk and Pyroptosis Contribute to N-Acetyl Cysteine and Chlorogenic Acid Protection against Cisplatin-Induced Nephrotoxicity

Abstract

Background: Cisplatin (Cp) is an antineoplastic agent with a dose-limiting nephrotoxicity. Cp-induced nephrotoxicity is characterized by the interplay of oxidative stress, inflammation, and apoptosis. Toll-4 receptors (TLR4) and NLPR3 inflammasome are pattern-recognition receptors responsible for activating inflammatory responses and are assigned to play a significant role with gasdermin (GSDMD) in acute kidney injuries. N-acetylcysteine (NAC) and chlorogenic acid (CGA) have documented nephroprotective effects by suppressing oxidative and inflammatory pathways. Therefore, the current study aimed to investigate the contribution of the upregulation of TLR4/inflammasomes/gasdermin signaling to Cp-induced nephrotoxicity and their modulation by NAC or CGA.

Methods: A single injection of Cp (7 mg/kg, i.p.) was given to Wistar rats. Rats received either NAC (250 mg/kg, p.o.) and/or CGA (20 mg/kg, p.o.) one week before and after the Cp injection.

Results: Cp-induced acute nephrotoxicity was evident by the increased blood urea nitrogen and serum creatinine and histopathological insults. Additionally, nephrotoxicity was associated with increased lipid peroxidation, reduced antioxidants, and elevated levels of inflammatory markers (NF-κB and TNF-α) in the kidney tissues. Moreover, Cp upregulated both TLR4/NLPR3/interleukin-1beta (IL-1β) and caspase-1/GSDMD-signaling pathways, accompanied by an increased Bax/BCL-2 ratio, indicating an inflammatory-mediated apoptosis. Both NAC and/or CGA significantly corrected these changes.

Conclusions: This study emphasizes that inhibition of TLR4/NLPR3/IL-1β/GSDMD might be a novel mechanism of the nephroprotective effects of NAC or CGA against Cp-induced nephrotoxicity in rats.

Keywords: N-acetylcysteine; NLPR3; TLR4; chlorogenic acid; cisplatin; gasdermin; inflammasomes; nephrotoxicity.

Figure 1

Histological Photomicrographs of the Kidney Specimens from Different Treatment Groups Stained with H and E. (A1,A2): Control group shows normal histological structure of the kidney glomeruli and renal tubules with the outer medullary region (A2); (B1,B2): Cisplatin-injected group shows severe tubular degenerative changes with cystic dilations and necrotic tubular segments of proximal and distal tubules (red star) and necrotic epithelial cells (red arrow) with mild interstitial inflammatory cells infiltrates; (C1,C2): NAC group (250 mg/kg/day p.o.) shows almost intact renal parenchyma and tubular epithelium (black arrow) with few focal records of degenerated tubular cells and pyknotic nuclei (red arrow); (D1,D2): CGA group (20 mg/kg/day p.o.) appears almost intact renal parenchyma and tubular epithelium (black arrow) with minimal records of dilated (red star) or degenerated tubular segments (red arrow), and minimal inflammatory cells infiltrate; (E1,E2): Combined group (NAC and CGA) shows apparent intact renal parenchyma and tubular epithelium (black arrow) with minimal dilatation of a few tubular segments, as well as occasional periglomerular inflammatory cells infiltrates (arrowhead), 400×. NAC: N-acetylcysteine; CGA: Chlorogenic acid.

Figure 2

Effects of NAC and/or CGA on Cisplatin-Mediated Changes in Renal Expressions of NF-kB and TNF-α. Control: normal rats; Cisplatin: Cisplatin (7 mg/kg, IP) injected rats; NAC (250 mg/kg): Cisplatin-injected rats treated with NAC (250 mg/kg/day p.o.) one week before and after cisplatin injection; CGA (20 mg/kg): Cisplatin-injected rats treated with CGA (20 mg/kg/day p.o.) one week before and after cisplatin injection; NAC + CGA: Cisplatin-injected rats treated with NAC (250 mg/kg/day p.o.) and CGA (20 mg/kg/day p.o.) one week before and after cisplatin injection. Values are mean ± SEM (n = 5). ^a: compared to the control group; ^b compared to the cisplatin group. For NF-kB (F = 5.58, df = 4, p = 0.0067) and for TNF-α (F = 5.56, df = 4, p = 0.0025). p values were evaluated using one-way ANOVA followed by Tukey–Kramer as a post-hoc test. NF-kB: Nuclear factor-κB; TNF-α: Tumor necrosis factor alpha; NAC: N-acetylcysteine; CGA: Chlorogenic acid.

Figure 3

Effect of NAC and/or CGA on the TLR4/NLPR3/IL-1β and caspase-1/GSDMD Signaling Pathway in Kidney Tissue of Rats. Representatives immunoblot of protein level of TLR4, NF-κB, NLPR3, caspase-1, IL-1β, and GSDMD in kidney tissues. Sample proteins were immunoblotted with the antibodies’ “upper panels” and after stripping with GAPDH antibody as a loading control “lower panel.” Control: normal rats; Cis: Cisplatin (7 mg/kg, IP) injected rats; Cis + NAC: Cisplatin injected rats treated with NAC (250 mg/kg/day p.o.) one week before and after cisplatin injection; Cis + CGA: Cisplatin-injected rats treated with CGA (20 mg/kg/day p.o.) one week before and after cisplatin injection; Cis + NAC + CGA: Cisplatin-injected rats treated with NAC (250 mg/kg/day p.o.) and CGA (20 mg/kg/day p.o.) one week before and after cisplatin injection. NAC: N-acetylcysteine; CGA: Chlorogenic acid; TLR4: Toll-4 receptors; NF-κB: Nuclear factor kappa B; NLPR3: Inflammasomes; IL-1β: Interleukin-1 beta; GSDMD: Gasdermin D.

Figure 4

Quantitative Results of Immunoblots of TLR4/NF-kB/NLPR3/IL-1β and Caspase1/GSDMD Signaling Pathway in Kidney Tissue of Rats. Densities were quantified using analysis software. (A–F) Quantitative results of the immunoblot of TLR4, NF-κB, NLPR3, caspase-1, IL-1β, and GSDMD. Protein levels are expressed as the ratio of protein/GAPDH. The relative quantities were normalized to the control and expressed as a fold of induction. Data are presented as the mean ± SEM (n = 3). ^a p ≤ 0.05 compared to the control group; ^b p ≤ 0.05 compared to the cisplatin group. p ≤ 0.05 using ANOVA followed by Tukey-Kramer as post-hoc test. Control: normal rats; Cis: Cisplatin-injected (7 mg/kg, i.p.) rats; Cis + NAC: Cisplatin-injected rats treated with NAC (250 mg/kg/day, p.o.) one week before and after cisplatin injection; Cis + CGA: Cisplatin-injected rats treated with CGA (20 mg/kg/day, p.o.) one week before and after cisplatin injection; Cis + NAC + CGA: Cisplatin-injected rats treated with NAC (250 mg/kg/day, p.o.) and CGA (20 mg/kg/day, p.o.) one week before and after cisplatin injection. For TLR4 (F = 238.8, df = 4, p < 0.0001), for NF-kB (F = 226, df = 4, p < 0.0001), for NLPR3 (F = 144, df = 4, p < 0.0001), for caspase-1 (F = 41, df = 4, p < 0.0001), for IL-1β (F = 125.3, df = 4, p < 0.0001), and for GSDMD (F = 50, df = 4, p < 0.0001). p values were evaluated using one-way ANOVA followed by Tukey–Kramer as a post-hoc test. NAC: N-acetylcysteine; CGA: Chlorogenic acid; TLR4: Toll-4 receptors; NF-κB: Nuclear factor kappa B; NLPR3: Inflammasomes; IL-1β: Interleukin-1 beta; GSDMD: Gasdermin D.

Figure 5

Effect of NAC and/or CGA on Cisplatin-Induced upregulation in Apoptotic Markers: (A): Effect of NAC and/or CGA on cisplatin-induced changes in Bax/Bcl-2 ratio, values are mean ± SEM, n = 5. (B): Effect of NAC and/or CGA on cisplatin-induced upregulation of caspase-3 using immunoblot. (C): Quantitative results of immunoblots of caspase-3, values are mean ± SEM, n = 3. Protein levels are expressed as the ratio of protein/GAPDH. The relative quantities were normalized to the control and expressed as a fold of induction (F = 19.84, df = 4, p < 0.0001). Control: normal rats; Cisplatin: Cisplatin-injected rats (7 mg/kg, i.p.); NAC (250 mg/kg): Cisplatin-injected rats treated with NAC (250 mg/kg/day p.o.) one week before and after cisplatin injection; CGA (20 mg/kg): Cisplatin-injected rats treated with CGA (20 mg/kg/day, p.o.) one week before and after cisplatin injection; NAC + CGA: Cisplatin-injected rats treated with NAC (250 mg/kg/day, p.o.) and CGA (20 mg/kg/day, p.o.) one week before and after cisplatin injection. ^a p < 0.05 compared to the control group; ^b p < 0.05 compared to the cisplatin group. p value was evaluated using one-way ANOVA followed by Tukey–Kramer as a post-hoc test. Bax/Bcl2: Bax/Bcl2 ratio; NAC: N-acetylcysteine; CGA: Chlorogenic acid.

Figure 6

Correlation of TLR4 and NLPR3 protein expression. TLR4: Toll-4 receptors, NLPR3: Inflammasomes.

Figure 7

Cisplatin effect is represented as red arrows. TLR4: Toll-4 receptors; NF-κB: Nuclear factor-αB; NLPR3: Inflammasomes; IL-1β, and TNF-α; Tumor necrosis factor-α.