Ghrelin/GHSR System in Depressive Disorder: Pathologic Roles and Therapeutic Implication

1. Introduction

Growth hormone-releasing peptide (ghrelin) is a multifunctional 28-amino acid peptide hormone, which was originally discovered in rat stomach but has also been found in a variety of other organs including bowels, hypothalamus, pituitary, and hippocampus[1]. Ghrelin's serine 3 (Ser3) is n-octastylated, a modification that allows for the cycling of both the acylated and des-acylated forms of ghrelin[2]. Acylated ghrelin is able to cross the blood-brain barrier (BBB) and binds to the central growth hormone-releasing peptide receptor (GHSR), which are widely found in the central nervous system (CNS) and peripheral tissues and has two isoforms, GHSR1a and GHSR1b[3]. Acylated ghrelin mainly binds to GHSR1a and exerts a variety of physiological and behavioral modulatory effects, such as regulation of glucose homeostasis, regulation of metabolism and energy homeostasis, modulation of blood pressure, and renal protection[4,5,6]. GHSR1b contains 289 amino acids and is a splice variant and dominantly inactivated form of GHSR1a[7]. Due to the absence of the 6th and 7th transmembrane chains, it is unable to bind ghrelin and does not have signaling ability. There is much evidence that GHSR1b does not bind ghrelin, but it can heterodimerize with GHSR1a to interfere with GHSR1a function[8]. In an experiment to study, the expression of two GHSRs cloned from black seabream in HEK293 cells, GHSR1b was found to inhibit the signaling activity of GHSR1a-mediated elevation of intracellular Ca²⁺ concentration[9]. GHSR1a has also been shown to form mixed heterodimers with dopamine receptor subtype 2 (D2R)[10], serotonin 2c receptors (5-HT2cR)[11], orexin 1 receptor (OX1R)[12], and the dimers can fine-tune the activity of the GHSR1a receptor.

Depression is a prevalent severe chronic mental illness, which is characterized by appetite disorders, weight changes, bipolar disorder and insomnia[13,14,15]. The above mentioned disorders may be attributed to decreased size and function of the hippocampus and amygdala in the limbic brain regions[16]. With the rise in survival pressures in contemporary society and the dramatic increase in prevalence and incidence in different parts of the country, depression is emerging as a complex disorder present in all stages of the population, including the pregnant population, the adolescent population, and the elderly population[17,18]. Although the exact pathogenesis of the disorder remains obscure, a large body of data strongly suggests that stress, and genetic predisposition are collectively involved in the progression of the disorder [19]. The pathophysiologic processes of depression include inflammation in the brain, reduced neurogenesis, monoamine neurotransmitter changes, and endocrine abnormalities[20,21,22]. However, the interrelationships between these processes remain unclear. Currently, several classes of drugs are available for the treatment of major depressive disorder (MDD) [23], and most of them exert their antidepressant effects by enhancing monoaminergic function, such as typical monoamine oxidase inhibitors (MAOIs) and selective 5-hydroxytryptamine reuptake inhibitors (SSRIs)[24]. In addition, scientists are now working on research beyond typical monoamine targets and pathways to develop antidepressants with novel mechanisms of action[25].

However, the efficacy and severe side effects of these antidepressants remain major issues to be addressed[26], and there are no therapeutic candidates shown to completely eliminate disease progression. In line with ghrelin’s effects in the CNS, there is increasing evidence for an important role of ghrelin in depression[27,28,29]. The majority of research has elucidated the anti-depressive mechanisms of ghrelin. Conversely, few studies reported a detrimental effect as ghrelin itself induces depressive tendencies and patterns in animal models. Here, we have attempted to elucidate the aforementioned link between ghrelin and the onset as well as the treatment of depression, and to elucidate the mechanisms and pathways that may be involved in the development of ghrelin in depression. We made such an explanatory work by conducting a comprehensive review of the existing literature (reviews and research articles) through keyword searches.

2. Mechanisms of Ghrelin/GHSR System in Depressive Disorder

Numerous studies have shown that ghrelin/GHSR system inhibits key pathways and mechanisms in the development of depression that have been found to correlate with certain factors, transmitters, and cells in the body in a variety of models of depression, which we will describe in more detail in the following paragraphs.

2.1. Links between Monoamine Neurotransmitter Receptors and Ghrelin

The monoamine hypothesis, long considered to be the most common hypothesis of depression, is centred on the fact that the concentration of monoamines (5-hydroxytryptamine, norepinephrine, and dopamine) in the synaptic gap is reduced in depressive states[30]. The expression of these monoamine neurotransmitters may be involved in the neuropathological process of ghrelin. It was found that in neurons co-expressing D1R and GHSR, ghrelin can amplify dopamine signalling by activating GHSR to enhance its mediated downstream pathways[31]. And reduced mRNA expression of dopaminergic receptors in the amygdala and the dorsal nucleus of the middle suture was found in studies of GHSR1a-/- mice[32], suggesting a potential regulation of dopamine neuron production by the ghrelin/GHSR system. In addition to this, another study showed that the central 5-hydroxytryptamine system is a target of ghrelin[32], and increased mRNA expression of some 5-hydroxytryptamine receptors in the amygdala and the dorsal nucleus of the intermediate suture was found in mice acutely administered ghrelin centrally. Conversely, unlike the ghrelin/GHSR system described above, which regulates the expression of monoamine neurotransmitters, norepinephrine has been shown to stimulate ghrelin secretion in mouse cells[33], and depletion of catecholamine-secreting neurons can also inhibit fasting-induced ghrelin secretion[34]. In conclusion. There are many unknown connections between the monoamine system and the ghrelin/GHSR system, and more research is needed to determine whether these monoamine receptors can be used as targets in the treatment of depression.

2.2. The Ghrelin/GHSR System Mediates the Inflammatory Response to Depression

In the CNS, IL-1β stimulates microglia and astrocytes to produce other cytokines, such as IL-6 and TNF-α, to promote inflammation in the brain[35]. Furthermore, pro-inflammatory cytokines such as IL-1β and TNF-α have been reported to play an important role in the onset of depression[36], and an increase in pro-inflammatory cytokines contributes to the development of depression[20]. And IL-6 is the most persistently elevated cytokine in the blood of MDD patients, so it may serve as a predictive biomarker and a potential target for the treatment of depression in humans[37]. These experimental conclusions show that the role of pro-inflammatory cytokines of the CNS in the pathogenesis of depression cannot be ignored.

Serum ghrelin concentrations have been reported to increase in inflammatory bowel disease (IBD) and are associated with the degree of activity[38]. Similar reports have been made in animal model experiments: Serum ghrelin concentrations were higher in rats with severe pancreatitis[39]and arthritis[35], as well as acute colitis[40]. It can be seen that serum ghrelin concentration is positively correlated with inflammation. Nonetheless, an experiment is at odds with the conclusion just mentioned, it is believed that rheumatoid arthritis patients and arthritis rats have lower levels of serum ghrelin concentrations[41]. This discrepancy may be due to unpredictable reasons such as rat nutritional status or experimental conditions, making it difficult to analyze the effect of inflammation on serum ghrelin levels. However, it is undeniable that ghrelin levels are elevated in inflammation as demonstrated in most experiments. On the whole, the causal relationship between inflammation and changes in serum ghrelin concentrations requires further study, although the effects of various conditions cannot be ruled out. Pursuant to the existing data, there are many reports that elevated serum ghrelin concentrations are the result of inflammation. Interestingly, experiments have found that elevated ghrelin is involved in regulating inflammatory response, downregulating neutrophil transport and the number of pro-inflammatory cytokines, significantly reducing cerebral ischemic injury and improving neurobehavioral function[42]. The same study also reported that exogenous ghrelin inhibits endothelial cell production of IL-1, IL-6, and IL-8 by regulating the release of pro-inflammatory cytokines, which play an important role in the pathological process of depression[43]. In addition, GHSR and ghrelin are expressed in human T lymphocytes and monocytes, and ghrelin specifically inhibits the expression of pro-inflammatory cytokines via GHSR[44]. Also, two other investigations displayed decreased expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL6 in rats with GHSR gene knockouts[45,46]. Thus, there is increasing evidence of the mediating role of the Ghrelin/GHSR system in regulating pro-inflammatory cytokine release events in depressive disorders.

On the other hand, we can note that inflammation reduces neuroplasticity by downregulating brain-derived neurotrophic factor (BDNF), which may be the basis of the pathophysiology of depression[47,48]. IL-6 has been shown to be a reliable positive predictor of BDNF in patients with melancholic MDD[49]. However, the correlation between inflammation and BDNF requires further study. And the Ghelin/GHSR system can also produce antidepressant effects through the regulation of BDNF, which we will elaborate below.

2.3. The Ghrelin/GHSR System Promotes Neurogenesis in Depression

Numerous clinical studies have shown that depression is closely associated with decreased size and function of the hippocampus and amygdala in limbic brain regions, and that patients with depression have reduced hippocampal volume and decreased neurogenesis[50,51]. Where neurogenesis is closely related to the treatment of depression. Studies have found that ablation of hippocampal nerve genesis in mice impairs the efficacy of antidepressants[21]. In animal experiments, antidepressant drugs promote hippocampal neurogenesis. Similarly, alterations in adult hippocampal neurogenesis mediate the effects of antidepressants, and chronic administration of antidepressants enhances adult hippocampal neurogenesis [52].

The neuroprotective effects of ghrelin as an neuropeptide have been widely demonstrated. In rat cortical neuronal damage induced by hypoxia and hypoglycaemia[53], ghrelin inhibited the neuronal damage process and the protective effect disappeared after administration of a GHSR-specific inhibitor. In addition to this, a reduction in oligodendrocyte and neuronal apoptosis was found in an experiment in which ghrelin was administered after spinal cord injury in rats[54]. Most notably, the antidepressant effects exerted by ghrelin in vivo can be dependent on its neuroprotective effects. Published literature has displayed that ghrelin directly increases hippocampal neurogenesis in the treatment of depression[55]. Further studies have identified hippocampal neuroprotection as the primary mechanism by which stress-induced elevation of ghrelin protects the organism from stress-induced worsening of associated depression[56]. In addition to the protective effects of ghrelin on hippocampal neurons against depression, activation of catecholaminergic neurons has also been identified as a possible mechanism for contributing to the antidepressant effects of ghrelin[57]. Although numerous studies have demonstrated that ghrelin exerts its antidepressant effects through neuroprotection, the mechanisms involved are currently not well understood. It has been found that in the hippocampus, ghrelin is able to cross the BBB and bind to GHSR1a to improve cognitive function and enhance hippocampal neurogenesis[58], and to enhance LTP[59]. Ghrelin also directly induces proliferation and differentiation of adult neural progenitor cells in the hippocampal subgranular zone, and in the case of ghrelin receptor knockout mice (GHSR1a-/-) systemic administration, it was found that the number of progenitor cells was reduced compared to wild-type controls[55]. In addition to this, another report concluded that ghrelin provides neuroprotection through activation of AMPK and enhances clearance of damaged mitochondria[60], and it has also been demonstrated that ghrelin mediates neuroprotection through inhibition of glial cell activation and release of pro-inflammatory mediators[61]. In addition to the above mentioned related mechanisms, it has been experimentally confirmed that BDNF plays an important role in the pathophysiology of depression[62], and ghrelin exerts an antidepressant effect by regulating the relevant expression of BDNF is also a hot topic of research.

2.4. Regulation of Astrocyte Physiology by the Ghrelin/GHSR System

The above-mentioned neuronal physiology is closely related to the onset of depression. In addition to this, as non-neurons, the significant reduction in density and massive damage of astrocytes after the onset of depression has been confirmed by a number of experiments[63,64,65]. A study elucidated that astrocytes show dysfunction and a significant reduction in number in the medial prefrontal cortex (mPFC) region in depressed patients[66]. In addition, significant glial cell pathology is present in patients with MDD and cells with chronic unpredictable mild stress (CUMS) can be reversed by antidepressant pharmacological modulation that promotes glial cell Glu clearance[67]. Another investigations have detected that long-term administration of the antidepressant fluoxetine reversed the stress-induced decline in the number of hippocampal glial cells in tree shrews, and the relevance of structural plasticity of astrocytes in stress and therapeutic support with antidepressants has been proposed[65]. These all suggest that antidepressant-mediated changes in astrocytes may be key to their effects. Taken together, these suggest that astrocytes may play an active role in brain function and that there is some connection to the process of depression.

Traditionally, astrocytes have often been thought of as brain glue, a class of cells that only provide metabolic and functional support to neurons. However, with the discovery of various neurotransmitter receptors and channels on the astrocyte membrane, our understanding of the function of astrocytes in the nervous system has fundamentally changed[68]. A large number of studies have found that the most prominent neurotransmitter receptors expressed on astrocyte membranes are the family of GPCRs, including metabotropic glutamate receptors, adrenergic receptors, GABAergic receptors, cholinergic receptors, histaminic receptors, dopaminergic receptors, and neurotrophic factor receptors[69,70,71,72,73,74]. Upon activation of these receptors by neurotransmitters released from presynaptic neurons, astrocytes can undergo "gliotransduction"[75], releasing gliotransmitters to feedback on neuronal excitability and synaptic transmission[76,77]. Thus, astrocytes are now considered to be active participants in neuronal communication.

GHSR1a has been shown to be expressed in astrocytes in the arcuate nucleus of the hypothalamus[78]and in the dentate gyrus in the hippocampus[79], and that astrocytes respond to ghrelin and its analogues. Thus, we can make the hypothesis that glial cells may act as mediators of ghrelin activity in the brain. It is interesting to see whether ghrelin, as an important mediator in the development of depression, also plays a role in depression in some way linked to astrocytes. A study found that the astrocytoma cell line C6 could respond to GHRP-6 by upregulating GHSR1a levels, increasing activation of the PI3K/Akt pathway and increasing its own proliferation[80]. And this effect can be inhibited by D-Lys3-GHRP-6, an antagonist of GHSR1a[79], so ghrelin may exert neuroprotective effects by stimulating astrocyte proliferation through GHSR1a to increase the expression of the PI3K/Akt pathway. However, another study found that ghrelin did not show a promotive effect on astrocyte proliferation, and it found that ghrelin reversed the activation and accumulation of astrocytes in hippocampal neurodegeneration following hippocampal excitotoxicity injury induced by sea manate[61]. This difference may be related to the activation of astrocytes and the increase in the concentrated release of pro-inflammatory cytokines in inflammatory and related conditions and the triggering of neuroinflammation[81]. In conclusion, the specific mechanisms associated with ghrelin and astrocytes need to be investigated more thoroughly. ghrelin may have a neuroprotective role in the development of depression through the regulation of astrocyte physiological activity by the receptor GHSR1a.

2.5. The Role of the Ghrelin/GHSR System in Endocrine Disruption in Depression

Depression has long been recognised as having a correlation with endocrine disruption, with overactivity of the hypothalamic-pituitary-adrenal (HPA) axis being the most common[82]. In addition, the hypothalamic-pituitary-thyroid (HPT) axis and the hypothalamic-pituitary-gonadal (HPG) axis are also disturbed in depressed patients [83,84]. Therefore, changes in the levels of related hormones in the body can provide ideas for research on the treatment of depression. For example, testosterone levels are reduced in men suffering from depression[85], and the vulnerability of perimenopausal women to depression is associated with changes in oestrogen[86]. Then we can consider testosterone and oestrogen as targets for further research on antidepressants.

There is growing evidence that ghrelin is involved in the regulation of endocrine disruption in depression. Ghrelin has been reported to inhibit the secretion of luteinising hormone (LH) and thyroid stimulating hormone (TSH) in patients with major depression[87]. Ghrelin also affects the activity of the HPT axis by decreasing TSH and increasing free thyroxine in plasma[88]. In addition, the β1-adrenergic receptor blocker atenolol exacerbates depressive-like behaviours in chronic social defeat stress (CSDS) mice by attenuating the elevation of plasma acylgrowth factor-releasing peptide[89]. In conclusion, the relevant role of ghrelin in endocrine disruption in depressed patients requires more research.

3. Signalling Pathways Induced by Ghrelin/GHSR1a System in Depression

3.1. cAMP-CREB-BDNF Signaling Pathway

Neurodegenerative and neuropsychiatric disorders can be caused by an inadequate supply of neurotrophic factors[90]. Among them, BDNF, as a member of the neurotrophic protein family, plays an extremely important role in promoting neuronal growth, survival, differentiation and in synaptic transmission and enhancing central plasticity[91,92,93], and slows down depressive progression. Relatedly, ghrelin increases total BDNF mRNA expression in the mouse hippocampus and synthesises different kinds of BDNF mRNAs by acting on different promoters in rats of different ages[94]. Different BDNF transcripts exhibit different subcellular localisations that selectively shape the proximal and distal compartments of the cytosol or dendrites[95]and play an important role in increasing neuronal plasticity[96].

Impaired cAMP signalling occurs in patients with major depression[97]. In addition, in the hippocampus and prefrontal cortex of patients, the levels of BDNF, CREB and p-CREB are significantly reduced[98,99,100,101]and the levels of BDNF mRNA in peripheral monocytes of this population are also significantly reduced[102]. Interestingly, administration of ghrelin through the centre normalised hippocampal BDNF levels[103]. In addition, exogenous ghrelin can improve depressive behaviour by upregulating CREB signaling through activation of ghrelin receptors and cAMP/PKA signaling pathway, and increasing BDNF expression downstream[27]. Thus, ghrelin-induced increases in BDNF in the hippocampus involve activation of the GHSR1a/cAMP/PKA/CREB signalling pathway (Figure 1).

3.2. p38-MAPK Signaling Pathway

Several studies have demonstrated that p38-MAPK is activated in response to various stressful stimuli and is involved in the pathological process of depression [104,105]. p38-MAPK can be activated by interferon and lipopolysaccharide to up-regulate the expression of the depression-related gene IDO[106,107], and it can also exacerbate oesophageal cancer-associated depression by directly enhancing the expression of the IDO gene[108]. Therefore, the expression status of p38-MAPK pathway-related substances could be a powerful tool for depression monitoring, while p38-MAPK itself could be a target for antidepressant research. In addition, activation of the p38-MAPK pathway phosphorylates the glucocorticoid receptor (GR), and GR phosphorylation is associated with reduced glucocorticoid sensitivity[109], which may be closely related to glucocorticoid resistance exhibited by depressed patients[110]. In contrast, ghrelin treatment of rats activates GHSR1a and decreases p38-MAPK phosphorylation, which in turn increases GR levels[111]. Furthermore, no significant increase in phosphorylation of p38 by CSDS in vector-treated mice was observed after ghrelin treatment. Additionally, hippocampal GHSR-deficient mice showed higher levels of p38 phosphorylation than control mice, suggesting that ghrelin may also mediate antidepressant mechanisms by inhibiting the p38-MAPK signalling pathway in the hippocampus[112]. Interestingly, social failure stress produces depressive-like behaviour in wild-type mice, but selective deletion of p38-MAPK in serotonergic neurons of the nucleus dorsalis of the mouse middle suture protects the mice under stress induction[113]. This suggests that p38-MAPK has the ability to specifically regulate selected downstream targets, and the role played by this pathway in antidepressant disorders requires further investigation.

3.3. PI3K/Akt Signaling Pathway

Depression is closely related to neurogenic hypoplasia[114], and PI3K/Akt is thought to be an important signal for the proliferation of adult hippocampal progenitor cells [115]. Akt can exert its utility in controlling cellular proliferation by activating the phosphorylation of its downstream targets (GSK-3β, mTOR, and p70^S6K), where GSK-3β is a pro-apoptotic protein whose activity plays an important role in neuropathology and psychiatric disorders [116]. β-catenin, as a transcription factor regulated by GSK-3β, undergoes nuclear translocation under conditions of GSK-3β inactivation [117], which is an indispensable step in its role in promoting cell survival. In addition, as downstream of PI3K/Akt, phosphorylation of mTOR and p70^S6K also promotes the proliferation of neural stem cells [118]. Interestingly, ghrelin could induce hippocampal neural stem cell (NSC) proliferation by activating the PI3K/Akt signaling pathway by binding to GHSR1a, and the stimulatory effects of ghrelin on GSK-3β, mTOR and p70^S6K phosphorylation were significantly inhibited by the treatment of GHSR1a-specific antagonist D-Lys-3-GHRP-6[119]. Furthermore, it has been demonstrated that ghrelin enhances the nuclear translocation of β-catenin, which in turn contributes to its anti-apoptotic effects[120]. Therefore, we may hypothesize that ghrelin may promote neuronal cell proliferation through activation of the PI3K/Akt pathway and subsequently play a role in the treatment of depression. Moreover, autophagy plays an important role in maintaining neuronal stem cells and adult neuronal plasticity, while ghrelin can stimulate autophagy by inhibiting PI3K/AKT/MTOR signaling pathway [121]. However, an experiment in a mouse model of corticosterone-induced depression found that overactive neuronal autophagy depleted BDNF and impaired adult hippocampal neurogenesis[92]. Therefore, it remains to be investigated whether ghrelin can regulate autophagy homeostasis in vivo through the PI3K/AKT/MTOR pathway and promote neurogenesis.

3.4. Jak2-STAT3 Signaling Pathway

The Jak2/STAT3 signaling pathway, like the aforementioned PI3K/Akt signaling pathway, has also been shown to play an important role in neuroprotection. Unlike single pathways that act independently, one study found that resveratrol may work together to exert neuronal protection by indirectly upregulating the PI3K/Akt/mTOR pathway through the activation of Jak2/STAT3 [122]. As shown in Figure 2, exposure of rat hippocampal NSCs to the Jak2/STAT3 inhibitor cucurbitacin I significantly blocked the proliferative effects of ghrelin on NSCs [119]. Thus, ghrelin would also be potent through activation of the Jak2/STAT3 pathway. Furthermore, this pathway not only plays an active role in neuroprotection and regeneration, its role in neuroinflammation has also been shown to be promoted. It has been found that inhibition of the Jak2/STAT3 pathway ameliorates neuroinflammation[123] and reduces neuronal senescence by suppressing the inflammatory response[124]. Since depression is closely related to decreased neuronal genesis and upregulation of inflammatory factors, and activation of the Jak2/STAT3 pathway was found to have completely opposite effects in these two aspects, whether this pathway can be a therapeutic target for depression needs to be investigated at a deeper level on the multifaceted mechanisms. The study of whether ghrelin regulates neuroinflammation when activating the Jak2/STAT3 pathway will be an important basis for comprehensively determining whether ghrelin can be used as a target for the treatment of depression.

4. Ghrelin/GHSR as a Therapeutic Target for Depressive Disorder

Many neuropeptides have been reported as targets for depression treatment. For example, nonselective glycopeptide receptor agonists (galnon) can exert antidepressant activity in preclinical models of depression[125,126]. VP antagonists have shown similar antidepressant behavior in preclinical studies[127,128]. Similarly, many studies have shown that ghrelin can be used as a powerful tool in the treatment of depression. It has been reported that ghrelin produces antidepressant effects in estrogen-deficient mice[103], and it may also counteract depressive symptoms caused by chronic stress[129]. In addition, many substances that exert antidepressant effects by modulating ghrelin/GHSR expression have been identified. For example, paeoniflorin (PF) significantly increased the expression of GHSR1a to mediate antidepressant effects[130]. The GHSR inhibitor JMV29282259 blocked saffronin-induced expression of neuroplasticity-related proteins[131]. Here, we briefly summarized the correlation between ghrelin/GHSR system and depression, as well as the research methodology, as shown in Table 1. Thus, ghrelin/GHSR has many potent functions in defense against depression-like symptoms. However, further studies are needed because of the unstable antidepressant behavioral effects of neuropeptides expressed in various tests and the two-sided nature of ghrelin's effects on depression in different models[132].

5. Conclusions and Future Direction

There is still ongoing debate about the role of ghrelin in depression. The majority of studies suggest that ghrelin has antidepressant effects, with few studies indicating depressogenic effects. Indeed, ghrelin/GHSR can exert antidepressant and neuroprotective effects by triggering multiple signaling pathways including cAMP/CREB/BDNF, PI3K/Akt, Jak2/STAT3, p38-MAPK et al. Moreover, GHSR1a can also form dimers with other GPCRs to exert antidepressant effects. So, ghrelin/GHSR system is becoming a new target for the treatment of depression. However, due to the two-sided claims of ghrelin's antidepressant potency from different experiments, ghrelin had no antidepressant effect on young rats, and the drug's neuropharmacology differed in adolescents and adults. Future experiments should therefore focus on coming to investigate the link between the pathogenesis of ghrelin in different depressive groups and the development of receptors biased drugs is also a good strategy. Moreover, more studies are necessary to determine the extent to which central and peripheral ghrelin signaling are functionally interconnected, as this understanding is crucial for the development of potential new ghrelin-based therapeutic agents. Finally, promising findings from animal studies necessitate further human-based research to ascertain the extent to which such results can be applied to human disorders.