Tryptophan in the diet ameliorates motor deficits in a rotenone‐induced rat Parkinson's disease model via activating the aromatic hydrocarbon receptor pathway

Abstract Background and purpose Parkinson's disease (PD), a common neurodegenerative disorder with motor and nonmotor symptoms, does not have effective treatments. Dietary tryptophan (Trp) supplementation has potential benefits for the treatment of multiple disorders. However, whether additional Trp in the diet could be beneficial for PD remains to beinvestigated. In the present study, the neuroprotective role of dietary Trp on a rotenone‐induced rat model of PD was determined. Methods The rotenone was injected to build the PD model, and then the rats were treated with Trp in the diet. And then, an open field test, western blot analysis, and enzyme linked immunosorbent assay (ELISA) were performed. Results We observed that dietary Trp significantly ameliorated impaired motor function, upregulated tyrosine hydroxylase expression, inhibited the nuclear transport of Nuclear factor‐kappa B (NF‐κB) in substantia nigra (SN), and downregulated the protein levels of IL‐1β, IL‐6, and TNF‐α in serum in rotenone‐treated rats. However, these patterns were reversed in response to treatment with ampicillin, an agent that can clean intestinal Trp metabolism flora. Moreover, after using CH223191, an inhibitor of the aromatic hydrocarbon receptor (AhR) pathway, dietary Trp could not exert neuroprotective roles in the rotenone‐induced rat model of PD. Conclusion These results suggest that Trp in the diet can protect against rotenone‐induced neurotoxicity to ameliorate motor deficits, which may be mediated through activating AhR pathway.

. A pro-inflammatory profile was detected in the microbiota of PD patients (Bedarf et al., 2017;Petrov et al., 2017) owing to the increased intestinal permeability to lipopolysaccharide (LPS, an endtoxin) (Forsyth et al., 2011). To date, drug treatments for PD provided only be symptomatic, without preventing the progressive loss of dopaminergic neurons in patients with PD (Athauda & Foltynie, 2015). In consequence, research on an agent that may target for the neuroinflammation to treat PD is necessary.
Attempts to target individual molecules that may decrease the pathological impact of PD have been made to counteract dopaminergic neuron death by the introduction of neuroprotective molecules.
One of these targets is tryptophan (Trp), an essential amino acid mainly obtained from dietary intake in humans and only in part from endogenous protein degradation (Le Floc'h et al., 2011). Trp is involved in the development of brain areas associated with behavioral functions (Zhang et al., 2006). Moreover, rats with decreased intake of tryptophan had higher running performance (Yamamoto & Newsholme, 2003). Trp metabolites acting on the metabolic crossroad interconnecting different organs exert various beneficial effects (Le Floc'h et al., 2011;Roager & Licht, 2018) in a multiple of disorders, including central nervous system disorders, autoimmune diseases, and cancer . Under inflammatory conditions, most Trp is diverted to produce Kyn and its metabolites kynurenine acid (KYNA) (Keszthelyi et al., 2012) with neuroprotective roles (Lim et al., 2017). Thus, dietary Trp may result in the generation of drugs for the treatment of multiple diseases (Platten et al., 2019), including PD.
However, since multiple underlying mechanisms are involved in PD, we explored other targets associated with dietary Trp that could ameliorate the pathology of PD. Aryl hydrocarbon receptor (AhR), binding to a variety of ligands, including xenobiotic ligands such as physiological compounds derived from the digestion of dietary components by commensal microbiota (Zhou, 2016) such as dietary Trp supplementation (Liang et al., 2018), was selected. The AhR, broadly expressed in immune cells (Lee et al., 2017), plays a pleiotropic role in maintaining both the innate and adaptive immune systems in multiple organs and has been shown to be a transcriptional regulator for the development and function of several immune cells (Stockinger et al., 2014). Also, AhR can inhibit the production of IL-6 in response to the treatment of LPS via interacting with NF-κB and STAT1 (Kimura et al., 2009). AhR signaling regulates multiple cellular processes, including immunomodulation, cell development, differentiation, proliferation, survival, and apoptosis (Stockinger et al., 2014). In cerebellar granule neuron precursors, disrupted expression of the AhR impairs neurogenesis via inhibiting precursor proliferation and increasing differentiation (Dever et al., 2016).
In addition, constitutive activity of AhR is essential for spontaneous movement in mice (Williams et al., 2014).
Given the key neuroprotective roles of Trp and AhR signaling under inflammatory conditions, we were interested in their relationship with PD, hypothesizing that Trp in the diet may play a neuroprotective role in PD by fostering neuroinflammation inhibition-associated signaling via AhR. To achieve this goal, rotenone-challenged rats were used as an in vivo model of PD, and the effect of dietary Trp on motor function, dopaminergic neuronal survival and inflammation, and the underlying mechanisms associated with the AhR signaling pathway inhibited by CH223191 were investigated. Here, we revealed a neuroprotective role of dietary Trp against rotenone-induced neurotoxicity via activating the AhR pathway.

Animals
Male Sprague-Dawley (SD) rats (200-220 g) of 6 weeks of age were purchased from Hunan SJA Laboratory Animal Co., Ltd. and maintained (n = 4 rats/cage) in an air-conditioned room (22 ± 1˚C) with a 12-h light/12-h dark cycle and water and food ad libitum. All experimental protocols performed on animals were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of University of South China.

Groups and treatments
Rats were randomly divided into six groups (n = 6 rats/group): Normal saline-treated group (Control, CTRL), rotenone + Trp-deficienttreated group (Rot+Trpdef), rotenone + Trp-treated group (Rot+Trp), rotenone + Trp + ampicillin-treated group (Rot+Trp+Amp), rotenone + Trp + vancomycin-treated group (Rot+Trp+Van), and rotenone + Trp The rats in the CTRL group were treated daily with 0.1 ml saline for 4 weeks by subcutaneous injection through the neck and fed with normal diet. The rats in the other groups were subcutaneously injected with rotenone at a concentration of 2 mg/kg in the neck. The rats in Rot+Trpdef group were fed with Trp-deficient forage. The left four groups were fed with Trp-rich forage. Treatments with 300 mg/kg ampicillin or 150 mg/kg vancomycin by oral administration were performed at week four after the injection of rotenone. The rats in Rot+Trp+AhRi group were treated with 10 mg/kg CH223191 (cat no. A8609; APeXBIO Technology LLC) by oral administration at the week 1 after rotenone injection.

Open field test
An automated open field apparatus (Accuscan Instruments Inc.) was used to measure the locomotor activity of rats, as described previously (Gordon et al., 2016). The movement of the rat within 5 min is observed, and the total distance of movement is calculated. Two independent investigators, who were blinded to the experimental groups, recorded and quantified the total distance traveled. No animals died during the survival time, after which the rats were sacrificed.

Tissue preparation
The tissues were prepared according to previous studies Li et al., 2017;Xu et al., 2017). For western blot analysis, rats were sacrificed by decapitation after 10% chloral hydrate anesthesia at a concentration of 350 mg/kg. Briefly, in the SNc area, three different levels (−4.8, −5.04, and −5.28 mm of bregma) were selected (Javed et al., 2020). SN tissues (n = 6/group) were collected and treated as fol-

Western blot analysis
Western blot analysis was performed according to previous studies (Chen et al., 2015;Chen et al., 2020a;Chen et al., 2020b;Jiang et al., 2016;Li et al., 2017;Tan et al., 2021;Yi et al., 2021). The tissue lysates mixed with a sample loading buffer was heated at 95˚C for 15 min.

Statistical analysis
All statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc.). Data were expressed as the mean ± SD and analyzed with one-way ANOVA followed by a post hoc Bonferroni test. p < 0.05 was considered to indicate a statistically significant difference.

Dietary Trp in the diet ameliorates impaired motor function in rotenone-treated rats
To investigate the protective effects of dietary Trp on motor function in rotenone-treated rats, an open field test was performed, and the total length and average speed were calculated.
As shown in Figure 1, in comparison with the CTRL group, Rot+Trpdef rats exhibited motor dysfunction, including shorter lengths and lower average speed. However, Rot+Trp rats displayed significantly improved performance compared with that of Rot+Trpdef rats (p = .039, p = .011). After using ampicillin, motor dysfunction was observed in the Rot+Trp+Amp group, but after using vancomycin, motor dysfunction was significantly improved in the Rot+Trp+Van group. Moreover, after using CH223191, an inhibitor of AhR, motor dysfunction was observed.

Dietary Trp in the diet decreases TH expression in the SN of rotenone-treated rats
To investigate the protective effects of dietary Trp on the degeneration of dopaminergic neurons in rotenone-treated rats, western blot analysis was performed, and the expression of tyrosine hydroxylase (TH) in the SN of rotenone-treated rats was evaluated. with Trp in the diet, the TH protein level was increased. After using ampicillin, the TH protein level was decreased in the Rot+Trp+Amp group. By contrast, after using vancomycin, the TH protein level was not significantly changed in the Rot+Trp+Van group. Moreover, after using CH223191, an inhibitor of AhR, the TH protein level was decreased.

Dietary Trp in the diet decreases the nuclear translocation of NF-κB in the SN of rotenone-treated rats
To evaluate the effect of dietary Trp on inflammation in the SN, western blot was performed, and the translocation of the p65 subunit of NF-κB from the cytoplasm into the nucleus was calculated.
As shown in Figure 3, in comparison with the CTRL group, the nuclear translocation of NF-κB (indicated by the ratio of nuclear NF-κB p65 protein levels to cytoplasmic NF-κB p65 protein levels) was increased in the Rot+Trpdef group. After treatment with dietary Trp, the nuclear translocation of NF-κB was decreased. After using ampicillin, the nuclear translocation of NF-κB was increased in the Rot+Trp+Amp group, whereas, after using vancomycin, the nuclear translocation of NF-κB was not significantly changed in the Rot+Trp+Van group. Moreover, after using CH223191,

F I G U R E 3
The effect of dietary tryptophan (Trp) on the nuclear translocation of NF-κB in rotenone-treated rats was investigated by open field test. Trp in the diet (a-b) inhibits the nuclear translocation of NF-κB (as indicated by the ratio of nuclear NF-κB p65 protein levels to cytoplasmic NF-κB p65 protein levels), but (c-d) cannot inhibit the nuclear translocation of NF-κB after inhibiting AhR, in rotenone-induced Parkinson's disease (PD) rats (**p < .01, *p < .05, n = 6/subgroup). NS, no significance F I G U R E 4 The effect of dietary tryptophan (Trp) on inflammation in the serum of rotenone-treated rats was investigated by enzyme linked immunosorbent assay (ELISA). Trp in the diet inhibited the expression levels of proinflammatory cytokines, including (a) TNF-α, (b) IL-1β, and (c) IL-6, in rotenone-induced Parkinson's disease (PD) rats (***p < .001, n = 6/subgroup). NS, no significance an inhibitor of AhR, the nuclear translocation of NF-κB was increased.

Dietary Trp decreases inflammation in the serum of rotenone-treated rats
To evaluate the effect of dietary Trp on inflammation in PD, the ELISA was performed, and TNF-α, IL-1β, and IL-6 levels in the serum were evaluated.
As shown in Figure 4, in comparison with the CTRL group, the IL-1β, IL-6, and TNF-αprotein levels were increased in the Rot+Trpdef group. After treatment with dietary Trp, the IL-1β protein levels were decreased. After using ampicillin, the IL-1β protein levels were increased in the Rot+Trp+Amp group, while, after using vancomycin, the IL-1β protein levels were not significantly changed in the Rot+Trp+Van group. Moreover, after using CH223191, an inhibitor of AhR, the IL-1β protein levels were increased.

DISCUSSION
In the present study, we revealed that Trp in the diet can ameliorate impaired motor function under rotenone-induced neurotoxicity via activating the AhR signaling pathway.
A previous study found that chronic exposure to rotenone was a risk factor for the development of PD (Betarbet et al., 2000). Administration of rotenone in rats can reproduce multiple PD-like behavioral characters, including rigidity and hypokinesia (Wang et al., 2017). In addition, increasing evidence suggests that using rotenone to construct a PD experimental model can mimic the behavioral and neuropathological conditions of PD through selecting the degeneration of dopaminergic neurons, thus offering more advantages than other models (Johnson & Bobrovskaya, 2015). In our previous study, rotenone was used to construct an in vitro cell model to mimic the condition of PD (He et al., 2020). In the present study, a rat model was successfully constructed using rotenone, and the influence of dietary Trp on the motor deficits of PD was evaluated.
In patients with PD, motor dysfunction commonly occurs. Neurological function assessment is commonly performed to evaluate the therapeutic effect of different strategies. In the present study, we observed that Trp in the diet can ameliorate motor deficits in the rotenoneinduced rat PD model.
PD is a slowly progressive neurodegenerative disease that is associated with the degeneration of dopaminergic neurons (Vogt Weisenhorn et al., 2016). This loss of dopamine (DA) accounts for many of the symptoms that accompany the disease, including motor dysfunction, mood alterations, and cognitive impairment (Olanow et al., 2003). In the present study, we observed that Trp in the diet can upregulate TH expression in the rotenone-induced rat PD model.
Abnormal neuronal inflammation can promote the loss of dopaminergic neurons (Yang et al., 2019). NF-κB p65, an oxidative stressresponsive transcription factor, modulates inflammation in multiple experimental models (Helenius et al., 1996). Under normal condition, NF-κB p65 is segregated in the cytoplasm (Karin & Delhase, 2000), but NF-κB p65 translocated into the nucleus to activate the downstream proinflammatory cytokines (Oeckinghaus & Ghosh, 2009). In the present study, we found that Trp in the diet can decrease the translocation of NF-κB in rats induced by rotenone via the AhR pathway.
Proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, are found in either cerebrospinal fluid (CSF) or in affected brain regions in PD (Nagatsu et al., 2000). Rotenone-injected animals displayed the occurrence of neuroinflammation (Javed et al., 2020). Previous studies have indicated that the survival of dopaminergic neurons could be protected via inhibiting neuroinflammatory responses (Furuyashiki, 2012). In the present study, we observed that Trp in the diet can inhibit the expression of NF-κB, IL-1β, IL-6, and TNF-α in rotenone-induced rats via the AhR pathway.

CONCLUSION
Taken together, these results may suggest that Trp in the diet modulates the AhR pathway to ameliorate impaired motor function in PD, laying the foundation for Trp to be a novel candidate for the treatment of PD.
Although the results in the current study look promising, this study still exhibited some limitations. More sophisticated approaches are no doubt needed to be conducted on the female animals to see whether Trp in the diet can also ameliorate motor deficits effectively.

This research was funded by Scientific Research Project of Hunan
Health Committee (grant nos. 20201911 and 20201963).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

AUTHOR CONTRIBUTIONS
Zijian Xiao and Heng Wu conceived and designed the experiments. Yilin Wang, Shuangxi Chen, Jian Tan, Yijiang Gao, Hongye Yan, Yao Liu, and Shanqing Yi performed the experiments and analyzed the data. Yilin Wang contributed to reagents/materials/analysis tools. Zijian Xiao, Heng Wu, and Shuangxi Chen wrote the paper.