Anti-neuroinflammatory effects of Ephedra sinica Stapf extract-capped gold nanoparticles in microglia

Abstract

Background: Combination therapy remains a promising strategy for treating neurodegenerative diseases, although green synthesis of gold nanoparticles for treating chronic neuroinflammation and studying their efficacy in treating neuroinflammation-mediated neurodegenerative diseases is not well assessed. Results: Here, Ephedra sinica Stapf (ES) extract was used as the reducing, capping, and stabilizing agent for gold nanoparticle synthesis. We developed ES extract-capped gold nanoparticles (ES-GNs) and investigated their anti-neuroinflammatory properties in microglia. ES-GNs displayed maximum absorption at 538 nm in ultraviolet-visible spectroscopy. Dynamic light scattering assessment revealed that ES-GN diameter was 57.6±3.07 nm, with zeta potential value of -24.6±0.84 mV. High resolution-transmission electron microscopy confirmed the spherical shape and average diameter (35.04±4.02 nm) of ES-GNs. Crystalline structure of ES-GNs in optimal conditions was determined by X-ray powder diffraction, and elemental gold presence was confirmed by energy-dispersive X-ray spectroscopy. Fourier transform-infrared spectroscopy confirmed gold nanoparticle synthesis using ES. Anti-neuroinflammatory properties of ES-GNs on production of pro-inflammatory mediators (nitric oxide, prostaglandin E₂, and reactive oxygen species) and cytokines (tumor necrosis factor-α, IL-1β, and IL-6) in lipopolysaccharide (LPS)-stimulated microglia were investigated by ELISA and flow cytometry. ES-GNs significantly attenuated LPS-induced production of pro-inflammatory mediators and cytokines, which was related to suppressed transcription and translation of inducible nitric oxide synthase and cyclooxygenase-2, determined by RT-PCR and western blotting. ES-GNs downregulated upstream signaling pathways (IκB kinase-α/β, nuclear factor-κB, Janus-activated kinase /signal transducers and activators of transcription, mitogen-activated protein kinase , and phospholipase D) of pro-inflammatory mediators and cytokines in LPS-stimulated microglia. Anti-neuroinflammatory properties of ES-GNs were mediated by ES-GNs-induced AMP-activated protein kinase)-mediated nuclear erythroid 2-related factor 2 /antioxidant response element signaling. Conclusion: Collectively, these findings provide a new insight on the role of ES-GNs in treating chronic neuroinflammation-induced neurodegenerative diseases.

Keywords: Ephedra sinica Stapf; gold nanoparticle; microglia; neuroinflammation.

Figure 1

Reducing power potential and antioxidant activity of ES extract and optimization of ES-GNs. DPPH radical scavenging activity (A) and ABTS radical scavenging activity (B) of ES extract. UV-Vis spectroscopy analysis of ES extract (C) and green-synthesized ES-GNs (D). Optimization of ES extract concentration (E), gold(III) chloride (chloroauric acid) solution concentration (F), and reaction times (G) for ES-GN production. Abbreviations: ES, Ephedra sinica Stapf; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); ES-GNs, ES extract–gold nanoparticles; UV-Vis, ultraviolet-visible.

Figure 2

DLS and HR-TEM images of ES-GNs showing the SAED pattern, HAADF, and EDS. The hydrodynamic size (A) and zeta potential (B) of ES-GNs were determined by DLS. (C–D) Particle shape of ES-GNs was confirmed by low- and high-magnification images and HR-TEM images. The SAED pattern (E), HAADF image (F, G), and EDS analysis (H) of ES-GNs. Abbreviations: DLS, dynamic light scattering; HR-TEM, high-resolution transmission electron microscopy; SAED, selected area electron diffraction; HAADF, high-angle annular dark field; EDS, energy dispersive spectroscopy.

Figure 3

XRD and FT-IR spectra of ES-GNs. XRD patterns of ES extract (A) and synthesized ES-GNs (B). FT-IR spectra of ES extract (C) and synthesized ES-GNs (D). Abbreviations: XRD, X-ray diffraction; FT-IR, Fourier transform-infrared spectroscopy.

Figure 4

Effect of ES extract and ES-GNs on microglia viability and pro-inflammatory cytokine production. Primary microglia (A) and BV-2 microglia cell viability (B) were determined using the MTT assay. Primary microglia (C) and BV-2 microglia cell cytotoxicity (D) were analyzed using the LDH assay. (E, F) The production of TNF-α, IL-1β, and IL-6 in the culture media was analyzed using a commercial ELISA kit. ROS content was determined using CM-H₂DCFDA. ^# P<0.01 relative to the control group. *P<0.05 and **P<0.01 relative to the LPS-treated group. Abbreviations: ES, Ephedra sinica; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor-α.

Figure 5

Effect of ES-GNs on iNOS and COX-2 activity via the IKK-α/β, NF-κB, JAK/STAT, ERK-1/2, p38 MAPK, JNK, and PLD signaling pathways in BV-2 microglia cell. NO content (A) was measured using the Griess reaction. PGE₂ (B) was measured using a commercial ELISA kit. The relative mRNA levels encoding iNOS (C) and COX-2 (D) were determined by real-time RT-PCR. Dual luciferase assay was performed to evaluate iNOS (E) and COX-2 (F) promoter activity. (G) Western blotting and semi-quantitative analysis were performed to estimate iNOS, COX-2, and α-tubulin protein levels. (H) Western blot analysis and semi-quantitative analysis were performed to evaluate the protein expression of p-IKK-α/β, IKK-α/β, p-IκB-α, IκB-α, and α-tubulin. (I) Western blot analysis and semi-quantitative analysis were performed to evaluate the protein expression of NF-κB p65, p-NF-κB p65, and TBP. (J) Dual luciferase assay was performed to evaluate NF-κB p65 promoter binding activity. (K) Western blotting and semi-quantitative analysis were performed to estimate protein levels of p-JAK-1, p-STAT-1, JAK-1, and STAT-1. (L) Western blotting and semi-quantitative analysis were performed to estimate the protein expression of p-ERK, p-JNK, p-p38, ERK, JNK, and p38. (M) Amplex Red PLD assay kit was used to estimate the PLD activity. ^# P<0.01 relative to the control group. *P<0.05 and **P<0.01 relative to the LPS-treated group. Abbreviations: NO, nitrite; PGE₂, prostaglandin E₂; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase; p-, phosphorylated; IKK, IκB kinase; TBP, TATA-binding protein; JAK, Janus-activated kinase; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; JNK, c-JUN N-terminal kinase; PLD, phospholipase D.

Figure 6

Effect of ES-GNs on anti-neuroinflammatory properties through the AMPK/Nrf2/ARE signaling pathway in BV-2 microglia cell. (A) Western blot and semi-quantitative analysis were performed to evaluate the protein expression levels of HO-1 and α-tubulin. (B) BV-2 microglia cells were transfected with HO-1 siRNA and treated 20 μg/mL ES-GNs followed by LPS and analyzed for HO-1 and α-tubulin protein levels. (C) BV-2 microglia cells were transfected with HO-1 siRNA and treated 20 μg/mL ES-GNs followed by LPS and analyzed for iNOS promoter activity. (D) NQO1 and α-tubulin protein levels were assessed by western blotting. (E) After transfection with NQO1 siRNA were pre-treated with 20 μg/mL ES-GNs and treated with LPS. NQO1 siRNA system was performed to evaluate the protein expression levels of NQO1 and α-tubulin. (F) After transfection with NQO1 siRNA were pre-treated with 20 μg/mL ES-GNs and treated with LPS. Dual luciferase assay was performed to evaluate iNOS promoter activity in NQO1 siRNA system. (G) Western blotting and semi-quantitative analysis were performed to evaluate Nrf2 and TBP protein levels. (H) After transfection with Nrf2 siRNA were pre-treated with 20 μg/mL ES-GNs and treated with LPS. Nrf2 siRNA system was performed to evaluate Nrf2, HO-1, NQO1, TBP, and α-tubulin protein levels. (I) After transfection with Nrf2 siRNA were pre-treated with 20 μg/mL ES-GNs and treated with LPS. Dual luciferase assay was performed to evaluate iNOS promoter activity in Nrf2 siRNA system. (J) Western blotting and semi-quantitative analysis were performed to evaluate p-AMPK and AMPK protein levels. (K) After transfection with AMPK siRNA were pre-treated with 20 μg/mL ES-GNs and treated with LPS. AMPK siRNA system was performed to evaluate p-AMPK, AMPK, Nrf2, HO-1, NQO1, TBP, and α-tubulin protein levels. (L) After transfection with AMPK siRNA were pre-treated with 20 μg/mL ES-GNs and treated with LPS. Dual luciferase assay was performed to evaluate iNOS promoter activity in the AMPK siRNA system. ^# P<0.01 relative to the control group. *P<0.05 and **P<0.01 relative to the LPS-treated group. Abbreviations: HO-1, heme oxygenase-1; NQO1, NAD(P)H dehydrogenase1; Nrf2, nuclear erythroid 2-related factor 2; TBP, TATA-binding protein; si, small interfering; AMPK, AMP-activated protein kinase.

Figure 7

Scheme illustrating the anti-neuroinflammatory properties of ES-GNs in microglia. Abbreviations: ES-GN, Ephedra sinica Stapf extract–gold nanoparticle; TNF-α, tumor necrosis factor-α; iNOS, Inducible nitric oxide synthase; COX-2, cyclooxygenase; p-, phosphorylated; Ikk, IĸB kinase; TBP, TATA-binding protein; JAK, Janus kinase; STAT, signal transducer of activation; MAPKs, Mitogen-activated protein kinases; HO-1, heme oxygenase-1; NQO1, NAD(P)H dehydrogenase1; Nrf2, nuclear factor erythroid-derived 2-related factor 2; TBP, TATA-binding protein; AMPK, AMP-activated protein kinase.