Myricetin alleviates diabetic cardiomyopathy by regulating gut microbiota and their metabolites

Diabetic cardiomyopathy (DCM) is a major complication of diabetes and a leading contributor to cardiovascular morbidity and mortality, with diabetes prevalence projected to affect 400 million people by 2030 and ~80% experiencing cardiovascular disease. DCM arises from multifactorial mechanisms including disordered energy metabolism, oxidative stress, inflammation, and mitochondrial dysfunction. Inflammatory activation accelerates cardiomyocyte hypertrophy and cardiac fibrosis, suggesting anti-inflammatory strategies as potential treatments. The gut microbiota plays essential roles in nutrient metabolism, immune modulation, and maintenance of the intestinal barrier. Dysbiosis is linked to cardiometabolic and autoimmune diseases and is altered in type 2 diabetes (T2D), with decreased butyrate-producing taxa (e.g., Roseburia intestinalis, Faecalibacterium prausnitzii) and increased conditional pathogens. Increased intestinal permeability in T2D allows microbial products such as LPS to enter circulation, activating inflammation via TLR4/MyD88 signaling and damaging cardiomyocytes. Myricetin, a dietary polyphenol with antioxidative and anti-inflammatory properties, improves DCM partly via NF-κB suppression, but its low bioavailability implies additional mechanisms, potentially through gut microbiota modulation, as seen in NAFLD models and with related compounds. The study tests the hypothesis that myricetin inhibits DCM progression by modulating gut microbiota to restore intestinal barrier integrity and reduce inflammatory signaling.

Prior work demonstrates gut microbiota alterations in T2D, including reduced butyrate-producing bacteria and enrichment of opportunistic pathogens, contributing to metabolic endotoxemia and systemic inflammation via LPS–TLR4/MyD88 signaling. TLR4 is expressed in cardiomyocytes and implicated in DCM pathogenesis. Polyphenols, including myricetin and its derivative dihydromyricetin, can modulate gut microbiota and reduce inflammation, with reports of increased SCFA-producing bacteria and reduced plasma LPS in metabolic disease models. These findings suggest microbiota-mediated pathways may underlie part of myricetin’s cardioprotective effects despite low bioavailability.

Study design: Two-phase mouse study. Phase I assessed direct myricetin effects in DCM; Phase II evaluated fecal microbiota transplantation (FMT). Chemicals: Myricetin (>98% purity by HPLC) from Chengdu Herbpurify; antibiotics (vancomycin, neomycin sulfate, metronidazole, ampicillin) from Beijing Vital River. Animals: Male C57BL/6J mice (4 weeks old) housed SPF, 22–26 °C, 12-h light/dark, standard chow and water. After 1-week acclimation, mice were randomized (n=10/group) into: CON (normal diet 4 weeks; vehicle STZ injections; vehicle gavage twice daily for 16 weeks), M (normal diet 4 weeks; vehicle STZ; myricetin gavage twice daily for 16 weeks), STZ (HFD 60% kcal fat for 4 weeks; STZ 50 mg/kg i.p. daily ×5; then vehicle gavage twice daily for 16 weeks under continued HFD), MSTZ (HFD 4 weeks; STZ 50 mg/kg i.p. ×5; myricetin gavage twice daily for 16 weeks under continued HFD). Diabetes confirmation: random blood glucose >16.7 mmol/L. DCM induction: continued HFD for 16 weeks. Monitoring: body weight and random blood glucose every 4 weeks. Sample collection: during final week, feces collected daily and stored at −80 °C; at endpoint, mice euthanized by CO2; serum prepared (5000 rpm, 5 min, 4 °C); heart and colon harvested, split for −80 °C storage and histopathology. Phase II (FMT): DCM mice (n=40) allocated to Vehicle (10% glycerol gavage q2d for 16 weeks), Vehicle+Abx (10% glycerol q2d for 16 weeks plus 2-week antibiotic pretreatment), M-FMT (2-week antibiotics, then gut contents from MSTZ donors gavaged q2d for 16 weeks), CON-FMT (2-week antibiotics, then gut contents from CON donors q2d for 16 weeks). Donor gut content prep: contents from MSTZ or CON donors homogenized to 200 mg/ml in saline with 10% glycerol, centrifuged 800 rpm 3 min, supernatant stored at −80 °C. HPLC (λ=352 nm) confirmed no residual myricetin in MSTZ donor suspensions prior to FMT. Antibiotic cocktail: ampicillin 1 g/L, vancomycin 0.5 g/L, neomycin sulfate 1 g/L, metronidazole 1 g/L. Outcomes and assays: - Cardiac structure/function: 2D echocardiography under isoflurane anesthesia; blinded analysis of LVEF, FS, LVIDd, LVIDs from three cardiac cycles per mouse. - Histology: Heart and colon (2–4 cm above cecum) paraffin sections. H&E for myocardial morphology and cardiomyocyte cross-sectional area (CSA); PAS for goblet cells; IHC for collagen I (primary ab ab90395, 1:500; antigen retrieval in citrate buffer pH 6; 8% goat serum block; DAB detection; Papanicolaou hematoxylin counterstain). Image analysis with Image-Pro 6.0. - Intestinal barrier: H&E, PAS (goblet cell counts per field at ×200), IHC for occludin (ab216327, 1:1000). - Serum LPS: ELISA kits (Nanjing Jiancheng Bioengineering) per manufacturer. - Gut microbiota: 16S rDNA sequencing of fecal DNA (Mag Pure Soil DNA LQ Kit); Illumina MiSeq; OTU clustering at 97% with UPARSE; chimera removal with UCHIME; taxonomy by RDP Classifier; analyses at multiple taxonomic levels; PLS-DA; LEfSe for discriminant taxa; rarefaction, Venn diagrams. - Western blotting: Heart and colon lysed in RIPA; protein quantification by BCA; antibodies: TLR4 (ab13556, 1:800), MyD88 (ab219413, 1:1000), NF-κB p65 total (ab32536, 1:10,000), NF-κB p65 phospho-T254 (ab131100, 1:800), occludin (ab216327, 1:1000), GAPDH/β-actin loading controls; densitometry with Image Pro Plus 6.0. - IHC in myocardium for TLR4 and p-p65. Statistics: GraphPad Prism 8.0; mean ± SEM; one-way ANOVA with Tukey’s post hoc; P<0.05 significant. Ethics: Animal Ethics Committee of Shantou University Medical College (SUMC2021-463).

• Metabolic status and cardiac remodeling (Phase I): STZ mice had higher blood glucose than CON (26.23 ± 0.93 vs 8.68 ± 0.47 mmol/L, P<0.05); no significant difference between STZ and MSTZ for glucose. Body weight showed no group differences. Heart weight (HW) and HW/BW increased in STZ vs CON and were reduced by myricetin: HW 121.67 ± 2.46 mg (STZ) vs 103.00 ± 1.75 mg (CON), P<0.05; HW/BW 4.55 ± 0.19 vs 3.78 ± 0.09 mg/g, P<0.05. Echocardiography showed impaired function in STZ (reduced LVEF, FS; increased LVIDd, LVIDs) that improved in MSTZ (all P<0.05). Histology revealed disordered myocardium, cardiomyocyte hypertrophy (CSA increased), and elevated collagen I in STZ; these were attenuated by myricetin (P<0.01). - Gut microbiota composition: 16S rDNA sequencing indicated distinct clustering (PLS-DA) of STZ vs CON, with MSTZ partially overlapping STZ. At phylum level, Firmicutes increased in STZ vs CON (P<0.01); Proteobacteria decreased in STZ vs CON (P<0.01); Bacteroidetes and Actinomycetes decreased in STZ and increased in MSTZ (P<0.05). LEfSe highlighted Bacilli as discriminant taxa (P<0.05). At genus level, SCFA-producing bacteria Roseburia, Faecalibaculum, and Bifidobacterium were increased in MSTZ vs STZ (P<0.05). - Intestinal barrier and endotoxemia: STZ mice showed disordered colon epithelium, reduced goblet cells (PAS), and decreased occludin (IHC); MSTZ improved epithelial morphology, increased goblet cells and occludin (P<0.05). Serum LPS increased in STZ vs CON/M, and decreased in MSTZ (P<0.01). - Cardiac inflammatory signaling: TLR4, MyD88, and phosphorylated p65 (p-p65) were elevated in STZ myocardium and reduced by myricetin (WB and IHC; P<0.05), while total p65 showed no significant detrimental increase after myricetin. - FMT outcomes (Phase II): Compared to Vehicle or Vehicle-Abx, M-FMT recipients had reduced HW (106.33 ± 2.70 vs 118.33 ± 2.12 mg, P<0.05) and HW/BW (3.83 ± 0.11 vs 4.55 ± 0.16 mg/g, P<0.05); CON-FMT did not significantly change HW or HW/BW vs controls. Both M-FMT and CON-FMT improved cardiac function (LVIDd, LVIDs, LVEF, FS; P<0.05), reduced myocardial hypertrophy (CSA) and collagen I (P<0.05), ameliorated colon epithelial disruption, increased goblet cells and occludin (P<0.05), lowered serum LPS (P<0.05), and decreased myocardial TLR4, MyD88, and p-p65 levels (WB and IHC; P<0.05).

The findings support the hypothesis that myricetin mitigates DCM progression primarily via modulation of gut microbiota and restoration of intestinal barrier integrity. In DCM, dysbiosis with decreased SCFA-producing taxa likely contributes to increased intestinal permeability and metabolic endotoxemia (elevated LPS), which activates myocardial TLR4/MyD88 signaling, promoting NF-κB activation, inflammation, cardiomyocyte hypertrophy, and fibrosis. Myricetin treatment increased beneficial SCFA-producing genera (Roseburia, Faecalibaculum, Bifidobacterium), improved epithelial barrier markers (occludin, goblet cells), reduced circulating LPS, and attenuated TLR4/MyD88/NF-κB signaling in the heart, culminating in better cardiac structure and function. FMT experiments corroborated a causal role for gut microbiota: transplanting microbiota from myricetin-treated or healthy control donors reproduced improvements in cardiac function, fibrosis, barrier integrity, and inflammatory signaling in DCM recipients, independent of glycemia or body weight changes. Collectively, these results indicate that augmenting beneficial gut microbes and decreasing endotoxemia are key to myricetin’s cardioprotective effects in DCM.

Myricetin prevents or attenuates diabetic cardiomyopathy by remodeling gut microbiota toward SCFA-producing taxa, restoring intestinal barrier function, reducing systemic LPS, and suppressing myocardial TLR4/MyD88/NF-κB signaling, thereby improving cardiac structure and function. Fecal microbiota transplantation from myricetin-treated or healthy donors similarly ameliorates DCM features, highlighting the central role of gut microbiota. These results provide mechanistic insight into myricetin’s benefits despite low bioavailability and suggest microbiota-targeted strategies (e.g., polyphenols, probiotics, FMT) as potential therapies for DCM. Future studies should validate these findings in clinical settings, define the specific microbial metabolites and pathways involved, and optimize dosing regimens and formulations to enhance myricetin’s bioactivity.