In this study, we found that subcutaneous administration of low-dose ghrelin for 8 weeks provided a neuroprotective effect in a mouse model of PD. These protective effects may stem from three major functions of this peptide. First, ghrelin enhanced energy intake that induced the increase of body weight. Second, ghrelin blocked microglial activation and inhibited the release of IL-6, both of which can mitigate neuro-inflammation and promote neuronal survivability. Last, ghrelin elevated the activity of SOD1 and increased the Bcl-2/Bax ratio, which prevented neurons from oxidative damage and inhibited apoptosis during the course of the progressive degeneration of dopaminergic neurons, respectively.
PD patients exhibit non-motor symptoms, including hyposmia, sleep disturbances and gastrointestinal dysfunction, 10–20 years before they experience motor impairment [10]. Indeed, approximately 98.6% of PD patients suffered at least one non-motor symptom [11]. Moreover, PD patients usually have a lower body weight [12]. Ghrelin, the "hunger hormone", functions as a neuropeptide in the central nervous system [13]. In addition to regulating appetite, ghrelin increases body weight and fat mass by triggering receptors in the arcuate nucleus [3, 14, 15]. In PD patients, plasma total and active plasma ghrelin levels were decreased, and postprandial ghrelin suppression and preprandial peak responses were both attenuated [7]. In this study, we also found homozygous A53T mice plasma ghrelin were decreased at 3 months age when non-motor symptom exhibited. Injections of ghrelin in both humans and rodents have been shown to increase plasma ghrelin level, and thus increase food intake and motivation to seek out food as well [16]. Here, we showed that the plasma total and active ghrelin levels were both recovered at the age of 3 months after treatment with ghrelin for 8 weeks. Ghrelin injections increased food intake in a dose-dependent manner, and ghrelin could only stimulate food intake when it was elevated much higher than normal levels. In our study, we first reported that nearly normal ghrelin levels rescued weight loss in homozygous A53T mice 3–6 months old. However, the plasma ghrelin level was lower in obese individuals than leaner individuals [16], suggesting that ghrelin did not contribute to obesity. In line with this research, ghrelin did not increase the body weight of normal mice, and there was “ghrelin resistance” in mice with adiposis [17]. Therefore, it could explain why normal physiological ghrelin dose treatment had no effect on WT mice body weight. Report has found that ghrelin increased the content of DA in the SN through its receptor [18]. Even though all ghrelin administrations were began at the age of 1 month, the administration process with 8 weeks rather than 4weeks, could recover the decreased plasma ghrelin and restrict both the progressive loss of dopaminergic neurons in the SN and the depletion of DA in the Str. The administration time was refer to Therefore, our results indicated that early intervention of ghrelin may delay the progression of PD or even prevent the onset of PD, and also suggested that the lasting time of drug delivery was an important factor for ghrelin clinical application.
Accumulating evidence has suggested a strong link between neurodegeneration and chronic inflammation resulting in the activation of microglia and astrocytes and the increased release of pro-inflammatory cytokines [19]. Elevated pro-inflammatory cytokines were observed in the brain, cerebrospinal fluid and serum of PD patients [20–23]. Lipopolysaccharides (LPS) or viral pathogens could also induce PD-like neuronal death in rodents [24–26]. This study found increased activation of microglia and increased release of IL-6 in the SN of homozygous A53T mice. Ghrelin markedly inhibited oligodendrocyte cell death in oligodendrocytes and LPS-stimulated BV-2 cell co-culture systems, which imitated microglial activation in vitro [27]. Ghrelin was reported to reduce microglial activation in an MPTP-induced PD model, inhibiting the increased expression of TNFα and IL-1β mRNA and inducible nitric oxide synthase in the SN [28]. Nevertheless, the anti-inflammatory effects of ghrelin may originate from the attenuation of the release of inflammatory cytokines, such as IL-6, TNFα, IL-1β and cyclooxygenase-2 [29–31]. However, no significant difference was found in the concentration of TNFα between the homozygous A53T and WT mice. In addition, in vitro and in vivo studies have revealed the neuroprotective activity of IL-10 on dopaminergic neurons when exposed to LPS and in a 6-OHDA PD model [32–34]. This finding is in accordance with results of the inhibition of microglia, decreased pro-inflammatory cytokine IL-6 and increased anti-inflammatory cytokine IL-10 in the SN of G-A53T mice, suggesting anti-inflammatory and neuroprotective roles of ghrelin on dopaminergic neurons.
Increased levels of oxidation and its products, including nucleic acids, proteins and lipids, were detected in post-mortem PD brains [35, 36], suggesting that oxidative stress plays a vital role in the loss of dopaminergic neurons. Superoxide dismutase Cu-Zn, also known as SOD1, can bind copper and zinc ions and act as a homodimer to destroy naturally occurring, but harmful, superoxide radicals in the body [37]. The upregulation of malonaldehyde and the downregulation of SOD1 and catalase have been found in MPTP/MPP+-induced PD models [38, 39]. Correspondingly, this study found that homozygous A53T mice exhibited lower SOD1 protein levels in the SN at the age of 6 months. Nevertheless, exogenous ghrelin could antagonize MPTP-induced oxidative stress by reversing the reduced SOD1 [39]. This study observed that ghrelin administration for 8 weeks boosts the decreased levels of SOD1 in the SN of homozygous A53T mice, indicating that ghrelin may exert a neuroprotective effect on dopaminergic neurons against oxidative stress. Additionally, ghrelin pre-treatment could reduce ROS generation and oxidative stress by increasing the activity of uncoupling protein 2 (an important mitochondrial protein in control of ROS production that acts as a sensor for mitochondrial oxidative stress), resulting in decreased dopaminergic neuron loss in the MPTP-induced PD model [18].
The present study found that homozygous A53T mice exhibited a lower Bcl-2/Bax ratio. Emerging evidence indicates that ghrelin has anti-apoptotic effects in multi-pathophysiological conditions, including ischaemia, inflammation, and nutrient deprivation [40–42]. Recently, it has been reported that the anti-apoptotic effects of ghrelin in neurons occur via multiple signalling pathways, including the PI3K/Akt, ERK1/2, GSK-3β, JNK and p38 pathways [4]. Report have shown that ghrelin could increase Bcl-2/Bax ratio, prevent of cytochrome c release and inhibit activation of caspase-3, thus promoting the survival of cortical neuronal cells [19]. Moreover, exogenous ghrelin also inhibited the activation of caspase-3, c-Jun and p-38 in oxygen-glucose deprivation-induced apoptosis [19, 43]. Our previous study demonstrated that ghrelin could also attenuate Bax expression and caspase-3 activation to promote the survival of dopaminergic neurons in a MPTP-induced PD mouse model [5]. Additionally, ghrelin administration for 8 weeks promoted a decreased Bcl-2/Bax ratio in the SN of homozygous A53T mice. These findings suggest that ghrelin exerts its neuroprotective effect on dopaminergic neurons by anti-apoptosis processes.
In summary, with the progression of disease in homozygous A53T mice, the plasma ghrelin levels decreased, and continuous ghrelin administration could restore the decreased plasma ghrelin to normal levels. Continuous dose of ghrelin did not lead to obesity in WT mice. With the recovery of the plasma ghrelin, the degeneration of dopaminergic neurons in homozygous A53T mice was improved significantly in terms of increased numbers of TH-positive cells and TH protein levels in the SN. Treatment with ghrelin for 4 weeks had no neuroprotective effects on dopaminergic neurons at the age of 3 months; however, treatment with ghrelin for 8 weeks had a neuroprotective effect on dopaminergic neurons in the SN at the age of 3 months, and this effect lasted through 6 months of age. This early intervention with ghrelin may delay the progression of disease in homozygous A53T mice by inhibiting apoptosis decreasing oxidative stress and inflammation. Therefore, ghrelin can improve the progression of neurodegenerative diseases, which will have important potential clinical value for preventing the onset of PD, especially familial PD.