Biomarker Exploration in Human Peripheral Blood Mononuclear Cells for Monitoring Sulforaphane Treatment Responses in Autism Spectrum Disorder

Abstract

Autism Spectrum Disorder (ASD) is one of the most common neurodevelopmental disorders with no drugs treating the core symptoms and no validated biomarkers for clinical use. The multi-functional phytochemical sulforaphane affects many of the biochemical abnormalities associated with ASD. We investigated potential molecular markers from three ASD-associated physiological pathways that can be affected by sulforaphane: redox metabolism/oxidative stress; heat shock response; and immune dysregulation/inflammation, in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with ASD. We first analyzed the mRNA levels of selected molecular markers in response to sulforaphane ex vivo treatment in PBMCs from healthy donors by real-time quantitative PCR. All of the tested markers showed quantifiability, accuracy and reproducibility. We then compared the expression levels of those markers in PBMCs taken from ASD patients in response to orally-delivered sulforaphane. The mRNA levels of cytoprotective enzymes (NQO1, HO-1, AKR1C1), and heat shock proteins (HSP27 and HSP70), increased. Conversely, mRNA levels of pro-inflammatory markers (IL-6, IL-1β, COX-2 and TNF-α) decreased. Individually none is sufficiently specific or sensitive, but when grouped by function as two panels, these biomarkers show promise for monitoring pharmacodynamic responses to sulforaphane in both healthy and autistic humans, and providing guidance for biomedical interventions.

Figure 1

Major signaling pathways for protective mechanisms against ASD by SF. (a) Keap1/Nrf2/ARE pathway, (b) NF-κB inflammatory pathway, (c) heat-shock responses. (modified from Liu et al.).

Figure 2

Cytoprotective gene expression in human PBMCs from healthy donors in response to SF ex vivo treatment. (a) Single dose. (b) Three consecutive, and lower daily doses. In each case assessment was made 6 hours following the final dose. Means ± SD are shown. Symbols *(P < 0.05) or **(P < 0.01) indicate statistical differences between treatments and controls by two-tailed Student’s t-test.

Figure 3

Pro-inflammatory gene expression in human PBMCs from healthy donors in response to LPS stimulation (a) and to SF pre-treatment plus LPS stimulation (b). Means ± SD are shown. Symbols *(p < 0.05) or **(p < 0.01) indicate statistical differences between treatments and controls by two-tailed Student’s t-test (a,b), and symbol ^##(p < 0.01) indicates statistical differences between LPS stimulation and SF pre-treatment plus LPS stimulation (b).

Figure 4

Basal gene expression levels in human PBMCs isolated from three blood samples of the same healthy donor. Means ± SD are shown. Symbols *(p < 0.05) or **(p < 0.01) indicate statistical differences among three blood draws by one-way ANOVA.

Figure 5

Consistency of gene expression in human PBMCs from healthy donors in response to ex vivo treatment among three blood samples of the same healthy donor. Means ± SD are shown. Symbols *(p < 0.05) or **(p < 0.01) indicate statistical differences among three blood draws by one-way ANOVA, and symbols ^##(p < 0.01) or ^###(p < 0.001) indicate statistical differences between treatments by two-tailed Student’s t-test.

Figure 6

Gene expression level changes in PBMCs from 6 patients with ASD after SF intervention. SF was orally administered daily at the dose of 2.2 μmol/kg body weight in the form of GR plus myrosinase for two weeks. Expression level of each gene immediately prior to treatment was set as control. (a) Cytoprotective genes. (b) Pro-inflammatory genes. (c) Grouped genes by broad functionality (e.g. cytoprotective or pro-inflammatory). Differences between pre-dose and post-dose for individual markers were analyzed by two-tailed paired t-test, and p-values for each marker are the numbers in parentheses above the top error bars (a,b). Differences of grouped markers from baseline were analyzed by one-way ANOVA, and symbol ** indicates highly significant statistical difference (p < 0.01) (c). Data points for each subject (a,b) or means of each group of genes (c) are plotted. Horizontal lines within boxes are means.