Investigating the potential cardioprotective properties of two novel microbiome derived compounds urolithin a and urolithin b against methylglyoxal induced cellular damage

Philadelphia, PA

Introduction: Diabetes puts patients at higher risk to develop cardiac, vascular, and neuronal complications due to accumulation of advanced glycation end products. Methylglyoxal (MG) is found raised in diabetic plasma and serves as a precursor of advanced glycation end products. There still is a lack of strategy to mitigate MG’s deleterious effects. Urolithin A (URO A) and Urolithin B (URO B) are two novel microbiome derived compounds that can be found plentiful in such sources as pomegranates, raspberries, and strawberries. There has been recent interest in their ability to provide protection against cellular damage. Their activity against MG induced cellular damage has yet to be investigated. This study aims to better understand their activity against MG damage.

Methods: Rat myoblast H9c2 cell line was used to evaluate MG-induced cell damage and URO A and URO B’s protective effects. Cell viability was assessed via cell counting kit-8 assay (CCK) after MG alone, or MG with URO A or URO B, or MG with a combination of URO A and URO B. Double staining with Calcein-AM and Propidium iodide was used to assess for cell membrane stability and esterase activity of living cells and dead cells, respectively, with MG alone or URO A or URO B with MG. Reactive oxygen species (ROS) production was assessed with Dichlorofluorescin Diacetate (DCFDA) assay at MG alone, or MG with URO A or URO B.

Results: MG reduced cell viability in a dose-dependent manner after cell incubation for 24 hours. Cell viability was 0.50±0.15 (n = 11) when compared to the non-treated control at MG 1200 µM. By contrast, URO A dose-dependently improved cellular viability (5-50 µM, p < 0.05). The best performing ratio of cellular viability was observed at URO A 50 µM (1.75±0.5 compared to MG 1200 µM, n = 8, p < 0.05). Lower dose URO A 10 µM (1.50±0.2, n = 8, p < 0.05) and URO A 5 µM (1.27±0.3, n = 8, p < 0.05) additionally improved cellular viability when compared to MG 1200 µM. URO B (5-50 µM, p < 0.05) mirrored the results of URO A with less efficacy. By comparing to MG 1200 µM, the highest cellular viability was observed at URO B 50 µM (1.56±0.3, n = 10, p < 0.05), with less cellular protection at URO B 25 µM (1.22±0.2, n = 10, p < 0.05). Lower dose URO B 10 µM (1.12±0.1, n = 10, p < 0.05) and URO B 5 µM (1.02±0.1, n = 10, p < 0.05), only showed a slight cellular protection when compared to MG 1200 µM. The various combinations of the two compounds provided statistically significant improvement in cellular viability. The combinations of URO A 10 µM + URO B 5 µM (1.64±0.3, n = 8, p < 0.05) showed better cellular viability than URO A 10 µM + URO B 10 µM (1.39±0.2, n = 8, p

Conclusions: URO A and URO B both improved cell viability against MG H9c2 cell damage, with URO A providing higher efficacy. The combination of URO A and URO B additionally provided a survival advantage, with the best performing combination at UROA 10 µM + URO B 5 µM, suggesting a potential additive relationship between the compounds. Additionally, URO A 25 µM and 50 µM provided membrane stabilization with both Calcein-AM and propidium iodide assay, which suggests another potential mechanism of protection against cell damage. URO A significantly attenuated MG-induced ROS production, which highlights one potential mechanism by which URO A provides better protection than URO B. This data warrants further investigation into these compounds.