Salvia miltiorrhiza Bunge (Danshen) and Bioactive Compound Tanshinone IIA Alleviates Cisplatin-Induced Acute Kidney Injury Through Regulating PXR/NF-κB Signaling

Abstract

Objective: The present study aims to provide evidence on the potential protective role of Salvia miltiorrhiza Bunge (Danshen) and its bioactive compound Tanshinone IIA (TanIIA) in AKI and to reveal the specific regulatory function of PXR/NF-κB signaling in AKI-induced renal inflammation. Methods: A network pharmacological analysis was used to study target genes and regulatory networks in the treatment of Salvia miltiorrhiza on AKI. Further experiments with in vivo AKI mouse model and in vitro studies were applied to investigate the renal protective effect of TanIIA in AKI. The mechanisms of TanIIA regulating PXR/NF-κB signaling in renal inflammation were also studied. Results: Network pharmacology had suggested the nuclear receptor family as new therapeutic targets of Salvia miltiorrhiza in AKI treatment. The in vivo studies had demonstrated that TanIIA improved renal function and inflammation by reducing necrosis and promoting the proliferation of tubular epithelial cells. Improved renal arterial perfusion in AKI mice with TanIIA treatment was also recorded by ultrasonography. In vitro studies had shown that TanIIA ameliorated renal inflammation by activating the PXR while inhibiting PXR-mediated NF-κB signaling. The results had suggested a role of PXR activation against AKI-induced renal inflammation. Conclusion: Salvia miltiorrhiza Bunge (Danshen) may protect the kidneys against AKI by regulating nuclear receptors. TanIIA improved cell necrosis proliferation and reduced renal inflammation by upregulating the expression of the PXR and inhibiting NF-κB signaling in a PXR-dependent manner. The PXR may be a potential therapeutic target for AKI treatment.

Keywords: NF-κB; Salvia miltiorrhiza; acute kidney injury; pregnane X receptor; renal inflammation; tanshinone IIA.

FIGURE 1

GO enrichment analysis of potential targets and biological networks involved in Salvia miltiorrhiza treating AKI. (A) Bar chart presenting the percentage of genes involved in different biological functions, and the numbers of related genes in these signaling pathways are intersected. (B) Brief view of the 7 GO pathways; all pathways have a p-value of < 0.01. (C) GO pathways are presented in the pie chart. Detailed information could be found in the supplementary files: Supplementary Table S1: eligible active ingredients; Supplementary Table S2: target proteins involved in the networks; Supplementary Table S3: significant target genes related to the proteins; Supplementary Table S4, Supplementary Figure S1: intersection genes of Salvia miltiorrhiza and AKI; Supplementary Figure S2: the KEGG pathways.

FIGURE 2

TanIIA treatment restores renal function in cisplatin-induced AKI. (A) Body weight (g) before and after cisplatin injection. (B) Serum creatinine at 72 h after cisplatin (20 mg/kg) injection. (C) Bar chart represents the serum level of NGAL. (D,E) mRNA level of NGAL, KIM-1 in the kidney. Data represent as mean ± SEM, *p < 0.05, **p < 0.01 compared with the control group (n = 5). #p < 0.05, ##p < 0.01 compared with AKI group (n = 5). One-way ANOVA followed by Turkey’s test for multiple comparisons was used for three or more groups; CTRL: 0.9% normal saline; TanIIA concentrations = 12.5 and 25 mg/kg/day; abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule 1.

FIGURE 3

TanIIA improves renal function by preventing cell necrosis and promoting proliferation. (A,B) Representative images and relative quantitative data of HE, PAS, and Masson’s trichrome staining in mouse kidney sections. The AKI model group observed tubular necrosis (arrowheads) and casts (asterisks). (C) Immunohistochemical staining of PCNA in indicated groups. Data represent as mean ± SEM, *p < 0.05, **p < 0.01 compared with the control group (n = 5). #p < 0.05, ##p < 0.01 compared with AKI group (n = 5). One-way ANOVA followed by Turkey’s test for multiple comparisons was used for three or more groups. Scale bar, 50 μm; abbreviations: PCNA, proliferating cell nuclear antigen.

FIGURE 4

TanIIA improves renal artery blood flow, reduces pro-inflammatory cytokine production, and inhibits the activation of NF-κB signaling. (A,B) Representative image of renal vasculature with color Doppler in a mouse kidney (right) and representative bar chart of pulsatility index (PI) in mice. (C–E) Immunohistochemical staining of NF-κB p105/p50 and IKKβ and relative quantitative data in indicated groups; (F,G) ELISA bar chart representing serum level of TNF-α and IL-6. (H–J) mRNA level of IL-6, IL-1β, and TGF-β in the kidney; (K–M) Protein expression and relative quantitative data of NF-κB p105/p50 and IKKβ. Data represent as mean ± SEM, *p < 0.05, **p < 0.01 compared with the control group (n = 5). #p < 0.05, ##p < 0.01 compared with AKI group (n = 5). One-way ANOVA followed by Turkey’s test for multiple comparisons was used for three or more groups. Scale bar, 50 μm; abbreviations: IKKβ, inhibitor of kappa B kinase beta; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-1β, interleukin-1 β; TGF-β, transforming growth factor-β.

FIGURE 5

TanIIA actives mouse PXR and inhibit NF-κB activity, and upregulates PXR-mediated Cyp3a11 expression in AKI model. (A–D) The protein expression and relative quantitative data of PXR, RXRα, phospho-NF-κB p65, and NF-κB p65 in kidneys; (E,F) mRNA expression of PXR and Cyp3a11 in kidneys; data represent as mean ± SEM, *p < 0.05, **p < 0.01 compared with the control group (n = 5). #p < 0.05, ##p < 0.01 compared with the AKI group (n = 5). Student’s t-test was used for paired data. One-way ANOVA followed by Turkey’s test for multiple comparisons was used for three or more groups; abbreviations: PP65, phospho-NF-κB p65; P65, NF-κB p65; Cyp3a11, cytochrome P450 3A11.

FIGURE 6

Effects of TanIIA in human PXR activation and human PXR-mediated NF-κB repression. (A) Cell viability assay of TanIIA on HK-2 cells; (B) TanIIA on PXR-mediated CYP3A4 transcriptional reporter assay. RIF = 10 µM **p < 0.01 compared with indicated groups. ##p < 0.01 compared with the control group. (C) TanIIA on PXR-mediated NF-κB repression reporter assay. HK-2 cells were transfected with/without hPXR small-interfering RNA (siRNA). All cells were transfected with NF-κB luciferase reporter plasmid. HK-2 cells were stimulated by TNF-α (20 ng/ml) to activate NF-κB signaling. **p < 0.01 compared between two groups. ##p < 0.01 compared with TNF-α-stimulated cells without human PXR plasmid transfection. (D) Immunofluorescence staining images were captured by a fluorescence microscope. Data were represented as mean ± SEM, Student’s t-test was used for paired data. One-way ANOVA followed by Turkey’s test for multiple comparisons was used for three or more groups. Scale bar, 25 μm; abbreviations: DMSO, dimethyl sulfoxide; P65, NF-κB p65; RIF, rifampin; TNF-α, tumor necrosis factor α.

FIGURE 7

Diagrams of the interaction of TanIIA with the crystal structure of PXR (1SKX). (A,B) Chemical and 3D structure of TanIIA; (C) the 3D crystal structure of human PXR and ligand-binding domain with an endogenous ligand (yellow) (PDB ID: 1SKX); (D) twenty poses of TanIIA docked into the endogenous ligand’s (yellow) active site of 1SKX; the binding model of TanIIA in PXR: at least four residues involved in the interactions in twenty random poses, H-bonds with SER²⁴⁷, GLN²⁸⁵, and π–π interaction with PHE²⁸⁸ and TRP²⁹⁹.