Honey and levodopa comparably preserved substantia nigra pars compacta neurons through the modulation of nuclear factor erythroid 2-related factor 2 signaling pathway in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease model

Abstract

Parkinson's disease (PD) affects about 8.5 million individuals worldwide. Oxidative and inflammatory cascades are implicated in the neurological sequels, that are mostly unresolved in PD treatments. However, proper nutrition offers one of the most effective and least costly ways to decrease the burden of many diseases and their associated risk factors. Moreover, prevention may be the best response to the progressive nature of PD, thus, the therapeutic novelty of honey and levodopa may be prospective. This study aimed to investigate the neuroprotective role of honey and levodopa against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced oxidative stress. Fifty-four adult male Swiss mice were divided into control and PD model groups of 27 mice. Each third of the control mice either received phosphate buffered saline, honey, or levodopa for 21 days. However, each third of the PD models was either pretreated with honey and levodopa or not pretreated. Behavioral studies and euthanasia were conducted 2 and 8 days after MPTP administration respectively. The result showed that there were significantly (P<0.05) higher motor activities in the PD models pretreated with the honey as well as levodopa. furthermore, the pretreatments protected the midbrain against the chromatolysis and astrogliosis induced by MPTP. The expression of antioxidant markers (glutathione [GSH] and nuclear factor erythroid 2-related factor 2 [Nrf2]) was also significantly upregulated in the pretreated PD models. It is thus concluded that honey and levodopa comparably protected the substantia nigra pars compacta neurons against oxidative stress by modulating the Nrf2 signaling molecule thereby increasing GSH level to prevent MPTP-induced oxidative stress.

Keywords: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Honey; Levodopa; Nrf2; Parkinson disease.

Fig. 1

Honey and levodopa pretreatment comparably preserved locomotion (A–C), motor coordination (D), and gait (E, F). ^a,b,c,dIndicate significant differences compared to PBS, MPTP, levodopa, and honey groups respectively, while *signifies significant difference between honey & MPTP and levodopa & MPTP. All at P<0.05; n=6. PBS, MPTP, levodopa, honey, honey & MPTP, levodopa & MPTP stands for the mice that received, PBS. MPTP, honey, honey & MPTP, and levodopa & MPTP respectively. PBS, phosphate buffered saline; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 2

Nissl substance of SNpc neurons. (A) CFV stained sections of the midbrain (scale bar=10 µm), slide; PBS (PBS group) showed neurons with intact Nissl bodies (black arrows); MPTP (MPTP group) exhibits neurons with pale cytoplasm, indicating Nissl body loss (red arrows); levodopa (levodopa group) displayed neurons with intact Nissl bodies (black arrows); honey (honey group) showed deeply stained neurons with intact Nissl bodies (black arrows); honey & MPTP (honey & MPTP group) depicts mostly intact Nissl bodies (black arrows), with a few lost ones; levodopa & MPTP (levodopa & MPTP group) presents neurons with well-stained cytoplasm indicating intact Nissl bodies (black arrows) and some pale neurons (red arrows). (B) Chromatolytic cell count. A neuron is deemed chromatolytic when it loses its Nissl bodies. ^a,b,dIndicate significant differences compared to PBS, MPTP, and honey groups respectively, at P<0.05; n=6. PBS, MPTP, levodopa, honey, honey & MPTP, levodopa & MPTP stands for the mice that received, PBS. MPTP, honey, honey & MPTP, and levodopa & MPTP respectively. SNpc, substantia nigra pars compacta; CFV, cresyl fast violet; PBS, phosphate buffered saline; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 3

Honey and levodopa preserved SNpc neurons (A) immunostaining for TH protein (scale bar=50 µm), slide: PBS (PBS group) showed numerous dopaminergic neurons (black arrow). MPTP (MPTP group) exhibited fewer dopaminergic neurons. Levodopa (levodopa group) showed a high number of dopaminergic neurons. Honey (honey group) also had numerous dopaminergic neurons. Honey & MPTP (honey & MPTP group) and levodopa & MPTP (levodopa & MPTP group) demonstrated more dopaminergic neurons than the MPTP slide. (B) TH+ cell count. ^a,bIndicate significant differences compared to PBS and MPTP groups respectively, at P<0.05; n=6. PBS, MPTP, levodopa, honey, honey & MPTP, levodopa & MPTP stands for the mice that received, PBS. MPTP, honey, honey & MPTP, and levodopa & MPTP respectively. SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; PBS, phosphate buffered saline; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 4

Astrogliosis of the SNpc. (A) immunostaining for GFAP in the midbrain (scale bar=50 µm), slide: PBS (PBS group) depicted fewer astrocytes; MPTP (MPTP group) exhibited notably numerous reactive astrocytes (red arrows); levodopa (levodopa group) and honey (honey group) showed only a few astrocytes; honey & MPTP (honey & MPTP group) and levodopa & MPTP (levodopa & MPTP group) displayed fewer reactive astrocytes compared to the MPTP group. (B) GFAP+ cell count. The cells identified as astrocytes are GFAP-positive (GFAP+ cells). ^a,b,dSignify significant differences compared to PBS, MPTP, and honey groups respectively, at P<0.05; n=6. PBS, MPTP, levodopa, honey, honey & MPTP, levodopa & MPTP stands for the mice that received, PBS. MPTP, honey, honey & MPTP, and levodopa & MPTP respectively. SNpc, substantia nigra pars compacta; GFAP, glial fibrillary acidic protein; PBS, phosphate buffered saline; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 5

Honey and levodopa pretreatment comparably prevented oxidative damage (A) oxidative stressors, (B–D) antioxidants, and (E) product of oxidative stress. ^a,b,c,dSignifies significant differences compared to PBS, MPTP, levodopa, and honey groups respectively, while *signifies significant difference between honey & MPTP and levodopa & MPTP. All at P<0.05; n=6. PBS, MPTP, levodopa, honey, honey & MPTP, levodopa & MPTP stands for the mice that received, PBS. MPTP, honey, honey & MPTP, and levodopa and MPTP respectively. PBS, phosphate buffered saline; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 6

Graphical summary of the proposed mechanism of action of the neuroprotective effects of honey and levodopa against MPTP-induced PD in adult male Swiss mice. MPTP induced oxidative stress, raising ROS levels. Honey and levodopa possibly activated Nrf2, boosting glutathione production, and countering ROS effects. This reduced malondialdehyde and astrogliosis, preserving SNpc neurons and motor functions. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; SNpc, substantia nigra pars compacta; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; DA, dopaminergic.