Systems pharmacology dissection of the anti-myocardial ischemia-reperfusion injury mechanism for Ganjiang Fuzi decoction

7 Background: As the commonest form of ischemic heart diseases, the 8 Myocardial Ischemia-Reperfusion injury (MI/RI) accounts for almost 50 9 percent of all deaths. The prevention and treatment of MI/RI while 10 reducing the mortality of myocardial infarction has become a raging topic 11 of research in the cardiovascular field. At present, there are no effective 12 drugs for the treatment of MI/RI. Hence, it becomes imperative to identify 13 or develop efficient lead compounds for treating MI/RI. It has been 14 reported that the Ganjiang Fuzi Decoction (GFD) could be used for the 15 effective treatment of MI/RI due to its promotion of vasodilation and 16 vascular endothelial cell proliferation besides reducing the oxidative 17 damage. 18 Methods: The network pharmacological methods were used in this study, 19 for analyzing the biological processes and the molecular mechanisms of 20 the GFD for MI/RI treatment. In vitro and in vivo experiments were 21 performed for verification of the results of the network pharmacological 22 predictions. 23 Results: Around 16 active components of GFD were discovered against 24 MI/RI, where aconitine, 6-ginger, mesaconitine, and hypaconitine were the 25 leading ones with regard to the degree value. Moreover, it was found that 26 88 MI/RI-related targets mainly involved six aspects, apoptosis, oxidative 27 stress, inflammation, mitochondrial energy metabolism, and vasodilation. 28 In vitro studies indicated the ability of the GFD to increase the survival rate, 29 decrease the apoptosis rate, reduce oxidative damage, and increase the 30 expression of HIF- 1α, VEGF, and eNOS in hypoxia/reoxygenation(H/R) 31 injured Rat Vascular Endothelial Cells (RVEC). The in vivo studies 32 illustrated the capacity of the GFD to reduce the myocardial tissue damage 33 and the infarction area, while increasing the expression of HIF- 1α, VEGF, 34 and eNOS in the MI/RI rats. 35 Conclusions: The results of this study confirmed the anti-MI/RI role of the 36 GFD through the activation of the HIF- 1α signaling pathway, promotion 37 of vascular proliferation and dilation, and the reduction in oxidative 38 damage. The findings of this study would further provide experimental 39 evidence for the application of the GFD in the treatment of MI/RI. 40

The first two cases are reversible, however, the last two are irreversible [7]. 58 Clinically, the PCI, the antiplatelet, and the anticoagulants are mainly used 59 to treat the latter two types of injuries, while this could maintain the 60 patency of the relevant coronary arteries [8]. Nonetheless, the efficacy of 61 surgery for MI/RI has not been found to be significant. Hence, the necessity 62 to develop a new strategy at the earliest for the treatment of MI/RI. injury caused by the myocardial ischemia-reperfusion in the rats [14]. 80 Nevertheless, the fundamental molecular action mechanisms of the GFD 81 have not yet been systematically explored. The bioactive compounds, the potential targets, and the related pathways of GFD remain relatively 83 unknown. 84 With the advent of bioinformatics, network pharmacology has become a 85 powerful tool for the exploration of TCM [15,16]. Network pharmacology 86 integrated systems biology and multi-aspect pharmacological thinking,    were kept under conducive conditions at 24°C ± 1°C temperature, 60%-183 70% relative humidity, and a 12-hour light/dark cycle. The experiment was 184 started once the rats had been subjected to adaptive feeding for one week.

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The rats were used for two sections of the experiment, (1) 20 rats were used 186 for preparing the drug-containing serum, while (2) 60 rats were used for in  and inactivated in a water bath at a temperature of 56℃ for 30 minutes.

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The serum was subsequently stored at -80℃ for later use. were cultured at 37C°with 5% CO2. Upon reaching an 80% confluence 207 level of the population, they were subjected to the experimental procedures.

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The optimal concentration of the drug-containing serum for the RVEC was The 60 rats were randomly segregated into six groups and with 10 rats 243 in each group, the sham group, the MI/RI group, the CDDP group, the GFD 244 low-dose group, the GFD medium-dose group, and the GFD high-dose 245 group. The GFD low-dose group, the GFD medium-dose group, and the 246 GFD high-dose group were administered with 1.4g/kg, 2.8g/kg, and

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The hearts of four rats were sampled randomly and washed using PBS.

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The anterior wall of the left ventricle's myocardium was precooled with 4% All data were expressed as the mean ± Standard Deviation (SD). The 298 differences amongst the multiple groups were evaluated using the one-way 299 analysis of variance (ANOVA). The difference between the means was 300 considered statistically significant at P < 0.05.

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Around 213 components of the GFD were obtained from the TCMSP    (B) Target-Pathway network. The ellipse represents the signaling pathway and the diamond represents the target.

Figure 5
Effects of ganjiang fuzi decoction (GFD) on morphology, number and survival rate of vascular endothelial cells in H/R injured rats(100x). The morphology of vascular endothelial cells in normal rats is like rice grains. After H / R injury, cell morphology became atrophied. Data were expressed as mean ± SD. N=5. #p< 0.05, ##p < 0.01 vs. control group. *p < 0.05, **p < 0.01 vs. H/R group.

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