A Natural Dietary Supplement with a Combination of Nutrients Prevents Neurodegeneration Induced by a High Fat Diet in Mice

Abstract

Obesity and metabolic disorders can be risk factors for the onset and development of neurodegenerative diseases. The aim of the present study was to investigate the protective effects of a natural dietary supplement (NDS), containing Curcuma longa, silymarin, guggul, chlorogenic acid and inulin, on dysmetabolism and neurodegeneration in the brains of high fat diet (HFD)-fed mice. Decrease in the expression of FACL-4, CerS-1, CerS-4, cholesterol concentration and increase in the insulin receptor expression and insulin signaling activation, were found in brains of NDS-treated HFD brains in comparison with HFD untreated-mice, suggesting that NDS is able to prevent brain lipid accumulation and central insulin resistance. In the brains of NDS-treated HFD mice, the levels of RNS, ROS and lipid peroxidation, the expression of p-ERK, H-Oxy, i-NOS, HSP60, NF-kB, GFAP, IL-1β, IL-6 and CD4 positive cell infiltration were lower than in untreated HFD mice, suggesting antioxidant and anti-inflammatory effects of NDS. The decreased expression of p-ERK and GFAP in NDS-treated HFD mice was confirmed by immunofluorescence. Lastly, a lower number of apoptotic nuclei was found in cortical sections of NDS-treated HFD mice. The present data indicate that NDS exerts neuroprotective effects in HFD mice by reducing brain fat accumulation, oxidative stress and inflammation and improving brain insulin resistance.

Keywords: HFD mice; insulin resistance; natural antioxidants; neurodegeneration; obesity.

Figure 1

Effects of NDS on the liver. (A) Liver sections of control (STD), HFD (untreated-HFD) and NDS-treated (treated-HFD) mice; (B) Intrahepatic cholesterol concentration; (C) Western blot of proteins extracted from livers of STD, untreated-HFD and NDS treated-HFD mice and incubated with anti-fatty acid-CoA ligase-4 (FACL-4) and anti-β-Actin (loading control); (D) Quantification of immunoreactivity was performed using densitometric analysis. Data are the mean values ± SEM (n = 8/group). * p ≤ 0.05 vs. STD; # p ≤ 0.05 vs. untreated-HFD.

Figure 2

Lipid accumulation in the brain. (A) Western blot of proteins extracted from brains of STD, untreated-HFD and NDS treated-HFD mice and incubated with anti-FACL-4 and anti-β-Actin (loading control); (B) Quantification of immunoreactivity was performed using densitometric analysis; (C) CerS-1 and CerS-4 transcript levels determined by quantitative Real-Time PCR; (D) Cholesterol concentration in the brain tissue; (E) Nile Red staining of brain homogenates; (F) Quantification of E fluorescence intensity; (G) Total brain lipid levels. Data are the mean values ± SD (n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.

Figure 3

NDS decreases the HFD-induced insulin signaling alterations in the brain. (A) Fasting plasma glucose concentration in STD, untreated-HFD and treated-HFD mice; (B) Western blot of proteins extracted from brains of STD, untreated-HFD and NDS treated-HFD mice and incubated with anti-Insulin Receptor (IR) and anti-β-Actin (loading control); (C) Quantification of immunoreactivity was performed using densitometric analysis; (D) Western blot of proteins extracted from brains of STD, untreated-HFD and NDS treated-HFD mice and incubated with anti-Akt, anti-phospho-Akt (p-Akt) and anti-β-Actin (loading control); (E) Quantification of Akt/p-Akt ratio. Data are the mean values ± SD (n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.

Figure 4

Nitric and oxidative stress and lipid peroxidation in the brains of HFD-mice were prevented by NDS treatment. (A) Nitrite concentration in STD, untreated-HFD and treated-HFD brains; (B) Levels of ROS in STD, untreated-HFD and treated-HFD brains (C) Lipid peroxidation levels in STD, untreated-HFD and treated-HFD brains; (D) Western blot of proteins extracted from brains of STD, untreated-HFD and treated-HFD mice and incubated with anti-p-ERK, anti-H-Oxy, anti-i-NOS, anti-HSP60 and anti-β-Actin (loading control); (E) Quantification of immunoreactivity was performed using densitometric analysis; (F) Immunofluorescence of superficial and deep cerebral cortex sections of STD, untreated-HFD and treated-HFD mice incubated with anti-phospho-ERK; (G) Schematic representation of superficial (i) and deep (ii) cerebral cortex positive areas; (H) Brain map indicating the levels of positive p-ERK staining. Representative images from 3 animals per group are shown. Bar 20 μm. Data are the mean values ± SD (n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.

Figure 5

NDS prevents HFD-induced inflammation and immunological response in the brain. (A) Western blot of proteins extracted from brains of STD, untreated-HFD and treated-HFD mice and incubated with anti-GFAP and anti-β-Actin (loading control); (B) Quantification of immunoreactivity was performed using densitometric analysis; (C) Immunofluorescence of superficial and deep cerebral cortex sections incubated with anti-GFAP; (D) Schematic representation of superficial (i) and deep (ii) cerebral cortex positive areas; (E) Brain map indicating the levels of positive staining of GFAP; (F) Western blot of proteins extracted from brains of STD, untreated-HFD and treated-HFD mice and incubated with anti-NFkB and anti-β-Actin (loading control); (G) Quantification of immunoreactivity was performed using densitometric analysis; (H,I) Levels of IL-6 and IL-1β in brains of STD, untreated-HFD and treated-HFD mice, quantified by ELISA assay. (L) Brain sections from STD, untreated-HFD and treated-HFD mice incubated with anti-CD4. Representative images from 3 animals per group are shown. Bar 20 μm. Data are the mean values ± SD (n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.

Figure 6

NDS inhibits neurodegeneration induced by HFD. (A) Brain sections from STD, untreated-HFD and treated-HFD mice incubated with Hoechst 33342. Fragmented apoptotic nuclei are indicated by the arrows; (B) The outlined area is enlarged in the squares; (C) TUNEL assay on superficial and deep cortical regions from STD, untreated-HFD and treated-HFD mice; (D) Number of apoptotic nuclei in the superficial cerebral cortex positive area; (E) Number of apoptotic nuclei in the deep cerebral cortex positive area. Representative images from 3 animals per group are shown. Bar 20 μm. Data are the mean values ± SD (n = 8/group). * p ≤ 0.05 vs. STD. # p ≤ 0.05 vs. untreated-HFD.