Withania somnifera as a potential candidate to ameliorate high fat diet-induced anxiety and neuroinflammation

Abstract

Background: The epidemic of obesity has reached alarming levels in both developing and developed nations. Excessive calorie intake and sedentary lifestyle due to technological advancements are the main causal factors for overweight and obesity among the human population. Obesity has been associated with a number of co-morbidities such as hypertension, type 2 diabetes mellitus, cardiovascular diseases, and neurodegeneration and dementia. The progression of neurological disorders in obese subjects has been mainly attributed to neuroinflammation. Withania somnifera has been used in numerous Ayurvedic formulations owing to its wide array of health-promoting properties. The current study was designed to test the hypothesis whether dry leaf powder of W. somnifera has anxiolytic and anti-neuroinflammatory potential in diet-induced obesity.

Methods: Young adult female rats were divided into four groups: low fat diet group (LFD) fed with regular chow feed, high fat diet group (HFD) fed with diet containing 30% fat by weight, low fat diet plus extract group (LFDE) fed with regular chow feed supplemented with dry leaf powder of W. somnifera 1 mg/g of body weight (ASH), and high fat diet plus extract group (HFDE) fed with diet containing 30% fat by weight and supplemented with ASH. All the animals were kept on respective feeding regimen for 12 weeks; following which, the animals were tested for their anxiety-like behavior using elevated plus maze test. The animals were sacrificed and used to study various inflammatory markers such as GFAP, Iba1, PPARγ, iNOS, MCP-1, TNFα, IL-1β, IL-6, and various markers of NF-κB pathway by Western blotting and quantitative real-time PCR. Serum levels of leptin, insulin and pro-inflammatory cytokines were also assayed.

Results: ASH treated rats showed less anxiety levels as compared to HFD animals. At molecular level, ASH ameliorated the HFD-induced reactive gliosis and microgliosis and suppressed the expression of inflammatory markers such as PPARγ, iNOS, MCP-1, TNFα, IL-1β, and IL-6. Further, ASH ameliorated leptin and insulin resistance and prevented HFD-induced apoptosis.

Conclusions: Dry leaf powder of W. somnifera may prove to be a potential therapeutic agent to attenuate neuroinflammation associated with obesity and may prevent its co-morbidities.

Keywords: Anxiety; Apoptosis; High fat diet; Inflammatory cytokines; Insulin; Leptin; Neuroinflammation; Obesity; Withania somnifera.

Conflict of interest statement

Ethics approval

Animal care and procedures were followed in accordance with the guidelines of the Institutional Animal Ethical Committee of Guru Nanak Dev University, Amritsar, India (Registration number 226/CPCSEA).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

ASH supplementation maintained body weight and suppressed anxiety-like behavior in HFD-induced obese rats. a Line graph representing the percentage change in body weight among different groups of animals over the period of 12 weeks (n = 10 ± 1 each group). b Histogram represents average time spent by the animals in open and closed arms of elevated plus maze. Among the four groups, HFD animals spent maximum time in the closed arm. The exploratory activity of animals of each group is shown by the number of entries (c) and number of crossings (d) in open and closed arms. HFDE animals showed more exploratory activity than the other groups. e Histogram represents the number of head dips by the animals of each group. Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats; #p ≤ 0.05 HFD versus HFDE rats; $p ≤ 0.05 HFD versus LFDE rats; @p ≤ 0.05 LFDE versus HFDE rats; ¤ statistically significant difference within group, Holm–Sidak method after one-way ANOVA in a, b and f and two-way ANOVA in c, d and e

Fig. 2

ASH suppressed inflammation at both transcriptional and translational levels. a Immunostaining of GFAP in the hippocampus and PC regions of LFD, HFD, LFDE, and HFDE rats (n = 3 each group). Insets show high magnification images of GFAP staining. Upregulation of GFAP in HFD rats was seen, which was ameliorated in HFDE rats. b, c Representative Western blot analysis for GFAP, Iba1, and PPARγ in the hippocampus and PC regions of LFD, HFD, and HFDE animals. Histograms represent percent change in intensity taking value in LFD rats as 100%. d Histograms representing fold change in mRNA expression of GFAP, ITGAM, iNOS, MCP-1, and COX2 in the hippocampus and PC regions of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, Holm–Sidak method after one-way ANOVA

Fig. 3

ASH suppressed the synthesis and secretion of pro-inflammatory cytokines. a Histograms represent the serum levels of TNFα, IL-1β, and IL-6 among LFD, HFD, LFDE, and HFDE rats (n = 8 each group). b, c Representative Western blot analysis for TNFα, IL-1β, and IL-6 in the hippocampus and PC regions of rat brain among the four groups of animals. Histograms represent percent change in intensity taking value in LFD rats as 100%. d Histograms representing fold change in mRNA expression of TNFα and IL-1β in the hippocampus and PC regions of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, @p ≤ 0.05 LFDE versus HFDE rats, Holm–Sidak method after one-way ANOVA

Fig. 4

ASH prevented hyperleptinemia and hyperinsulinemia. a Histograms represent the serum levels of leptin and insulin among LFD, HFD, LFDE, and HFDE rats (n = 8 each group). b Representative Western blot analysis for OB-Rb in the hippocampus and PC regions of rat brain. Histograms represent percent change in intensity taking value in LFD rats as 100%. c, d Histograms representing fold change in mRNA expression of OB-Rb, SOCS1, SOCS3, JAK2, STAT3, IRS1, and IRS2 in the hippocampus (c) and PC (d) regions of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, @p ≤ 0.05 LFDE versus HFDE rats, Holm–Sidak method after one-way ANOVA

Fig. 5

ASH modulated NF-κB pathway and prevented apoptosis. a, c Representative Western blot analysis for markers of NF-κB pathway in the hippocampus and PC regions of brain among the LFD, HFD, and HFDE animals. Histograms represent percent change in intensity taking value in LFD rats as 100%. b, d Representative Western blot analysis for apoptotic markers AP-1 and Bcl-xL in the hippocampus and PC regions of brain among the LFD, HFD, and HFDE rats. Histograms represent percent change in intensity taking value in LFD rats as 100%. e Histograms representing fold change in mRNA expression of markers of NF-κB pathway and apoptotic markers in the hippocampus and PC regions of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, @p ≤ 0.05 LFDE versus HFDE rats, Holm–Sidak method after one-way ANOVA

Fig. 6

ASH suppressed inflammation caused by DIO in the hypothalamus. a, b Representative Western blot analysis for Iba1, TNFα, and IL-1β in the hypothalamus region of LFD, HFD, and HFDE animals. Histograms represent percent change in intensity taking value in LFD rats as 100%. c Histograms representing fold change in mRNA expression of iNOS, COX2, TNFα, and IL-1β in the hypothalamus region of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, @p ≤ 0.05 LFDE versus HFDE rats, Holm–Sidak method after one-way ANOVA

Fig. 7

ASH modulated leptin signaling pathway in the hypothalamus. a, b Representative Western blot analysis for OB-Rb in the hypothalamus region of rat brain. Histogram represents percent change in intensity taking value in LFD rats as 100%. c Histograms representing fold change in mRNA expression of OB-Rb, JAK2, STAT3, and SOCS3 in the hypothalamus region of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, @p ≤ 0.05 LFDE versus HFDE rats, Holm–Sidak method after one-way ANOVA

Fig. 8

ASH modulated NF-κB signaling pathway in the hypothalamus. a, b Representative Western blot analysis for markers of NF-κB pathway in the hypothalamus region of the brain among the LFD, HFD, and HFDE animals. Histograms represent percent change in intensity taking value in LFD rats as 100%. c Histograms representing fold change in mRNA expression of NF-κB and Bcl-xL in the hypothalamus region of rat brain among the four groups of animals (n = 3–4 each group). Values are expressed as mean ± SEM. * p ≤ 0.05 LFD versus HFD, LFDE, and HFDE rats, #p ≤ 0.05 HFD versus HFDE rats, $p ≤ 0.05 HFD versus LFDE rats, @p ≤ 0.05 LFDE versus HFDE rats, Holm–Sidak method after one-way ANOVA