Walnut Prevents Cognitive Impairment by Regulating the Synaptic and Mitochondrial Dysfunction via JNK Signaling and Apoptosis Pathway in High-Fat Diet-Induced C57BL/6 Mice

Abstract

This study was conducted to evaluate the protective effect of Juglans regia (walnut, Gimcheon 1ho cultivar, GC) on high-fat diet (HFD)-induced cognitive dysfunction in C57BL/6 mice. The main physiological compounds of GC were identified as pedunculagin/casuariin isomer, strictinin, tellimagrandin I, ellagic acid-O-pentoside, and ellagic acid were identified using UPLC Q-TOF/MS analysis. To evaluate the neuro-protective effect of GC, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2',7'-dichlorodihydrofluorecein diacetate (DCF-DA) analysis were conducted in H₂O₂ and high glucose-induced neuronal PC12 cells and hippocampal HT22 cells. GC presented significant cell viability and inhibition of reactive oxygen species (ROS) production. GC ameliorated behavioral and memory dysfunction through Y-maze, passive avoidance, and Morris water maze tests. In addition, GC reduced white adipose tissue (WAT), liver fat mass, and serum dyslipidemia. To assess the inhibitory effect of antioxidant system deficit, lipid peroxidation, ferric reducing antioxidant power (FRAP), and advanced glycation end products (AGEs) were conducted. Administration of GC protected the antioxidant damage against HFD-induced diabetic oxidative stress. To estimate the ameliorating effect of GC, acetylcholine (ACh) level, acetylcholinesterase (AChE) activity, and expression of AChE and choline acetyltransferase (ChAT) were conducted, and the supplements of GC suppressed the cholinergic system impairment. Furthermore, GC restored mitochondrial dysfunction by regulating the mitochondrial ROS production and mitochondrial membrane potential (MMP) levels in cerebral tissues. Finally, GC ameliorated cerebral damage by synergically regulating the protein expression of the JNK signaling and apoptosis pathway. These findings suggest that GC could provide a potential functional food source to improve diabetic cognitive deficits and neuronal impairments.

Keywords: JNK/NFκB pathway; Juglans regia; cognitive function; high-fat diet; inflammation; insulin resistance; walnut.

Figure 1

UPLC Q-TOF/MS^E chromatography in negative ion mode of ethyl acetate fraction from Gimcheon 1ho (GC) cultivar walnut (Juglans regia).

Figure 2

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia): (a) cell viability from H₂O₂-induced cytotoxicity in PC12 cells; (b) cell viability from high-glucose-induced cytotoxicity in PC12 cells; (c) cell viability from H₂O₂-induced cytotoxicity in HT22 cells; (d) cell viability from high glucose-induced cytotoxicity in HT22 cells. Results shown are mean ± SD (n = 3). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 3

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia): (a) ROS production from H₂O₂-induced cytotoxicity in PC12 cells; (b) ROS production from high glucose-induced cytotoxicity in PC12 cells; (c) ROS production from H₂O₂-induced cytotoxicity in HT22 cells; (d) ROS production from high glucose-induced cytotoxicity in HT22 cells. Results shown are mean ± SD (n = 3). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 4

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) in HFD-induced mice: (a) fasting glucose; (b) oral glucose tolerance test (OGTT) at 19 weeks old; (c) area under the curve (AUC) of OGTT. Results shown are mean ± SD (n = 5). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 5

Protective of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) in HFD-induced mice: (a) a number of arm entries; (b) spontaneous alternation behavior; (c) 3D moving routes in Y-maze test; (d) latency during habituation; (e) step-through latency in passive avoidance test; (f) escape latency in the hidden test; (g) retention time in W zone; (h) path tracing of each group in Morris water maze (MWM) test. Results shown are mean ± SD (n = 5). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 5

Protective of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) in HFD-induced mice: (a) a number of arm entries; (b) spontaneous alternation behavior; (c) 3D moving routes in Y-maze test; (d) latency during habituation; (e) step-through latency in passive avoidance test; (f) escape latency in the hidden test; (g) retention time in W zone; (h) path tracing of each group in Morris water maze (MWM) test. Results shown are mean ± SD (n = 5). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 6

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) on malondialdehyde (MDA) contents in HFD-induced mice biochemical changes related with antioxidant system: (a) hepatic MDA level; (b) cerebral MDA level Results shown are mean ± SD (n = 5). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 7

Protective of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) in HFD-induced mice: (a) serum level of FRAP; (b) serum level of AGEs formation. Results shown are mean ± SD (n = 5). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05. Bold line indicates mean.

Figure 8

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) on HFD-induced mice: (a) ACh level; (b) AChE activity; (c) representative western blots for total protein and expression of AChE, ChAT and β-actin; (d) protein expression levels of AChE; (e) protein expression levels of ChAT. Results shown are mean ± SD (a,b: n = 5, c–e: n = 3). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 9

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) on mitochondrial dysfunction in HFD-induced mice: (a) cerebral ROS contents; (b) cerebral MMP levels Results shown are mean ± SD (n = 5). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; ** and ^## p < 0.01. Bold line indicates mean.

Figure 10

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) on HFD-induced synaptic disorders and neuronal apoptosis in mice brain tissues: (a) representative Western blots for total protein and expression; (b) protein expression levels of p-JNK, p-Akt, p-tau, IDE, Aβ, BAX, caspase-3. Results shown are mean ± SD (n = 3). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 11

Protective effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) on HFD-induced neuroinflammation in mice brain tissues: (a) representative Western blots for total protein and expression; (b) protein expression levels of TNF-α, IL-1β, p-NFκB, caspase-1, and HO-1. Results shown are mean ± SD (n = 3). Data are statistically represented with * = significantly different from the NC group, and ^# = significantly different from PM group; * and ^# p < 0.05, ** and ^## p < 0.01. Bold line indicates mean.

Figure 12

A schematic illustration shows the immunomodulatory effect of 80% ethanolic extract from Gimcheon 1ho (GC) cultivar walnut (Juglans regia) on HFD-induced neuroinflammation in mice brain tissues.