Total Extracts of Abelmoschus manihot L. Attenuates Adriamycin-Induced Renal Tubule Injury via Suppression of ROS-ERK1/2-Mediated NLRP3 Inflammasome Activation

Abstract

Abelmoschus manihot (L.) Medik. (Malvaceae) is a herb used in traditional Chinese medicine to treat some kidney diseases. To date, the detailed mechanisms by which A. manihot improves some kinds of renal disease are not fully understood. In this study, we established Adriamycin-induced NRK-52E cells, the normal rat kidney epithelial cell line, injury, and Sprague-Dawley rats with Adriamycin-induced nephropathy to evaluate the role and mechanisms of total extracts of A. manihot flower (TEA) both in vitro and in vivo. We found that TEA ameliorated Adriamycin-induced cellular morphological changes, cell viability, and apoptosis through the suppression of protein oxidation and ERK1/2 signaling. However, this anti-oxidative stress role of TEA was independent of ROS inhibition. Adriamycin activated ERK1/2 signaling followed by activation of NLRP3 inflammasomes. TEA suppressed NLRP3 inflammasomes via inhibition of ERK1/2 signal transduction; decreased proteinuria and attenuated renal tubule lesions; and inhibited the expression of NLRP3 in tubules in rats with Adriamycin nephropathy. Collectively, TEA protects renal tubular cells against Adriamycin-induced tubule injury via inhibition of ROS-ERK1/2-NLRP3 inflammasomes.

Keywords: Abelmoschus manihot L.; Adriamycin nephropathy; ERK1/2; NLRP3 inflammasome; oxidative stress; renal tubular cell; total extract.

FIGURE 1

Fingerprint analysis of TEA by HPLC. (A) Chromatograms of mixed standards. (B) TEA samples.

FIGURE 2

Adriamycin elicited renal tubular cell injury. (A) Role of Adriamycin (ADR) in morphological changes. NRK-52E cells were treated with different concentrations of ADR (0, 0.5, 1.0, 1.5 μg/mL) for 24 h. Cell morphology was analyzed using phase-contrast microscopy (magnification, ×100). (B) Effects of ADR on cell viability. NRK-52E cells in 96-well plates were exposed to the different concentrations (0, 0.5, 1.0, 1.5 μg/mL of ADR for 24 h. Cell viability was evaluated using a CCK-8 assay. Data are expressed as the percentages of living cells versus the control (Ctrl) (means ± SD, n = 5). ^∗∗P < 0.01 versus Ctrl. (C) The effects of ADR in flow cytometry assay. NRK-52E cells in 6-well plates were treated with different concentrations of ADR (0, 0.5, 1.0, 1.5 μg/mL) for 24 h and the apoptotic cells were evaluated by flow cytometry to detect labeled Annexin V-FITC/PI. Flow cytometry analysis of apoptosis is shown at the bottom. ^∗∗P < 0.01 versus Ctrl.

FIGURE 3

TEA ameliorates ADR-elicited tubular cell injury. (A) Role of TEA in ADR-induced morphological changes. NRK-52E cells were pretreated with TEA (100 μg/mL) for 1 h and challenged with ADR for 24 h. Cell morphology was analyzed using phase-contrast microscopy (magnification, ×100). (B) Effects of TEA on cell viability. NRK-52E cells in 96-well plates were pretreated with TEA (100 μg/mL) for 1 h and challenged with ADR for 24 h. Cell viability was evaluated by the CCK-8 assay. Data are expressed as the percentages of living cells versus Ctrl (means ± SD, n = 5). ^∗∗P < 0.01 versus Ctrl. ##P < 0.01 versus ADR in Ctrl. (C) Apoptosis staining of NRK-52E cells. NRK-52E in 48-well plates were pretreated with TEA (100 μg/mL) for 1 h and challenged with ADR for another 24 h. Apoptotic cells were evaluated by TUNEL and DAPI staining. Data on the right are expressed as the percentages of dead cells compared with the Ctrl (means ± SD, n = 5; ^∗∗P < 0.01 versus Ctrl. ##P < 0.01 versus ADR in Ctrl). (D) The effects of TEA in flow cytometry assay. NRK-52E cells in 6-well plates were pretreated with TEA (100 μg/mL) for 1 h and challenged with ADR for another 24 h. The apoptotic cells were detected by flow cytometry to detect labeled Annexin V-FITC/PI. Flow cytometry analysis of apoptosis is shown on the right. ^∗∗P < 0.01 versus Ctrl; ##P < 0.01 versus ADR in Ctrl.

FIGURE 4

Oxidative stress underlies ADR-induced cell injury. (A) Effects of ADR on superoxide and ROS production. NRK-52E in 96-well plates were incubated with ADR for different periods and loaded with superoxide and ROS detection reagents for 1 h. The cells were analyzed by fluorescence microscopy (magnification, ×400). Quantitative measurements of the fluorescence intensities were conducted using ImageJ software. Data are expressed as the relative intensities against zero point control (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl). (B) Effects of TEA and NAC on superoxide and ROS production triggered by ADR. NRK-52E in 96-well plates were pretreated with TEA (100 μg/mL) or NAC (5 mM) for 1 h, challenged with ADR for 3 h, then loaded with superoxide and ROS detection reagent for another 1 h. The cells were subsequently analyzed by fluorescence microscopy (magnification, ×400). Quantitative measurements of the fluorescence intensities are shown at the bottom (means ± SD, n = 3; ^∗∗ P < 0.01 versus Ctrl; ##P < 0.01 versus ADR in Ctrl). (C) Role of TEA and NAC on oxidative modification of proteins induced by ADR. NRK-52E cells were pretreated with TEA (100 μg/mL) and NAC (5 mM) in 12-well plates for 1 h and incubated with ADR for another 4 h. Thereafter, cellular lysates were analyzed by OxyBlot Protein Oxidation Detection Kit and immunodetection of carbonyl groups. β-actin was used as an internal control. Densitometric analysis of protein oxidation is shown at the bottom (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl; ##P < 0.01 versus ADR in Ctrl). (D) Effects of TEA and NAC on caspase 3 cleavage induced by ADR. NRK-52E cells in 12-well plates were pretreated with TEA (100 μg/mL) and NAC (5 mM) for 1 h and incubated with ADR for 24 h. Cellular lysates were analyzed by western blots targeting caspase 3 and cleaved-caspase 3. Densitometric analysis of cleaved-caspase 3 is shown at the bottom (means ± SD, n = 3; ^∗∗ P < 0.01 versus Ctrl; ## P < 0.01 versus ADR in Ctrl). (E) Effects of TEA and NAC on cell viability. NRK-52E cells in 96-well plates were pretreated with TEA or NAC for 1 h and challenged with ADR for 24 h. Cell viability was evaluated by CCK-8 assay. Data are expressed as percentages of dead cells versus Ctrl (means ± SD, n = 5). p < 0.01 versus Ctrl; ## P < 0.01 versus ADR in Ctrl.

FIGURE 5

TEA regulates ERK1/2 signaling. (A) Induction of p38 and ERK1/2 phosphorylation by ADR. NRK-52E cells in 12-well plates were incubated with ADR for 1, 3, 6, and 12 h Cellular lysates were analyzed by western blot for phosphorylated p38 and ERK1/2. Statistical analyses of phosphorylated ERK1/2 and p38 are shown at the bottom (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl). (B) Effects of TEA and NAC on ERK1/2 and p38 phosphorylation induced by ADR. NRK-52E cells in 12-well plates were pretreated with TEA (100 μg/mL) and NAC (5 mM) for 1 h and incubated with ADR for another 6 h. Lysates were analyzed by western blots targeting phosphorylated p38 and ERK1/2. Statistical analysis of phosphorylated ERK1/2 and p38 are shown at the bottom (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl; ## P < 0.01 versus ADR in Ctrl). (C) Effects of TEA on JNK phosphorylation induced by ADR. NRK-52E cells in 12-well plates were pretreated with TEA (100 μg/mL) and NAC (5 mM) for 1 h and challenged with ADR for another 6 h. Lysates were analyzed by western blot targeting phosphorylated JNK. Statistical analyses of phosphorylated JNKs are shown at the bottom.

FIGURE 6

ADR induces oxidative stress-mediated NLRP3 inflammasome activation. (A) Induction of ERK1/2 phosphorylation and NLRP3 protein levels by ADR. NRK-52E cells in 12-well plates were incubated with ADR for 1, 3, 6, and 9 h, respectively. Statistical analyses of phosphorylated ERK1/2 and NLRP3 are shown at the bottom (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl). (B) Effects of TEA and NAC on NLRP3 induced by ADR. NRK-52E cells in 12-well plates were pretreated with TEA or NAC for 1 h and incubated with ADR for 9 h. Statistical analyses of NLRP3 are shown at the bottom (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl; ##P < 0.01 versus ADR in Ctrl). (C) Effects of TEA and U0126 on cell viability. NRK-52E cells in 96-well plates were pretreated with TEA (100 μg/mL) or U0126 (20 μM) for 1 h and challenged with ADR for 24 h. Cell viability was evaluated by CCK-8 assay. Data are expressed as percentages of living cells versus control (means ± SD, n = 5). ^∗∗P < 0.01 versus control; ## P < 0.01 versus ADR in Ctrl. (D) Effects of NLRP3 siRNA on ADR-induced cell injury. NRK-52E cells were transfected with either NLRP3 siRNA or control siRNA for 24 h. The transfected cells were incubated with ADR for 24 h. Cell viability was evaluated using a CCK-8 assay. Data are expressed as the percentages of living cells compared with the siRNA controls (means ± SD, n = 4; ^∗∗P < 0.01 versus siRNA control; ## P < 0.01 versus ADR in Ctrl).

FIGURE 7

TEA regulates ERK1/2 mediated NLRP3 inflammasome activation. (A,B) Effect of TEA, U0126, INF9, and NLRP3 siRNA on ERK1/2 and NLRP3 inflammasome activation. NRK-52E cells were transfected with either NLRP3 siRNA or control siRNA for 24 h. Thereafter transfected cells were incubated with ADR for 24 h. Wild type NRK-52E cells in 12-well plates were pretreated with TEA (100 μg/mL), U0126 (20 μM), or INF39 (100 μM) for 1 h and challenged with ADR for 24 h. Statistical analysis of targeted proteins is shown at the bottom (means ± SD, n = 3; ^∗∗P < 0.01 versus Ctrl; ##P < 0.01 versus ADR in Ctrl). (C) Apoptosis staining of NRK-52E cells. NRK-52E in 48-well plates were pretreated with TEA (100 μg/mL) and INF39 for 1 h and challenged with ADR for another 24 h. Apoptotic cells were evaluated by TUNEL and DAPI staining. Data at the bottom are expressed as the percentages of dead cells compared with the Ctrl (means ± SD, n = 5; ^∗∗P < 0.01 versus Ctrl. ##P < 0.01 versus ADR in Ctrl).

FIGURE 8

TEA attenuates proteinuria and improves hypoproteinemia in rats with Adriamycin nephropathy. (A) Experimental procedure. The ADR and TEA groups underwent right nephrectomy on day 7 and ADR was injected through the tail vein on days 14 and 21. The rats were sacrificed on day 28. TEA was given by gavage once a day at a dosage of 1.5 g/kg/d in the treatment group. Rats in the model group received 2 mL normal saline by gavage daily from day 14. Comparisons of urinary protein (B), blood albumin (C), and serum creatinine (D) levels among the 3 groups (means ± SD) (^∗P < 0.05 between groups).

FIGURE 9

TEA improves renal pathology in rats with Adriamycin nephropathy. (A) Macroscopic morphology of the kidneys. (B) Hematoxylin and Eosin staining: Renal tubular epithelial cells in the normal group were in alignment and had normal shapes. In the ADR group, the renal tubular epithelial cells were shed, and some of the bare membranes were visible, while some of them had regenerated (arrows). Protein casts are indicted as (Δ). Granular degeneration can be seen in proximal tubular cells. Renal tubular epithelial cells were detached (arrows) in the treatment group, which was at a lower level compared with the ADR group. Masson staining: Capillary vasospasm and buccal segmental adhesion (^∗) were detected in the Adriamycin group. Renal tubular epithelial cells were detached, and bare membranes (arrow) were visible. There was no obvious fibrosis in the renal interstitium. Scale bar: 100 μm. (C) Representative images of sections assayed by immunohistology using NLRP3, Caspase 1, and IL-1β antibody. Scale bar: 100 μm.

FIGURE 10

Diagram of the mechanism by which TEA regulates the NLRP3 inflammasome. Adriamycin induces oxidative stress by increasing the overproduction of ROS. ERK1/2 kinases mediate ROS-triggered activation of the NLRP3 inflammasome. TEA reduces NLPR3 via suppression of ERK1/2 kinases and subsequently inhibits the NLRP3 inflammasome activation induced by Adriamycin. Thus, TEA ameliorates Adriamycin-induced renal tubule injury via suppression of ROS-ERK1/2-mediated NLRP3 priming process.