ABSTRACT
Aim: The Shexiang Baoxin Pill (SBP) has been extensively used to treat cardiovascular diseases in China for four decades, and its clinical efficacy has been widely approved. However, the mechanism by which this is achieved remains largely unexplored. Research attempting to understand the underlying mechanism is ongoing, but the findings are controversial. Here, we aimed to explore the possible mechanism of SBP in myocardial ischemia-reperfusion (I/R) injury using heart single-nucleus and spatial ribonucleic acid (RNA) sequencing. Methods: We established a murine myocardial I/R injury model in C57BL/6 mice by ligating and recanalizing the left coronary artery anterior descending branch. Subsequently, single-nucleus RNA-seq and spatial transcriptomics were performed on mice cardiac tissue. We initially assessed the status of cell types and subsets in the model administered with or without SBP. Results: We used single-nucleus RNA sequencing to comprehensively analyze cell types in the cardiac tissue of sham, I/R, and SBP mice. Nine samples from nine individuals were analyzed, and 75,546 cells were obtained. We classified the cells into 28 clusters based on their expression characteristics and annotated them into seven cell types: cardiomyocytes, endothelial cells, fibroblasts, myeloid cells, smooth muscle cells, B cells, and T cells. The SBP group had distinct cellular compositions and features than the I/R group. Furthermore, SBP-induced cardioprotection against I/R was associated with enhanced cardiac contractility, reduced endocardial cell injury, increased endocardial-mediated angiogenesis, and inhibited fibroblast proliferation. In addition, macrophages had active properties. Conclusion: SBP improves the early LVEF of I/R mice and has a cardioprotective effect. Through sequencing analysis, we observed that SBP can increase the gene expression of Nppb and Npr3 in the infarct area of the heart. Npr3 is related to vascular generation mediated by endocardial cells and requires further research. In addition, SBP increases the number of fibroblasts, inhibits the expression of genes related to fibroblast activation and proliferation, and increases the transformation of endothelial cells into fibroblasts. These findings will help to indicate directions for further research.
ABSTRACT
BACKGROUND: Many studies have shown effective protection from myocardial ischemia-reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard. METHODS: In vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3ß silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively. RESULTS: SJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3ß/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3ß/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3ß/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3ß/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR). CONCLUSION: SJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3ß/mTOR pathway, thanks to its two major constituents TMP and BOR.
ABSTRACT
Aim: The present study aimed to explore the potential herb-drug interactions (HDI) between Shengmai injection (SMI) and losartan potassium (LOS) based on the expression profiles of cytochromes P450 (CYP450) and drug transporters in rat and in vitro. Methods: Different concentrations of SMI were used to explore the influence of SMI on the antihypertensive efficacy of LOS in the hypertension rat model established by N (omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks. Subsequently, the serum concentration levels of LOS and losartan carboxylic acid (EXP3174) were determined by Liquid Chromatography Mass Spectrometry (LC-MS) and pharmacokinetic analysis. Human liver microsomes, human multidrug resistance protein 1 (MDR1/P-gp), and breast cancer resistance protein (BCRP) vesicles, human embryonic kidney 293 cell line with stable expression of the organic anion transporting polypeptide 1B1 (HEK293-OATP1B1 cells) and mock-transfected HEK293 (HEK293-MOCK) cells were used to verify the effects of SMI on CYP450 enzymes and drug transporters in vitro. Results: Low, medium, and high concentrations of SMI increased the antihypertensive efficacy of LOS to varying degrees. The high dose SMI increased the half-life (t 1/2 ), the maximum plasma concentration (C max), the area under the plasma concentration-time curve (AUC) from time zero to the time of the last measurable plasma concentration (AUC 0-t ), AUC from time zero to infinity (AUC 0-∞ ), and mean residence time (MRT) values of LOS and decreased its apparent volume of distribution (Vd) and clearance (CL) values. The AUC 0-t , AUC 0-∞ , and MRT of LOS were increased, whereas the CL was decreased by the medium concentration of SMI. In addition, the high, medium, and low doses of SMI increased the relative bioavailability (Frel) of LOS. SMI exhibited no significant effects on the pharmacokinetics of EXP3174. In vitro, SMI exhibited different suppressive effects on the enzyme activity levels of CYP1A2 (6.12%), CYP2B6 (2.72%), CYP2C9 (14.31%), CYP2C19 (12.96%), CYP2D6 (12.26%), CYP3A4 (3.72%), CYP2C8 (10.00-30.00%), MDR1 (0.75%), OATP1B1(2.03%), and BCRP (0.15%). Conclusion: In conclusion, SMI improved the antihypertensive efficacy of LOS in the L-NAME-induced hypertension rat model by increasing the concentration of LOS, while leaving the concentration of EXP3174 intact. SMI affected the pharmacokinetic properties of LOS by decreasing the elimination of LOS. These effects might partly be attributed to the inhibition of the activities of CYP3A4, CYP2C9, and of the drug transporters (P-gp, BCRP, and OATP1B1) by SMI, which need further scrutiny.
ABSTRACT
Following an acute myocardial infarction (AMI), thrombolysis, coronary artery bypass grafting and primary percutaneous coronary intervention (PPCI) are the best interventions to restore reperfusion and relieve the ischemic myocardium, however, the myocardial ischemia-reperfusion injury (MIRI) largely offsets the benefits of revascularization in patients. Studies have demonstrated that autophagy is one of the important mechanisms mediating the occurrence of the MIRI, while non-coding RNAs are the main regulatory factors of autophagy, which plays an important role in the autophagy-related mTOR signaling pathways and the process of autophagosome formation Therefore, non-coding RNAs may be used as novel clinical diagnostic markers and therapeutic targets in the diagnosis and treatment of the MIRI. In this review, we not only describe the effect of non-coding RNA regulation of autophagy on MIRI outcome, but also zero in on the regulation of non-coding RNA on autophagy-related mTOR signaling pathways and mitophagy. Besides, we focus on how non-coding RNAs affect the outcome of MIRI by regulating autophagy induction, formation and extension of autophagic vesicles, and the fusion of autophagosome and lysosome. In addition, we summarize all non-coding RNAs reported in MIRI that can be served as possible druggable targets, hoping to provide a new idea for the prediction and treatment of MIRI.
ABSTRACT
ETHNOPHARMACOLOGY RELEVANCE: Suxiao jiuxin pill (SJP) is a Chinese medical drug with anti-inflammatory, anti-apoptotic, and vasodilatory function. It is widely used in combination with other drugs for the treatment of coronary heart disease (CHD) and angina. Nevertheless, the effect of SJP on Cytochrome P450 (CYP450) enzymes and transporters' activity related to drug metabolism is rarely studied. OBJECTIVE: The aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. MATERIALS AND METHODS: Human primary hepatocytes were used in present study. Probe substrates of CYP450 enzymes were incubated in human liver microsomes (HLMs) with and without SJP while IC50 values were calculated. The inhibitory effect of SJP on the activity of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. The inducing effect of SJP on the activity of CYP1A2, 2B6 and 3A4 was accessed. The inhibition of SJP on human OATP1B1 was investigated through cell-based assay. The inhibition of SJP on human MDR1 and BCRP was also estimated by means of the vesicles assay. RESULTS: The results showed that the SJP under the concentration of 1000 µg/mL could inhibit the activity of CYP1A2, 2B6, 2C19, and 3A4, with IC50 values of 189.7, 308.2, 331.2 and 805.7 µg/mL, respectively. There was no inhibitory effect found in the other 3 liver drug enzyme subtypes. In addition, SJP showed no induction effect on CYP1A2, 2B6 and 3A4, however it had a significant inhibitory effect on human-derived OATP1B1 at the concentration of 100 and 1000 µg/mL, with the IC50 value of 21.9 µg/mL. Simultaneously, the SJP inhibited BCRP at high concentration of 1000 µg/mL but did not affect human MDR1. CONCLUSIONS: Based on these research results above, it is suggested that the SJP can affect some of the CYP450 enzymes and transporters' activity. When used in combination with related conventional drugs, potential herb-drug interactions should be considered.
