| Objective:Cardiac β1-adrenergic receptors(β1-ARs) are important regulators of sympathetic nervous system and are thought to play a critical role in cardiac function. Stimulation of cardiac β1-ARs leads to improved force of contraction, increased heart rate, disorder of cardiac energy substrate metabolism, shortage of ATP, which having a negative impact on cardiac efficiency and function. Therefor, the aim of the present study was to develop a high expressed β1-AR cell membrane chromatography(β1-AR/CMC) offline with ultra-performance liquid chromatography and mass spectrometry(UPLC/MS) method for screening active ingredients from Evodia rutaecarpa(Juss) Benth. Furthermore, the present study was to explore the effect of the active components on myocardial ischemia injury-induced impairment on myocardial structure and cardiac function, changes in myocardial energy substrate metabolism and the possible implication of the energy metabolism modulation. Methods:First of all, the CMC column which was prepared by the Chinese hamster ovary cells with high expression level of β1-AR(β1-AR/CHO-S), was connected to UPLC/MS system to develop the β1AR/CMC-Offline-UPLC/MS analytical method. A drug diazepam(DZ) and a metoprolol were injected into the β1-AR/CMC column to determine the validation. Extracts of Evodia rutaecarpa(Juss) Benth were prepared by 95% ethanol, petroleum ether, dichloromethane, and ethylacetate. The four extracts were carried out using this β1AR/CMC system for seperation, and then the obtained fractions to be analyzed by UPLC/MS system.Later, the CHO-S cells were treated with isoprenaline(ISO), and determinated the level of cyclic adenosine monophosphate(c AMP) and protein kinase A(PKA) after drug intervention to estimate the inhibitory effect on β1-AR.Last but not least, we explored the protective role of active components in myocardial ischemia–reperfusion injury. After reperfusion, myocardial infarct size was assessed after reperfusion by Evans blue-TTC staining; blood was sampled from the abdominal aorta and serum was isolated for determination of glucose, lactic acid(LD), lactate dehydrogenase(LDH) and free fatty acids(FFA) according to the reagent kit’s instructions; The content of ATP of myocardium, PPARα content and Ca2+-ATPase activity were assessed with ELISA. Then we make a preliminary investigation of active components on myocardial ischemia/reperfusion injury. Results:The results of the validation of β1AR/CMC-Offline-UPLC/MS method shows that the selective β1-AR antagonist metoprolol had better retention characteristics(tR=7.3 min), the RSD(%) of the retention time(tR) of the metoprolol peak was 7.84% when changing β1-AR/CMC columns(n=3) and he reproducibility of one β1-AR/CMC column was tested by injecting metoprolol 6 times, and the RSD(%) of the retention time(tR) of the metoprolol peak was 1.52%(n=6). The screening results of 95% ethanol, petroleum ether, dichloromethane, and ethylacetate using the β1AR/CMC-Offline-UPLC/MS method shows that 95% ethanol extract of Evodia rutaecarpa(Juss) Benth were significantly retained on the β1-AR/CMC, and the obtained fraction was identified as evodiamine(Evo) and rutaecarpine(Rut) by UPLC/MS.The results of in vitro and in vivo experiments:(i) concentrations of c AMP and PKA in Rut and Evo groups were lower than in the ISO group(P< 0.01).(ii) Infarct area was prevented significantly by treatment with Evo(0.1 mg/kg) and Rut(0.1 mg/kg), respectively; Serum glucose, LD and FFA levels in Rut and Evo groups were lower than that in I/R group; Treated with Rut and Evo, the ATP content and the activity of Ca2+-ATPase significantly improved compared to I/R group; there was a greatly decline of the PPARα content with treatment of Rut and Evo. The results above showed that Rut and Evo improved the energy metabolism of I/R heart between FFA and glucose. Conclusions:In conclusion, our β1AR/CMC-offline-UPLC/MS method could be used as a sensitive, specific, stable, quick alternative for screening new type of β1-AR antagonists from TCMs such as Evodia rutaecarpa(Juss) Benth. What’s more, we talked about Evo and Rut prevent cardiac ischemia–reperfusion injury via energy modulation. We argued that Evo and Rut may regulate the glycolipid substrate metabolism via blocking signaling pathways of β1-AR and effect the PPARα function to reduce the accumulation of free fatty acids and lactic acid to protect cardiac myocytes and delay heart I/R injury development process. The results provided a useful method for drug discovery and a novel treatment strategy against I/R-induced myocardial injury. |
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