Chemical Profile of XBW by UPLC-QTOF-MS
A specific UPLC-QTOF-MS method was used to identify the chemicals in XBW. As shown in Figure 2 and Table 1, a total of 37 compounds were identified by comparing their retention time with that of reference compounds or comparing their retention behaviors, empirical molecular formula and proposed fragmentations with that in literatures (11-25), including ginsenoside Rg1-3, Rb1-3, Re, Ro, gypenoside XVII, aconine, mesaconine, isotalatizidine, scopolamine, arenobufagin, ect. The chemical components were derived from Fuzi, Panax ginseng, Panax notoginseng, Flos Daturae, CCP, and toad (Chansu) in XBW.
Effect of XBW against myocardial ischemia-reperfusion injury in rat model in vivo and oxygen glucose deprivation-reperfusion (OGD/R) cell model in vitro
LAD ligation-induced MI/RI rat model was used to investigate the cardioprotective effect of XBW. As shown in Figure 3A and 3B, XBW administration significantly reduced infarct size. CK-MB is one of biomarker in serum for MI/RI, and XBW administration decreased the level of CK-MB induced by MI/RI (Figure 3C). Echocardiography in Figure 3D exhibited that XBW improved cardiac function. H&E staining showed that XBW administration attenuated inflammatory cell infiltration, and disordered myocardial fiber induced by MI/RI (Figure 3E). In vitro study, XBW protected H9c2 cell against OGD/R injury in a dose-dependent manner (Figure 4A-B). These results suggested that XBW ameliorated MI/RI.
Target Identification and Network Analysis
Using TargetNet database and literatures reported targets, we obtained 246 targets of 37 compounds in XBW (Figure 5A). The network contained 283 nodes and 462 edges, and its average number of neighbors was 3.216. After crossing with MI/RI targets, 50 targets were identified and a compound-target network was constructed in Figure 5B and 5C.
After GO enrichment analysis of the targets by the String database, the top 15 enrichment results listed in biological processes (BP), molecular functions (MF) and cellular components (CC) are shown in Figure 6A, which indicated that XBW may regulate the apoptosis and stress response of cardiomycytes via protein binding, enzyme binding, transcription factor binding, protein kinase binding, extracellular space, CHOP-ATF4 complex, etc to attenuate MI/RI. To clarify the underlying pathways of XBW on MI/RI, KEGG pathway analysis was performed in Figure 6B, which exhibited the top 20 related signaling pathways excluding the specific cancer related pathways. HIF-1 signaling pathway, PI3K-Akt signaling pathway, autophagy, FoxO signaling pathway, apoptosis, etc. Based on the protein-protein interactions (PPI) analysis (Figure 6C), CASP3, MTOR (apamycin), SIRT1, HIF1A, ATF4, GRP78 (BIP, glucose regulated protein 78) and ATG7 were identified with high-degree targets, which played important roles in apoptosis, autophagy and endoplasmic reticulum (ER) stress.
Experimental validations of the molecular mechanisms of XBW against MIRI.
Cardiomyocyte apoptosis is a key factor in the pathological process of MI/RI. As shown in Figure 7A, after 24 h reperfusion, TUNEL staining showed the percentage of apoptosis-positive cells in MI/RI group increased, while decreased in XBW treatment group. In addition, the apoptosis related proteins were detected. XBW increased the ratio of Bcl2/Bax expression and decreased caspase 3 expression (Figure 7B-D).
During MI/RI, autophagy was also activated following MI/RI in cardiomyocytes to involve in the apoptosis of myocardial cells (26, 27). Bclin-1 and LC3II expressions were detected (Figure 8A-C). The expressions of Bclin-1 and LC3II in cardiac tissue were markedly increased in MI/RI group, while significantly decreased in XBW administration group. In vitro study, XBW could also decrease Beclin-1 expression induced by OGD/R in H9c2 cells (Figure 8D and 8E). Cardiomyocytes open the unfolded protein response triggered by ER stress as a defensive mechanism at early stage of MI/RI, however, excessive ER stress induced cell apoptosis or even death. As shown in Figure 8F and 8G, the expression of BIP, a marker of ER stress, was significantly increased in MI/RI group compared with that in the Sham group, however, XBW administration reduced the elevation of BIP expression. Taken together, these data demonstrated that the cardioprotective effect of XBW against MI/RI was associated with the attenuation of ER stress and autophagy.