Artemisia indica Willd. Extract Regulate NLRP3 Inflammasome and ENaC Trafficking in Angiotensin II-Stimulated Renal Tubular Cells

Figures

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  Figure 1. Cytoprotective effects of AAE against Ang II-induced cell damage. (a) Quantification of NRK52E cells treated with various concentrations of AAE (0, 5, 10, 20, 50, and 100 μg/mL) for 24 h. (b) Quantification of NRK52E cells involved various concentrations of Ang II (0, 1, 2, and 10 μΜ) for 24 h. (c) Quantification of NRK52E cells involved various concentrations of ICAC (0, 1, 5, 10, 20 and 50 μΜ) for 24 h. (d) NRK52E cells pretreated with AAE (10 and 20 μg/mL) and ICAC (8.24 µM) for 1 h, then a combination of Ang II (1 μΜ). Cell viability was assessed using propidium iodide (PI) staining and analyzed with a Muse™ Cell Analyzer. (e) The ROS production was assessed using an oxidative stress detection kit and analyzed by flow cytometry. The quantitative data were presented as the mean ± SD, and each dot represents an independent experimental replicate. ## p < 0.01 vs. control; ** p < 0.01 vs. Ang II, analyzed by one-way ANOVA with Tukey’s post hoc test.

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  Figure 2. Protective effects of AAE and ICAC against Ang II-induced mitochondrial dysfunction and inflammation. (a) Mitochondrial membrane potential was evaluated using the JC-1 assay and analyzed with a Muse™ Cell Analyzer. The x-axis represents mitochondrial membrane potential (MitoPotential dye), while the y-axis represents cell viability (7-AAD staining). (b) Quantitative analysis of the ratio of total depolarization intensity. (c) The inflammatory cytokines, including IL-6, IL-18 (d), and IL-1β (e) were detected by ELISA. The quantitative data were shown as mean ± SD, and each dot represents an independent experimental replicate (n ≥ 3). ## p < 0.01 vs. control; * p < 0.05 and ** p < 0.01 vs. Ang II, analyzed by one-way ANOVA with Tukey’s post hoc test.

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  Figure 3. AAE inhibits Ang II-induced PKC/NF-κB activation and NLRP3 inflammasome signaling. Protein expression levels of AT1R, p-PKCα, PKCα, and NF-κB (a); NLRP3, ASC, and caspase-1 (b); and GSDMD and IL-1β (c) were determined using Western blot analysis. β-actin served as an internal control. The quantitative data were shown as mean ± SD (n ≥ 3), derived from at least three independent biological replicates. # p < 0.05 and ## p < 0.01 vs. control; * p < 0.05 and ** p < 0.01 vs. Ang II, analyzed by one-way ANOVA with Tukey’s post hoc test.

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  Figure 4. Ang II modulation of Na-K-ATPase and SGK1 in NRK52E cells. (a) Quantification of Na-K-ATPase activity. The protein levels of SGK1, NEDD4-2, and α-ENaC (b) were analyzed by Western blotting. β-actin served as an internal control. The quantitative data were shown as mean ± SD, and each dot represents an independent experimental replicate (n ≥ 3). ## p < 0.01 vs. control; * p < 0.05 and ** p < 0.01 vs. Ang II, analyzed by one-way ANOVA with Tukey’s post hoc test.

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  Figure 5. Regulation of AAE treatment on α-ENaC expression and ubiquitination in NRK52E cells. (a) Image of immunocytochemistry staining with α-ENaC and quantitation. Scale bar, 50 μm. (b) The protein levels of ubiquitin and α-ENaC were analyzed by IP. The cell extracts were IP with α-ENaC. The quantitative data were shown as mean ± SD, and each dot represents an independent experimental replicate (n ≥ 3). ## p < 0.01 vs. control; * p < 0.05 and ** p < 0.01 vs. Ang II, analyzed by one-way ANOVA with Tukey’s post hoc test.

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  Figure 6. Molecular docking analysis of ICAC with α-ENaC. Two-dimensional molecular docking images illustrate the predicted binding mode and structural interactions of ICAC within the extracellular domain of α-ENaC, as obtained using Discovery Studio. The interaction map depicts hydrogen bonds, π-related interactions, and van der Waals contacts between ICAC and surrounding amino acid residues, highlighting a putative ligand–protein interaction interface. Colors and dashed lines indicate interaction types: pale green, van der Waals; green, conventional hydrogen bonds; light green, carbon hydrogen bonds; orange, π–anion interactions; pink, π–π T-shaped interactions; and light pink, π–alkyl interactions.

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  Figure 7. Proposed mechanism by which Artemisia indica Willd. aqueous extract (AAE) and isochlorogenic acid C (ICAC) attenuate Ang II-induced renal epithelial cell injury. Angiotensin II (Ang II) activates the angiotensin II type 1 receptor (AT1R), leading to protein kinase C (PKC) activation and increased reactive oxygen species (ROS) production, which subsequently activates nuclear factor kappa B (NF-κB) and promotes the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In parallel, Ang II induces NLRP3 inflammasome activation, resulting in caspase-1 activation, gasdermin D (GSDMD) cleavage, and the release of interleukin-1β (IL-1β) and interleukin-18 (IL-18), leading to pyroptosis. Ang II also disrupts sodium homeostasis by enhancing epithelial sodium channel (ENaC) activity and Na+/K+-ATPase function. Serum/glucocorticoid-regulated kinase 1 (SGK1) inhibits neural precursor downregulated protein 4-2 (Nedd4-2), thereby reducing ENaC ubiquitination and promoting sodium reabsorption. AAE and ICAC attenuate these pathological processes by suppressing AT1R/PKC/NF-κB signaling, reducing ROS production, inhibiting NLRP3 inflammasome…