Shikonin alleviates rotenone-induced Parkinson's disease neuroinflammation by targeting PKM2-mediated glycolytic MG-Hs production

Abstract

Background: In Parkinson's disease (PD), microglial activation is driven by metabolic reprogramming toward aerobic glycolysis, a shift regulated by pyruvate kinase M2 (PKM2). While the environmental toxin rotenone is a recognized PD risk factor, the precise glycolytic mechanism linking it to microglial neuroinflammation remains unclear, and the therapeutic potential of targeting this axis is largely unexplored.

Purpose: We sought to elucidate the specific glycolytic pathway by which rotenone induces microglial activation and to investigate whether shikonin, a natural PKM2 inhibitor, could attenuate neuroinflammation by targeting this metabolic mechanism.

Methods: Using rotenone (250 nM)-treated BV2 microglia, we assessed glycolytic function (lactate production, glucose consumption) and quantified the formation of methylglyoxal-derived hydroimidazolones (MG-Hs), key pro-inflammatory glycation adducts. NF-κB pathway activation and inflammatory cytokine release were evaluated. The inhibitory effects of shikonin on this cascade were systematically examined.

Results: We identified a novel mechanistic pathway: rotenone promotes PKM2-mediated glycolytic flux, leading to accumulation of the cytotoxic metabolite methylglyoxal (MG) and its derived MG-Hs. These MG-Hs function as critical signaling mediators that directly activate the NF-κB pathway, fueling neuroinflammation. Shikonin effectively disrupted this cascade at its source by inhibiting PKM2, thereby normalizing glycolytic activity, reducing MG-Hs formation, and subsequently suppressing NF-κB activation and the release of pro-inflammatory factors.

Conclusion: This study delineates a complete PKM2-glycolysis-MG-Hs-NF-κB axis as a fundamental mechanism in rotenone-induced neuroinflammation. Our results provide compelling preclinical evidence that shikonin exerts its neuroprotective effects by specifically targeting this metabolic-inflammatory pathway, positioning it as a highly promising disease-modifying therapeutic candidate for PD.

Keywords: Glycolysis; Microglial activation; Neuroinflammation; Pyruvate kinase M2; Shikonin.

Fig. 1

Rotenone induces cytotoxic and pro-inflammatory responses in BV2 microglia. A Cell viability was assessed using the sulforhodamine B (SRB) assay after 24 h of treatment with 250 nM rotenone (Rot). B Extracellular levels of TNF-α, IL-1β, and IL-6 were measured by ELISA in culture supernatants from BV2 microglia exposed to 250 nM Rot for 24 h. Data are presented as mean ± SD (n = 5 biologically independent experiments). ^***P < 0.001

Fig. 2

Rotenone promotes inflammatory responses via glycolytic activation in BV-2 microglia. A Representative immunoblots of glycolytic enzymes (PFKFB3, PKM2, LDHA) and β-actin (loading control) after 24 h treatment with 250 nM Rot. B Quantitative analysis of protein levels in (A) normalized to β-actin (n = 5). C Glycolytic parameters (glucose consumption, lactate production, lactate/glucose ratio) measured after Rot treatment (n = 5). D Representative immunoblots of MG-Hs and phospho-NF-κB p65 (p-p65) with β-actin. E Quantification of MG-Hs and p-p65 levels from (D) normalized to β-actin (n = 5). F Representative immunoblots of NF-κB p65 in cytoplasmic (β-actin control) and nuclear (Lamin B1 control) fractions. G Quantitative analysis of cytoplasmic p65 (normalized to β-actin) and nuclear p65 (normalized to Lamin B1) (n = 5). H Glycolytic parameters following pretreatment with 20 µM 2-DG for 2 h before Rot exposure (n = 5). I Representative immunoblots of MG-Hs and p-p65 with β-actin after 2-DG and Rot treatment. J Quantification of protein levels in (I) normalized to β-actin (n = 5). K Representative immunoblots of NF-κB p65 in cytoplasmic and nuclear fractions after intervention. L Quantitative analysis of p65 levels in both fractions (n = 5). M Extracellular levels of TNF-α, IL-1β, and IL-6 measured by ELISA (n = 5). Data are presented as mean ± SD; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 3

MG-Hs mediate rotenone-induced inflammatory responses in BV2 microglia. A Representative immunoblots of MG-Hs and phospho-NF-κB p65 (p-p65) in cells pretreated with 100 µM AG for 2 h followed by 250 nM Rot for 24 h (β-actin loading control). B Quantification of MG-Hs and p-p65 levels normalized to β-actin (n = 5). C Representative immunoblots of NF-κB p65 in cytoplasmic (β-actin control) and nuclear (Lamin B1 control) fractions. D Quantification of cytoplasmic (normalized to β-actin) and nuclear (normalized to Lamin B1) p65 levels (n = 5). E Extracellular levels of TNF-α, IL-1β, and IL-6 measured by ELISA (n = 5). F Representative immunoblots of MG-Hs and p-p65 in cells pretreated with 25 µM MG for 2 h followed by 250 nM Rot for 24 h (β-actin loading control). G Quantification of MG-Hs and p-p65 levels normalized to β-actin (n = 5). H Representative immunoblots of NF-κB p65 in cytoplasmic and nuclear fractions. I Quantification of p65 levels in both fractions (n = 5). J Extracellular cytokine levels (TNF-α, IL-1β, IL-6) measured by ELISA (n = 5). Data are presented as mean ± SD; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 4

Shikonin attenuates rotenone-induced hyperglycolysis and MG-Hs formation in BV2 microglia. A Representative immunoblots of glycolytic enzymes (PFKFB3, PKM2, LDHA) and β-actin (loading control) following pretreatment with 50 nM shikonin (SK) for 1 h and subsequent treatment with 250 nM rotenone (Rot) for 24 h. B Quantitative analysis of glycolytic enzyme expression normalized to β-actin (n = 5). C-E Metabolic parameters measured by colorimetric assays: (C) glucose consumption, (D) lactate production, and (E) lactate-to-glucose ratio (n = 5). F Representative immunoblots of MG-Hs with β-actin loading control.G Quantitative analysis of MG-Hs levels normalized to β-actin (n = 5). Data represent mean ± SD from five biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey's post-hoc test ^***P < 0.001

Fig. 5

Shikonin attenuates rotenone-induced neuroinflammation in BV2 microglia by inhibiting PKM2-mediated NF-κB signaling. A Representative immunoblots of phospho-NF-κB p65 (p-p65) and β-actin in cells pretreated with 50 nM SK for 1 h followed by 250 nM rotenone (Rot) for 24 h. B Quantitative analysis of p-p65 levels normalized to β-actin (n = 5). C Extracellular TNF-α, IL-1β, and IL-6 levels measured by ELISA (n = 5). D Representative immunoblots of NF-κB p65 in cytoplasmic (β-actin control) and nuclear. (Lamin B1 control) fractions from cells with control (OE-Con) or PKM2 overexpression (OE-PKM2), after SK and Rot treatments. E Quantification of NF-κB p65 levels in cytoplasmic and nuclear fractions (n = 5). F Cytokine secretion under corresponding conditions (n = 5). Data are shown as mean ± SD from five independent experiments. ^***P < 0.001

Fig. 6

Shikonin attenuates microglia-mediated neuronal injury in a rotenone-induced Parkinson’s disease model. A Experimental design schematic: PC12 neurons differentiated with NGF were treated for 24 h with conditioned medium from BV2 microglia exposed to either: (i) 250 nM rotenone (Rot-MCM) or (ii) 250 nM rotenone plus 50 nM shikonin (Rot + SK-MCM). B Neuronal viability assessed by SRB assay (n = 5). C Representative immunoblots of cleaved PARP apoptosis marker with β-actin loading control. D Quantitative analysis of cleaved PARP expression normalized to β-actin (n = 5). E TUNEL staining (red, scale bar = 50 μm). F Representative flow cytometry dot plots of annexin V/PI staining. G Quantification of viable (PI⁻/annexin V⁻) and apoptotic (PI⁺/annexin V⁺ and PI⁻/annexin V⁺) cell populations (n = 5). Data represent mean ± SD from five biologically independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 7

Proposed mechanism of shikonin-mediated neuroprotection against rotenone-induced neuroinflammation. The schematic illustrates shikonin’s dual action in attenuating microglia-mediated neuroinflammation through: (1) direct inhibition of PKM2, suppressing glycolytic flux and subsequent MG-Hs formation; and (2) downstream reduction of NF-κB p65 activation and pro-inflammatory cytokine release. This metabolic intervention disrupts the vicious cycle between microglial hyperactivation and neuronal injury in Parkinson’s disease models