Coumarin-chalcone derivatives as dual NLRP1 and NLRP3 inflammasome inhibitors targeting oxidative stress and inflammation in neurotoxin-induced HMC3 and BE(2)-M17 cell models of Parkinson's disease

Abstract

Background: In Parkinson's disease (PD) brains, microglia are activated to release inflammatory factors to induce the production of reactive oxygen species (ROS) in neuron, and vice versa. Moreover, neuroinflammation and its synergistic interaction with oxidative stress contribute to the pathogenesis of PD.

Methods: In this study, we investigated whether in-house synthetic coumarin-chalcone derivatives protect human microglia HMC3 and neuroblastoma BE(2)-M17 cells against 1-methyl-4-phenyl pyridinium (MPP⁺)-induced neuroinflammation and associated neuronal damage.

Results: Treatment with MPP⁺ decreased cell viability as well as increased the release of inflammatory mediators including cytokines and nitric oxide in culture medium, and enhanced expression of microglial activation markers CD68 and MHCII in HMC3 cells. The protein levels of NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α were also increased in MPP⁺-stimulated HMC3 cells. Among the four tested compounds, LM-016, LM-021, and LM-036 at 10 μM counteracted the inflammatory action of MPP⁺ in HMC3 cells. In addition, LM-021 and LM-036 increased cell viability, reduced lactate dehydrogenase release, ameliorated cellular ROS production, decreased caspase-1, caspase-3 and caspase-6 activities, and promoted neurite outgrowth in MPP⁺-treated BE(2)-M17 cells. These protective effects were mediated by down-regulating inflammatory NLRP1, IL-1β, IL-6, and TNF-α, as well as up-regulating antioxidative NRF2, NQO1, GCLC, and PGC-1α, and neuroprotective CREB, BDNF, and BCL2.

Conclusion: The study results strengthen the involvement of neuroinflammation and oxidative stress in PD pathogenic mechanisms, and indicate the potential use of LM-021 and LM-036 as dual inflammasome inhibitors in treating both NLRP1- and NLRP3-associated PD.

Keywords: MPP+; Parkinson's disease; human HMC3 and BE(2)-M17 cells; neuroinflammation; oxidative stress; therapeutics.

Figure 1

LM compounds. (A) Structure and formula of LM-009,−016,−021, and−036. (B) Molecular weight (MW), hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), calculated octanol-water partition coefficient (cLogP), polar surface area (PSA), and blood–brain barrier (BBB) permeation score of LM compounds. (C) Cytotoxicity of LM compounds against human HMC3 and BE(2)-M17 cells examined by MTT assay. Cells were treated with LM compound (1–100 μM) and cell viability was measured the next day (n = 3). To normalize, the relative viability of untreated cells was set at 100%. Shown below were cytotoxicity IC₅₀ values. (D) DPPH free radical scavenging activity (10–160 μM) and (E) oxygen radical absorbance capacity (4–100 μM) of LM compounds and quercetin (as a positive control; n = 3). Shown below were EC₅₀ or trolox equivalent values.

Figure 2

MPP⁺ induced inflammation of HMC3 microglia and anti-inflammatory potential of LM compounds. (A) Cytotoxicity of MPP⁺ against HMC3 cells by MTT assay. Cells were treated with MPP⁺ (0–8 mM) and cell viability was measured the following day (n = 3). (B) Experimental flow chart of anti-inflammatory test. HMC3 cells were plated on day 1. After 20 h, cells were pre-treated with LM compounds (1–10 μM) for 8 h, followed by MPP⁺ (4 mM) treatment for 20 h. On day 3, the HMC3 cells were examined for cell viability and NO release in culture medium (C). In addition, cells with 10 μM compound treatment were examined for CD68 and MHCII expression by ICC staining (D), IL-1β, IL-6, and TNF-α in culture medium by ELISA (E), and NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α expression by Western blotting (F) (n = 3). The relative cell viability as well as CD68, MHCII, NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α expression levels in MPP⁺-untreated cells were normalized as 100%. In CD68 (yellow) and MHCII (green) staining cells, nuclei were counterstained with DAPI (blue). GAPDH was used as a loading control in immunoblotting. p-values: comparisons between MPP⁺-untreated and treated cells (^##p < 0.01, ^###p < 0.001), or with and without compound addition (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Reduction of cytotoxicity and ROS of LM compounds in MPP⁺-treated BE(2)-M17 cells. (A) Cytotoxicity of MPP⁺ against BE(2)-M17 cells by MTT assay. Cells were treated with MPP⁺ (0–5 mM) and cell viability was measured after 48 h (n = 3). The relative cell viability in MPP⁺-untreated cells was set at 100%. MPP⁺ at 0.62 mM concentration (80% cell viability) was selected for testing anti-inflammatory potential of LM-016, LM-021, and LM-036. (B) Experimental flow chart. On day 1, BE(2)-M17 cells were plated in the presence of retinoic acid (RA, 5 μM). On day 5, after removing retinoic acid, cells were treated with LM compounds (1–10 μM) and MPP⁺ (0.62 mM). (C) cell viability, LDH release, and (D) ROS production (H₂DCF-DA stain) were measured on day 7 (n = 3). The relative cell viability, LDH release, and ROS production in MPP⁺-untreated cells was set as 100%. Cellular ROS were analyzed by assessing fluorescence of DCF (green) and cell nuclei counterstained with Hoechst 33342 (blue). p-values: comparisons between MPP⁺-untreated and treated cells (^###p < 0.001), or with and without compound addition (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4

Neuroprotective effects of LM compounds on MPP⁺-treated BE(2)-M17 cells. Retinoic acid-differentiated BE(2)-M17 cells were treated with LM-016, LM-021, or LM-036 (10 μM) and MPP⁺ (0.62 mM) on day 5. On day 7, (A) tyrosine hydroxylase (TH, green) expression, (B) caspase-1, caspase-3 and caspase-6 activities, as well as (C) neurite length, process and branch were measured (n = 3). Shown were TUBB3-stained images and images of cells outlined with multi-colored mask to assign each outgrowth to a cell body for neurite outgrowth quantification. Processes and branches in LM-036-treated cells were marked with red and white arrows, respectively. To normalize, TH expression and caspase activities in MPP⁺-untreated cells were set at 100%. p-values: comparisons between MPP⁺-untreated and treated cells (^#p < 0.05, ^##p < 0.01), or with and without compound addition (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

Neuroinflammatory, antioxidative, and neuroprotective effects of LM compounds on MPP⁺-treated BE(2)-M17 cells. Cells were treated as Figure 4 described. (A) Neuroinflammatory NLRP1, IL-1β, IL-6, and TNF-α, (B) antioxidative NRF2, NQO1, GCLC, and PGC-1α, and (C) neuroprotective CREB, BDNF, and BCL2, and pro-apoptotic BAX were examined (n = 3). GAPDH was included as a loading control. To normalize, the relative protein levels in MPP⁺-untreated cells were set at 100%. p-values: comparisons between MPP⁺-untreated and treated cells (^#p < 0.05, ^##p < 0.01, ^###p < 0.001), or with and without compound addition (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6

Graphical summary of coumarin-chalcone derivatives LM-021 and LM-036 as dual NLRP1 and NLRP3 inhibitors in MPP⁺-induced HMC3 and BE(2)-M17 cell models of PD.