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. 2022 Jul 7;7(1):207.
doi: 10.1038/s41392-022-01027-6.

Berberine treats atherosclerosis via a vitamine-like effect down-regulating Choline-TMA-TMAO production pathway in gut microbiota

Affiliations

Berberine treats atherosclerosis via a vitamine-like effect down-regulating Choline-TMA-TMAO production pathway in gut microbiota

Shu-Rong Ma et al. Signal Transduct Target Ther. .

Abstract

Trimethylamine-N-oxide (TMAO) derived from the gut microbiota is an atherogenic metabolite. This study investigates whether or not berberine (BBR) could reduce TMAO production in the gut microbiota and treat atherosclerosis. Effects of BBR on TMAO production in the gut microbiota, as well as on plaque development in atherosclerosis were investigated in the culture of animal intestinal bacterial, HFD-fed animals and atherosclerotic patients, respectively. We found that oral BBR in animals lowers TMAO biosynthesis in intestine through interacting with the enzyme/co-enzyme of choline-trimethylamine lyase (CutC) and flavin-containing monooxygenase (FMO) in the gut microbiota. This action was performed by BBR's metabolite dihydroberberine (a reductive BBR by nitroreductase in the gut microbiota), via a vitamine-like effect down-regulating Choline-TMA-TMAO production pathway. Oral BBR decreased TMAO production in animal intestine, lowered blood TMAO and interrupted plaque formation in blood vessels in the HFD-fed hamsters. Moreover, 21 patients with atherosclerosis exhibited the average decrease of plaque score by 3.2% after oral BBR (0.5 g, bid) for 4 months (*P < 0.05, n = 21); whereas the plaque score in patients treated with rosuvastatin plus aspirin, or clopidogrel sulfate or ticagrelor (4 months, n = 12) increased by 1.9%. TMA and TMAO in patients decreased by 38 and 29% in faeces (*P < 0.05; *P < 0.05), and 37 and 35% in plasma (***P < 0.001; *P < 0.05), after 4 months on BBR. BBR might treat atherosclerotic plaque at least partially through decreasing TMAO in a mode of action similar to that of vitamins.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
BBR decreased the production of TMA/TMAO in gut microbiota. a Chemical structures of berberine (BBR), dihydroberberine (dhBBR), trimethylamine (TMA) and trimethylamine-N-oxide (TMAO). b The levels of TMA and TMAO in faeces or plasma of the HFD-fed hamsters were significantly reduced 6, 12, and 24 h after oral administration of BBR (100 mg/kg, n = 6, *P < 0.05, **P < 0.01 and ***P < 0.001). c BBR, dhBBR (0.03, 0.06 mM) inhibited the production of TMA in the intestinal bacteria from HFD-fed SD rat, and the inhibitory effect of dhBBR was stronger than that of 3,3-dimethyl-1-butanol (DMB, 0.12, 0.3 mM), a positive control, after 12 h incubation (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001). d A 4.6-fold increase of TMA was observed after addition of choline; but the phenomena was not seen with carnitine (40 μg/mL, n = 5, ***P < 0.001). e TMA level in the cutC-E.coli (transformed with pET28a-cutD) was elevated as compared to that in the cutC-E.coli (transformed with pET28a) (**P < 0.01) after 8 h incubation (**P < 0.01) (n = 6). f dhBBR (0.06 mM) inhibited the transformation from choline to TMA in a reaction system heterologously expressed choline-TMA lyase (cutC) (8 h, n = 6, **P < 0.01). g The effect of BBR (0.03 mM) on TMA production in 15 intestinal bacterial strains in vitro, and of the 15 strains, P. mirabilis, S. boydii and B. fragilis showed a significant decrease in TMA level after BBR treatment (n = 5, *P < 0.05, **P < 0.01). h BBR, dhBBR (0.03 mM) and DMB (a competitive inhibitor of choline-TMA lyase, 0.1 mM) decreased the level of TMA in P. mirabilis after 12 h incubation (n = 6, **P < 0.01, ***P < 0.001). i TMA level in P. aeruginosa did not change after BBR/dhBBR treatment (n = 6, 0.03 mM; NS, not significant). j DhBBR (0.06 mM) inhibited the decomposition of choline in the gut microbiota from the HFD fed rat (n = 6, ***P < 0.001). Data are expressed as mean ± SD and analysed with two-tailed student’s t test
Fig. 2
Fig. 2
Putative mechanisms of dhBBR on inhibiting bacterial choline-TMA lyase (CutC). a The 3D docking results of dhBBR and bacterial CutC by CDOCKER tool showed that multiple interactions might mediate the binding of dhBBR into the pocket. b BBR inhibited TMA production through its characteristic metabolite dhBBR from gut microbiota, and the inhibition was on CutC rather than CCAT. c Assumed mechanism of dhBBR in down-regulating CutC
Fig. 3
Fig. 3
Putative mechanisms of dhBBR on inhibiting bacterial flavin-containing monooxygenase (FMO). a The virtual docking of dhBBR with bacterial FMO by CDOCKER tools and the possible interactions. b BBR might inhibit TMAO production through dhBBR’s inhibitory activity on FMO in the gut bacteria
Fig. 4
Fig. 4
DhBBR inhibited TMAO generation by targeting bacterial flavin-containing monooxygenase (FMO) in gut. a Both BBR and dhBBR (0.03, 0.06 mM) inhibited TMAO production in intestinal bacteria (n = 5, ***P < 0.001), and the known FMO inhibitors (imipramine and methimazole) showed an inhibition as well. b The effect of BBR (0.03 mM) on TMAO production in vitro was tested in 15 intestinal bacterial strains, of which TMAO was detected in P. mirabilis, P. aeruginosa, P. anaerobius and E. aerogenes. In P. aeruginosa and P. anaerobius, TMAO showed a decrease after BBR treatment (n = 5, **P < 0.01). c BBR, dhBBR (0.03 mM) and imipramine (inhibitor of FMO, 0.1 mM) decreased TMAO level in P. aeruginosa after incubation for 12 h (n = 6, *P < 0.05, **P < 0.01). d The conversion from TMA to TMAO increased in the FMO-expressed reaction system (pET28a-fmo) after 4 h incubation (n = 4, ***P < 0.001). e BBR and dhBBR (0.06 mM) inhibited the transformation from TMA to TMAO in a heterologously FMO-expressed reaction system (E. coli with pET28a-fmo transformation) at 4 h after incubation (n = 4, *P < 0.05). f The inhibition ration (%) of TMAO production in the FMO-reaction system (in E. coli with pET28a-fmo) was dose-dependent (n = 4). g, h DhBBR (0.06 mM) inhibited the production of TMAO (g) and increased the level of TMA (h) in the FMO-expression reaction system (E. coli pET28a-fmo, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001). i DhBBR was transformed into BBR in the FMO reaction system (E. coli pET28a-fmo), n = 6. Data shown are mean ± SD and analysed with two-tailed student’s t test
Fig. 5
Fig. 5
Anti-atherosclerotic effects of BBR in hamsters fed with high-fat diet. a Oil red O and HE staining of arcus aortae in atherosclerotic hamsters after BBR treatment for 3 months. Group N, the normal control group, n = 8; Group H, the atherosclerosis model group fed with HFD, n = 7; Group BL, the low-dosage BBR group (oral, 100 mg/kg/d), n = 7; Group BH, the high-dosage BBR group (oral, 200 mg/kg/d), n = 4; Group A, the group treated with antibiotics (oral, terramycin 300 mg/kg/d, erythromycin 300 mg/kg/d and cefadroxil 100 mg/kg/d, 3 months), n = 5; Group AB, the group treated with BBR and the combination of antibiotics (oral, terramycin 300 mg/kg/d, erythromycin 300 mg/kg/d, cefadroxil 100 mg/kg/d, and BBR 200 mg/kg/d; 3 months), n = 5. Red arrows: the location of plaques. b The maximum intima-media thickness (IMTmax) measurement of arcus aortae in hamsters showed that BBR significantly alleviated the severity of atherosclerosis after 3 months treatment (*P < 0.05, **P < 0.01). c 16 s rDNA Gene copies in hamster faeces at the point of two months. The number of colonies was decreased after the treatment with combination of antibiotics for two months (−54%) or the treatment with BBR (−27%, 200 mg/kg), in comparison with the HFD model group. d BBR significantly inhibited the production of TMA and TMAO in faeces, and the effects were weakened after intervention with antibiotics (2 months, ***P < 0.001). e Oral administration of BBR significantly lowered the levels of TMA and TMAO in plasma after 2 months therapy (**P < 0.01, ***P < 0.001). f The levels of FBG, TG, TC and LDL-C in serum were significantly lowered by oral administration of BBR for 2 months (*P < 0.05, **P < 0.01, ***P < 0.001). Data in bf are expressed as the mean ± SD and analysed with two-tailed student’s t test
Fig. 6
Fig. 6
BBR reduced the plaque size in patients with atherosclerosis. a The ultrasonic images of atherosclerotic plaques (circle in white) of the patients at different positions of carotid arteries before (1) and after (2) 4-month BBR therapy are shown, including the common carotid artery (A: participate No. 1, plaque No. 1), the carotid bifurcation (B: participate No. 16, plaque No. 38), the internal carotid artery (C: participate No. 6, plaque No. 19) and the subclavian artery (D: participate No. 19, plaque No. 44). Also, please see Table S9 in the supplemental material. b The average plaque score in patients was decreased by 3.2% after oral BBR (0.5 g, bid) for 4 months (*P < 0.05, n = 21); the score value was slightly increased (by +1.9%) after treatment with conventional drug combination, including rosuvastatin, aspirin, as well as clopidogrel sulfate or ticagrelor (n = 12) if needed. c The average carotid intima-media thickness was reduced by 3.2% after oral treatment with BBR (P = 0.067), and the value was slightly increased (by +2.0%) in the combination therapy. d The average carotid plaque length in patients was decreased by 2.2% in either oral BBR (0.5 g/bid) group or combination drug group after 4 months on therapy. e TMA and TMAO level in faeces and plasma samples of patients went down after 4 months BBR treatment; the TMA/TMAO levels in plasma were lowered by 37% / 35% (***P < 0.001; *P < 0.05), and TMA/TMAO levels in faeces decreased by 38%/29% (*P < 0.05; *P < 0.05). Data shown are the mean ± SEM and analysed with one-tailed paired t test
Fig. 7
Fig. 7
Berberine (BBR) treats atherosclerosis through its vitamin-like effect down-regulating TMAO production in the gut microbiota

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