Whole-genome sequencing shows modulation of neurodegenerative genes by Withania somnifera in human SK-N-SH cells

Abstract

Background: Aging is driven by several primary and secondary hallmarks that manifest with age, of which neurodegenerative diseases are important manifestations. The ability to decelerate or reverse aging, and promote healthy aging, has garnered great interest in recent times. In traditional medicine, Withania somnifera (WS) or Ashwagandha has been recognized for its adaptogenic and rejuvenative effects.

Methods: To investigate WS-modulated global gene expression profiles, we performed whole-genome sequencing of WS-treated human neuroblastoma SK-N-SH cells at different doses (50 and 100 μg/mL) and time points (3 h and 9 h) and validation by quantitative real-time PCR (qRT-PCR) and immunoblotting. Disease enrichment analysis for brain-related disorders was performed by DisGeNET.

Results: Using differential gene expression analyses, we identified 19,945 WS-modulated genes. Of these, 2,403 and 177 genes were significantly (p ≤ 0.05) upregulated and downregulated, respectively, by WS treatment. Interestingly, different patterns of gene expression were exhibited in dose-dependent (9 upregulated, 1 downregulated, 100 μg/mL 3 h vs. 50 μg/mL 3 h; 21 upregulated, 86 downregulated, 100 μg/mL 9 h vs. 50 μg/mL 9 h) and temporal kinetics (210 upregulated, 6 downregulated, 50 μg/mL 9 h vs. 50 μg/mL 3 h; 8 upregulated, 49 downregulated, 100 μg/mL 9 h vs. 100 μg/mL 3 h). Furthermore, qRT-PCR experiments validated the RNA-seq results. WS-modulated genes were implicated in Alzheimer's disease, migraine, Parkinson's disease, bipolar disorder, cognition, stress, anxiety, forgetfulness, sleep disorders, and substance abuse among others.

Conclusion: Taken together, our transcriptomic profiling study revealed for the first time that WS may modulate key genes in neurodegenerative disorders with potential beneficial implications for brain-related disorders and healthy aging.

Keywords: Withania somnifera; aging; ashwagandha; healthy aging; neurodegenerative disease; whole genome sequencing.

Figure 1

Differential gene expression (DGE) mRNA analyses: (A) up- or downregulation of genes in WS-treated human neuroblastoma SK-N-SH cells at various treatments; (B) sample clustering dendrogram; (C) heatmap of top variable 2000 genes; (D) sample-to-sample correlation; (E) PCA plot of sample group.

Figure 2

Volcano plots showing upregulated or downregulation of genes at different time points and dose concentrations: (A) 100 μg/mL_3h vs. 50 μg/mL_3h; (B) 100 μg/mL_9h vs. 50 μg/mL_9h; (C) 50 μg/mL_9h vs. 50 μg/mL_3h; (D) 100 μg/mL_9h vs. 100 μg/mL_3h.

Figure 3

Heatmap showing differentially expressed genes at different time points and dose concentrations.

Figure 4

Disease-specific enrichment analysis of WS-treated neuroblastoma cells at different time points and concentration: (A) 50 μg/mL_3h vs. control_3h; (B) 50 μg/mL_9h vs. control_9h; (C) 100 μg/mL_3h vs. control_3h; (D) 100 μg/mL_9h vs. control_9h; (E) 100 μg/mL_9h vs. 50 μg/mL _9h; (F) 100 μg/mL_9h vs. 100 μg/mL _3h; (G) 50 μg/mL_9h vs. 50 μg/mL _3h.

Figure 5

Disease ontology analysis of WS-treated SK-N-SH cells at different time points and concentration: (A) 50 μg/mL_3h vs. control_3h; (B) 50 μg/mL_9h vs. control_9h; (C) 100 μg/mL_3h vs. control_3h; (D) 100 μg/mL_9h vs. control_9h; (E) 100 μg/mL_3h vs. 50 μg/mL _3h; (F) 100 μg/mL_9h vs. 50 μg/mL _9h; (G) 100 μg/mL_9h vs. 100 μg/mL _3h; (H) 50 μg/mL_9h vs. 50 μg/mL _3h.

Figure 6

Validation of RNA-sequencing data with qRT-PCR data of brain-related disorders genes. Fold changes in gene expression measured by qRT-PCR for each sample in triplicate (n = 3) were plotted against fold change in sequencing data (coefficient of determination, r² = 0.9).

Figure 7

(A) Western blot analysis of p-NF-κB, NF-κB, TNF-α, and HO-1. Representative of three independent experiments. (B) Densitometric analysis of p-NF-κB expression. All data are represented as means ± SD (**p < 0.01 as compared to the control). (C) Densitometric analysis of TNF-α expression. All data are represented as means ± SD (*p < 0.05, **p < 0.01, and ***p < 0.005 as compared to the control). (D) Densitometric analysis of HO-1 expression. All data are represented as means ± SD (**p < 0.01 and ***p < 0.005 as compared to the control). (−) represents cells treated with DMSO; (+) represents cells treated with DMSO and LPS.