Oral berberine improves brain dopa/dopamine levels to ameliorate Parkinson's disease by regulating gut microbiota

Abstract

The phenylalanine-tyrosine-dopa-dopamine pathway provides dopamine to the brain. In this process, tyrosine hydroxylase (TH) is the rate-limiting enzyme that hydroxylates tyrosine and generates levodopa (L-dopa) with tetrahydrobiopterin (BH₄) as a coenzyme. Here, we show that oral berberine (BBR) might supply H^• through dihydroberberine (reduced BBR produced by bacterial nitroreductase) and promote the production of BH₄ from dihydrobiopterin; the increased BH₄ enhances TH activity, which accelerates the production of L-dopa by the gut bacteria. Oral BBR acts in a way similar to vitamins. The L-dopa produced by the intestinal bacteria enters the brain through the circulation and is transformed to dopamine. To verify the gut-brain dialog activated by BBR's effect, Enterococcus faecalis or Enterococcus faecium was transplanted into Parkinson's disease (PD) mice. The bacteria significantly increased brain dopamine and ameliorated PD manifestation in mice; additionally, combination of BBR with bacteria showed better therapeutic effect than that with bacteria alone. Moreover, 2,4,6-trimethyl-pyranylium tetrafluoroborate (TMP-TFB)-derivatized matrix-assisted laser desorption mass spectrometry (MALDI-MS) imaging of dopamine identified elevated striatal dopamine levels in mouse brains with oral Enterococcus, and BBR strengthened the imaging intensity of brain dopamine. These results demonstrated that BBR was an agonist of TH in Enterococcus and could lead to the production of L-dopa in the gut. Furthermore, a study of 28 patients with hyperlipidemia confirmed that oral BBR increased blood/fecal L-dopa by the intestinal bacteria. Hence, BBR might improve the brain function by upregulating the biosynthesis of L-dopa in the gut microbiota through a vitamin-like effect.

Fig. 1

BBR increased dopa/dopamine production in the gut microbiota in vivo. a Dopa/dopamine significantly increased in the feces of ICR mice at 0, 6, 12, and 24 h after oral administration of BBR (100 and 200 mg/kg, respectively; mean ± SD, *P < 0.05, **P < 0.01 for dopa and ***P < 0.001 for dopamine). b Dopa/dopamine showed a dose- and time-dependent increase in plasma after BBR treatment (mean ± SD, *P < 0.05 and **P < 0.01 for dopa; *P < 0.05, ***P < 0.001 for dopamine). c Dopa/dopamine showed a similar dose- and time-dependent increase in the brain after BBR treatment (mean ± SD, *P < 0.05 and **P < 0.01 for dopa; *P < 0.05, **P < 0.01, and ***P < 0.001 for dopamine). d Intraperitoneal injection (i.p.) of BBR into the ICR mice did not increase the levels of dopa/dopamine in the feces, plasma, and brain at any of the study time points, which was different from the results of BBR in oral administration (mean ± SD, NS no significance). e Mass spectra of ¹⁵N-dopamine and dopamine in mouse brains. f Intestinal ¹⁵N-Tyr was the raw material for the synthesis of ¹⁵N-dopamine in brain. Pretreating the mice with antibiotics for 3 days attenuated the effect of BBR on ¹⁵N-dopamine production (mean ± SD, **P < 0.01, ***P < 0.001), and oral administration of BBR significantly increased ¹⁵N-dopamine in the brain (*P < 0.05)

Fig. 2

BBR stimulated intestinal bacteria to produce dopa/dopamine. a Levels of dopa/dopamine increased significantly at 0, 6, 12, and 24 h after BBR treatment (10 and 20 μg/mL, respectively) in the intestinal bacteria of SD rats in vitro (mean ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001, for dopa and dopamine). b Effect of BBR (10 μg/mL) in ten intestinal bacterial strains in vitro. Out of the ten strains, four bacterial strains (E. faecalis, E. faecium, P. mirabilis, and L. acidophilus) showed a significant increase in dopa after BBR treatment (10 and 20 µg/mL for 12 h), six had almost no change in dopa production. Of note, the increase in dopa by E. faecalis and E. faecium showed a dose-dependent manner with BBR. The BBR-induced dopamine production profile in the ten strains was different from that of dopa, that is, three bacterial strains (E. faecalis, E. faecium, and S. epidermidis) showed a significant increase in dopamine after BBR treatment (10 µg/mL for 12 h), seven had almost no change on dopamine production. Of note, the increase in dopamine by E. faecalis and E. faecium showed a similar dose-dependent manner with BBR. c Changes in the intestinal bacterial composition as a result of BBR treatment. The heat map of the top 50 bacterial genera exhibited the most substantial change in abundance after exposure to BBR; the proportion of dopa/dopamine-producing bacteria increased, including Enterococcus, Escherichia–Shigella, Pseudomonas, and Lactobacillus (labeled with the red “*”)

Fig. 3

BBR activated the pathway for dopa/dopamine synthesis in the gut microbiota. a The activity of TH and DDC increased after treating the gut bacteria of SD rats with BBR at 12 h (10 μg/mL, *P < 0.05, **P < 0.01), but weakened when TH inhibitor (BLMA5, 100 μM) or the DDC inhibitor (benserazide, 100 μM) was incubated with BBR as a whole (mean ± SD, *P < 0.05, **P < 0.01). The TH inhibitor BLMA or DDC inhibitor benserazide did not change the abundance of the intestinal bacteria at 50 or 100 μM. The activity of TH and DDC increased after treating the gut bacteria of SD rats with BBR at 12 h (10 μg/mL, *P < 0.05, **P < 0.01), but weakened when the TH inhibitor (BLMA5, 100 μM) or DDC inhibitor (benserazide, 100 μM) was incubated with BBR as a whole (mean ± SD, *P < 0.05, **P < 0.01). b Levels of BH₄ (the coenzyme of TH) increased significantly at 6 and 12 h after BBR or dhBBR treatment (10 and 20 μg/mL, respectively) in SD gut bacteria in vitro (mean ± SD, +23%↑, +31%↑ for BBR at 20 μg/mL at 6 and 12 h, **P < 0.01, ***P < 0.001; +26%↑, +34%↑ for dhBBR at 20 μg/mL at 6 and 12 h, **P < 0.01). In addition, the level of DDC coenzyme VB₆ was enhanced in the intestinal bacteria at 12 h (mean ± SD, +16%↑ for BBR at 20 μg/mL, ***P < 0.001; +19%↑ for dhBBR at 20 μg/mL, **P < 0.01). c The TH inhibitor (BLMA5, 100 μM) decreased the production of dopa in SD rat gut bacteria when treated with BBR for 6 and 12 h (*P < 0.05, **P < 0.01); DDC inhibitor (benserazide, 100 μM) also lowered the dopamine generation significantly when incubated with BBR (mean ± SD, **P < 0.01, ***P < 0.001). d The DDC inhibitor (benserazide, 100 μM) increased the generation of dopa in SD rat gut bacteria when treated with BBR (mean ± SD, *P < 0.05). e Levels of dopa/dopamine increased significantly at 6 hr after dhBBR treatment in the brain homogenate of mice in vitro (mean ± SD, **P < 0.01, ***P < 0.001). f Coincubation of mouse dopaminergic primary cells with BBR or dhBBR (10 μg/mL), significantly increased dopamine levels in the dopaminergic primary cells incubated with dhBBR (*P < 0.05). g Levels of TH/DDC increased significantly at 6 h after dhBBR treatment in the brain homogenate of mice in vitro (mean ± SD, **P < 0.01, ***P < 0.001). h Levels of BH₄/VB₆ increased significantly at 6 h after dhBBR treatment in the brain homogenate of mice in vitro (mean ± SD, **P < 0.01, ***P < 0.001). i BH₂ decreased significantly (*P < 0.05) with increasing BH₄ (*P < 0.05), when dhBBR was added to the mixture of BH₂ (as substrate) and dihydrobiopterin reductase at 37 °C for 2 h. j Levels of dopa/dopamine decreased significantly at 12 h after the treatment with DHFR inhibitor (methotrexate 100 μM) in the intestinal bacteria (mean ± SD, *P < 0.05)

Fig. 4

The dhBBR-mediated chemical reaction for the conversion from tyrosine to dopa. BH₂ obtained H^• from the dhBBR–BBR system, and its reduction was activated, in which H^• from dhBBR (N₇–C₈) moved to the N₃=C₄ bond in BH₂, and N₃=C₄ was then transformed into N₃–C₄ (BH₂ was converted into BH₄). In the meanwhile, dhBBR lost H^•, and was oxidized into BBR. The increased BH₄ in intestinal bacteria accelerates the transformation from tyrosine to l-dopa, in the presence of ROS (H₂O₂, etc.) as well as Fe²⁺. Accordingly, BH₄ is converted into tetrahydrobiopterin–4α-carbinolamine

Fig. 5

E. faecalis and E. faecium regulated dopa/dopamine and improved brain function in PD animals in the presence of BBR. a Treating mice with both MPTP (20 mg/kg/d, s.c.) and P (200 mg/kg/d, i.p.) for 7 days successfully generated the damage to brain function (LD, ***P < 0.001; LFR, ***P < 0.001) in the model mice. Carbidopa (1 mg/kg, s.c.) in combination with l-dopa (10 mg/kg/d, s.c.) for 6 days (from days 2 to 7) decreased the LD, cylinder test value (***P < 0.001, **P < 0.01), and increased the LFR (***P < 0.001); BBR (200 mg/kg/d, orally, days 2 to 7) significantly reduced the LD, cylinder test value (***P < 0.001, *P < 0.05), and elevated the LFR (*P < 0.05). Intravenous injection of BBR (10 mg/kg, for 6 days) did not cause a significant change in the LD, LFR, or cylinder test. b Levels of dopa/dopamine in the brain on day 7 of the experiment (mean ± SD, *P < 0.05 for dopa; **P < 0.01, ***P < 0.001 for dopamine). c Number of colonies from the mice treated with the antibiotics was significantly lower than that from the normal mice (mean ± SD, ***P < 0.001). d–f Translation of E. faecalis and E. faecium into the mice treated with antibiotics significantly increased dopa (L)/dopamine (R) levels in ICR mouse brains (d, *P < 0.05, **P < 0.01, NS no significance), blood (e, *P < 0.05, **P < 0.01), and feces (f, (L), (R), *P < 0.05, **P < 0.01, ***P < 0.001). g The relative abundance of Enterococcaeae in the fecal samples of clinical subjects increased after the two-month BBR treatment. Data are represented as the mean ± SD

Fig. 6

BBR stimulated the production of dopamine in striatum in mice transplanted with E. faecalis or E. faecium: TMP-TFB-derivatized MALDI-MS images. a The relative abundance and distributions of dopamine with a spatial resolution of 200 µm. b Rescaled image from a with baseline subtraction and total ion count normalization. c The relative abundance and distributions of dopamine with a spatial resolution of 100 µm. d The relative abundance and distributions of dopamine in half brains from the same mice in c with a spatial resolution of 200 µm. All of them showed elevated levels of dopamine after BBR addition. (group 1: normal group; group 2: antibiotic treated only; group 3: antibiotics and E. faecalis; group 4: antibiotics, E. faecalis, and BBR (200 mg/kg); group 5: antibiotics, E. faecium; and group 6: antibiotics, E. faecium, and BBR (200 mg/kg)). Blue represents the decreased level and red represents the increased level. The striatal dopamine level in the mice receiving both E. faecalis and BBR was higher than that treated with E. faecalis only (group 3 vs. group 4). The striatal dopamine in the mice receiving both E. faecium and BBR was higher than that treated with E. faecium only (group 5 vs. group 6)

Fig. 7

Dopa/dopamine might be the chemical link for the crosstalk between gut and brain. The regulation of Phe–Tyr–dopa–dopamine biosynthesis in the gut microbiota by BBR could improve brain function