PD is a neurodegenerative disorder for which levodopa has been the treatment of choice for many years; its main mechanism of action being the increase in concentrations of striatal dopamine, which is affected in this disease due to the death of dopaminergic neurons, preferably in the SNpc. Despite its beneficial effects, after 5–6 years of use this treatment is associated with complications that affect the patients’ quality of life [5]. In the present work, considering the results previously obtained with the administration of a selected combination of LAB, its neuroprotective potential was evaluated on the chronic parkinsonism model. This model, as explained previously, is one that most reflects the characteristics of PD and allowed the analysis of LAB in the presence of one of the most used conventional treatments, such as levodopa-benserazide.
Recent studies analysed the possibility that changes in the intestinal microbiota may be associated not only with the appearance of PD, but also with the complications related with treatments, especially with levodopa, although the mechanisms are not fully elucidated [8]. In this sense, even if the model used, according to the literature, does not depend on the age of the animals, taking into account that PD is more common in elderly individuals and that aging is accompanied by changes both at the level of the intestinal microbiota and the host's immune response, 1-year-old animals (aged mice) were used.
As expected, treatment with levodopa-benserazide improved the motor skills of animals affected by the MPTP neurotoxin. Similar to what was observed in the previous work [9], the administration of the selected LAB also improved the times in which the mice completed the different motor tests, even better than when the commercial vitamin mixture was given as a supplement. The evaluation of this LAB MIX together with levodopa-benserazide treatment showed to be effective, since this group of animals was the one that presented the best results in terms of motor skills. This was associated with diminished loss of dopaminergic neurons in the SNpc, this combination being better than that of levodopa-benserazide with vitamins. This results are similar to those published in a rat model of PD in which no significant differences were observed for the TH + terminals in the striatum of animals treated with levodopa, when compared to those untreated [16].
Considering the immunomodulatory effect previously observed with the LAB MIX in other experimental models [11, 17] and that, similar to patients with PD, the presence of inflammation accompanies the neurodegeneration caused by MPTP [18], different cytokines were evaluated in serum and brain. At systemic level, mice from MPTP group showed a pro-inflammatory profile with significant increases of IL-6, plus increases of TNF-α in some animals, and without significant modification for the anti-inflammatory cytokine IL-10. It was also observed that the treatment with levodopa-benserazide kept the mice with cytokine levels similar to the healthy control, showing that, at least during the period used in this model, it prevented the inflammation associated with MPTP. This effect was maintained with the administration of the vitamins. In contrast, the administration of LAB showed a different effect on the immune response, increasing cytokine concentrations, but maintaining an immunomodulatory effect since along with the increase in a pro-inflammatory cytokine such as TNF-α, increases in IL-10, a cytokine with anti-inflammatory properties, were also observed. Similarly, studies carried out with the same bacteria combination in other animal models such as a breast cancer model under chemotherapy treatment, showed that it was associated with a cytokine response at systemic level, different from treatments with specific drugs, and modulated the response with the production of pro- and anti-inflammatory cytokines [17]. In this sense, it is also important to highlight that peripheral inflammation is presented as a potential risk for PD, showing the importance of maintaining a balanced/modulated immune response [19].
The analysis at the brain level showed that the highest production of cytokines was observed in the MPTP group, which would be a result of the microglia activated by the neurotoxin. This activation depends on complex interactions between neurons, microglia and astrocytes, similar to those that occur in patients with PD [20]. Unlike what was observed in serum, at brain level, the administration of LAB in the presence of MPTP maintained the concentrations of the studied cytokines similar to those of the healthy control. Treatment with levodopa-benserazide was associated with increases in the anti-inflammatory cytokine IL-10, which was maintained in the presence of the LAB MIX, showing potential to control the inflammation caused by the neurotoxin.
In order to analyse other mechanisms that may be involved in the neuroprotective effect of the bacterial MIX, alterations at the intestinal level were evaluated. The intestinal epithelium forms a barrier that prevents the passage of harmful substances and at the same time allows the absorption and secretion of nutrients or drugs. Increased intestinal permeability, called “leaky gut,” was described in PD patients, in whom the intestinal barrier function impaired due to inflammatory processes and increased oxidation [21]. This environment further promotes the accumulation and aggregation of α-synuclein in the enteric nervous system [22], generating a vicious cycle of amplification of these toxic species that spread to the central nervous system (CNS) through the vagus nerve [23]. Furthermore, the increased expression of inflammatory cytokines and glial markers, also detected in biopsies of the gastrointestinal tract of PD patients, was positively correlated with the progression and severity of the disease [24]. In our model, similarly, MPTP was associated with inflammatory infiltrates and alterations at the intestinal level, with decreased absorption capacity by presenting shorter villi, and changing the relationship between the length of the villi and the depth of the crypts. Although the different treatments evaluated improved this situation, the administration of the LAB showed the best effect at the intestinal level.
Finally, taking into account that the composition of the intestinal microbiota affects the correct functioning of this barrier [25], and that it was also shown that alterations in the composition of the intestinal microbiota can accelerate the aggregation of α-synuclein, partly through the secretion of bacterial amyloid [26], the study of the microbiota, using faecal samples from the different groups, was carried out. In this sense, it is highlighted that although it was not possible to determine whether the changes in the intestinal microbiota were a cause or a consequence of the pathogenesis of the disease, it was established that a healthy microbiota can reduce the risk of developing PD [27]. The results obtained in our model showed that richness and abundance (alpha diversity), studied with different indices, was greater in the MPTP group than in the healthy control; while the different treatments showed intermediate values. The groups receiving the bacterial MIX were those that most resembled the healthy animals in the evaluated indices. Similar results were recently published in an article that compared two models of MPTP, one acute and one sub chronic; the latter being the one in which higher values were observed for the evaluated alpha diversity indices compared to the control [28]. Comparable results were also described in PD patients, whose samples had greater overall richness, as indicated by a significantly greater number of species observed, suggesting that this greater diversity could derive from a decrease in the abundance of dominant species and an increase in rare species [29].
At the phylum level, the dominant phyla in the samples from our model were Firmicutes and Bacteroidota; the latter being the one that increased significantly in the MPTP group, with a decrease in Firmicutes, which agrees with the results described in a subchronic model [28]. The different treatments did not reverse these modifications compared to the control healthy mice. However, it is notable that the groups that received the LAB MIX increased the Campylobacterota phylum, whose decrease was associated with MPTP [28]. This increase was even significant compared to the Control. At the family level, the MPTP group showed decrease for Lactobacillaceae, and levodopa-benserazida treatment was associated with increases of this family (without reach the healthy control values), being the group that received both the treatment and the LAB (MPTP/LEVO/MIX group) the one with the higher relative abundance. Regarding the Muribaculaceae family, a family in the order of the Bacteroidales which were described as decreased in PD patients [30], increased in the groups that received some treatment, highlighting its higher increase in the MPTP/MIX group. Finally, the analysis of the genera that presented the highest abundance in the samples under study showed that the parkinsonism model is associated to decreases in the Lactobacillus genus; similar to the results obtained when comparing the microbiota of patients with PD and healthy ones [30]. The treatments evaluated in the present work were associated with increases for this genus, and even though they did not recover the levels of the Control, the MPTP/LEVO/MIX group was the one that presented the greatest increases. These results are also in agreement with recent work in which expression of α-synuclein in a transgenic rat model led to changes in the composition of the intestinal microbiota with a decrease in the Lactobacillus genus and increase of Alistipes genus [31]. In this latter model, Alistipes abundance was associated with the inflammation in the colon of the transgenic rats. In our model, this genus was not significant increased in the MPTP group compared to the Control. The abundance of this genus in the healthy control could be related with the age of the animals; it was reported that Alistipes was the most significantly overrepresented taxon within middle-aged and older mice [32]. However, this genus decreased with all treatment compared with both MPTP and Control groups. Finally, another increased genus associated with most of the treatments was Prevotellaceae UCG-001. In this sense, using a Dcf1−/− knockout (KO) mouse model, which showed a behavioural and pathological change typical of PD, it was observed that they presented increases of Proteobacteria (phylum level) and decrease of Prevotellaceae (family level), more specifically of Prevotellaceae UCG-001 [33], similar to the situation reported in patients with PD [30].
In conclusion, the neuroprotective effect associated with the administration of previously selected LAB (MIX) was observed in a chronic parkinsonism model. This effect was related to the modulation of the immune response in the animals, altered by the presence of the neurotoxin. Furthermore, the effect of levodopa-benserazide treatment was maintained and in some cases improved in the presence of the LAB MIX. The administration of this LAB MIX showed the best results in terms of intestinal inflammation associated with the model. Finally, the results showed an intestinal dysbiosis associated with the parkinsonism model, which is similar in some phyla, families and genera to those observed in patients with PD. Treatment with levodopa-benserazide was able to partially modulate this dysbiosis, highlighting that when it was accompanied by the administration of the LAB MIX the microbiota was closer to the profiles observed in healthy controls. Thus, different mechanisms of action would be related to the protective effect of the LAB MIX, without interfering with levodopa-benserazide treatment. Future studies with appropriate specific models will be necessary to know if the administration of these selected LAB could also reduce the adverse effects associated with the treatment (such as levodopa-induced dyskinesia).