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. 2025 Mar 18;26(6):2745.
doi: 10.3390/ijms26062745.

Identification of a Novel Antagonist of BRS-3 from Natural Products and Its Protective Effects Against H2O2-Induced Cardiomyocyte Injury

Jihong Lu  1 , Lehao Wu  1 , Jianzheng Zhu  2 , Han Zhou  3 , Mingzhu Fang  1 , Hongshuo Liang  1 , Miao Guo  2 , Mo Chen  1 , Yuhang Zhu  4 , Jixia Wang  3 , Hua Xiao  2 , Yan Zhang  1
Affiliations

Identification of a Novel Antagonist of BRS-3 from Natural Products and Its Protective Effects Against H2O2-Induced Cardiomyocyte Injury

Jihong Lu et al. Int J Mol Sci. .

Abstract

The identification of exogenous ligands from natural products is an alternative strategy to explore the unrevealed physiological functions of orphan G-protein-coupled receptors (GPCRs). In this study, we have successfully identified and pharmacologically characterized licoisoflavone A (LIA) as a novel selective antagonist of BRS-3, an orphan GPCR. Functional studies showed that pretreatment with LIA ameliorated hydrogen peroxide (H2O2)-induced cardiomyocyte injury. Furthermore, LIA pretreatment significantly restored the activities of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), as well as lactate dehydrogenase (LDH) levels, in H9c2 cells following H2O2 exposure. The protective effect of LIA was also evident in primary cardiomyocytes from rats and mice against H2O2-induced cell injury but was absent in primary cardiomyocytes derived from bombesin receptor subtype-3 knockout (Brs3-/y) mice, strongly confirming the mechanism of LIA's action through BRS-3 antagonism. Proteomics studies further revealed that LIA exerted its protective effects via activating the integrin/ILK/AKT and ERK/MAPK signaling pathways. Complementary findings from Bantag-1, a well-recognized antagonist of BRS-3, in human embryonic kidney 293 mBRS-3 (HEK293-mBRS-3) stable cells and B16 cell lines, which demonstrated resistance to H2O2-induced damage, further supported the pivotal role of BRS-3 in oxidative stress-induced cell injury. Our study contributes to expanding our understanding of the potential pharmacological functions of BRS-3, unveiling previously unknown pharmacological functionality of this orphan receptor.

Keywords: BRS-3; antagonist; cardiomyocyte; licoisoflavone A; orphan GPCR.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Pharmacological characterization of LIA as a novel antagonist of BRS-3. Dose–inhibition curves of LIA for 50 nM MK-5046-induced calcium mobilization in HEK293-mBRS-3 cells (A), IP-1 accumulation in H1299-hBRS-3 cell (B), and DMR responses in HEK293-mBRS-3 cells (C). The predicted binding model of LIA with human BRS-3 (D). The ability of LIA to reverse 1 nM Bomb-induced calcium mobilization in HEK293 expressing GRPR and NMBR cells (E). Data shown are means ± SD, n = 3. Dose–response curves were fitted using the log(agonist) vs. response using GraphPad Prism 8.0 software.
Figure 2
Figure 2
The protective effects of LIA on H2O2-induced injury in H9c2 cells. Cell viability at different concentrations of H2O2 (A). Protective effect of LIA on 200 μM H2O2-induced cell injury (B). Protective effect of LIA on the activities of SOD (C), CAT (D), and the levels of MDA (E) and LDH (F). The values are represented as means ± SD, n = 3. * p < 0.05, ** p < 0.01, **** p < 0.001. “ns” stands for “not significant”, indicating p > 0.05.
Figure 3
Figure 3
Proteomics analysis of the effect of LIA in H9c2 cells induced by H2O2. Pearson correlation (A) and Volcano plots (B). The red dots represent proteins that were upregulated (p < 0.05), and the blue dots represent proteins that were downregulated (p < 0.05). The black dots represent proteins with fold change < 1.5 or without significant changes (p > 0.05). “Canonical pathway” enrichment (C), the dash line represents p = 0.05. The effects of LIA on the protein expressions of ILK, p-P70S6K, p-ERK1/2, and p-AKT (Ser473) were detected by Western blot (D). The values are represented as means ± SD, n = 3. * p < 0.05.
Figure 4
Figure 4
Roles of ILK/AKT and ILK/ERK signaling pathways in the protective effects of LIA against H2O2-induced H9c2 cell injury. Effects of LIA on cell viability in H9c2 cells pretreated with specific inhibitors (A). The protein expressions of p-ERK1/2 and p-AKT (Ser473) after treatment with ILK inhibitor (B). The values are represented as means ± SD, n = 3. * p < 0.05, *** p < 0.005, **** p < 0.001. “ns” stands for “not significant”, indicating p > 0.05.
Figure 5
Figure 5
The protective effects of LIA against H2O2-induced injury in primary rat and mouse cardiomyocytes. The effect of LIA on cell viability in primary rat and mouse cardiomyocytes (A). LIA alters the protein expressions of ILK, p-P70S6K, p-ERK1/2, and p-AKT (Ser473) against H2O2-induced injury in primary rat cardiomyocytes (B). The comparative analysis of the effects of LIA on cell viability against H2O2-induced injury in primary cardiomyocytes from wild-type (WT) and Brs3−/y (KO) mice (C). The values are represented as means ± SD, n = 6 for (A), n = 3 for (B), and n = 5 for (C). * p < 0.05, **** p < 0.001. “ns” stands for “not significant”, indicating p > 0.05.
Figure 6
Figure 6
Validation of target for the BRS-3 regulation of H2O2-induced cell injury. Effect of Bantag-1 on cell viability in H9c2 cells (A). Cell viability in different concentrations of H2O2 in HEK293 and HEK293-mBRS-3 stable cells (B). Effect of Bantag-1 and LIA on cell viability against H2O2-induced injury in HEK293 and HEK293-mBRS-3 stable cells (C). Cell viability in different concentrations of H2O2 in B16 (B16-PX459) cells and B16-KO cells (D). Comparative analysis of the effects of Bantag-1 on cell viability against H2O2-induced injury in B16 cells and B16-KO cells (E). The values are represented as means ± SD, n = 3 for (A–D) and n = 4 for (E). * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001. “ns” stands for “not significant”, indicating p > 0.05.
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