Data_Sheet_2.xls

<p>Shuxuening injection (SXNI) is a widely prescribed herbal medicine of Ginkgo biloba extract (EGB) for cerebral and cardiovascular diseases in China. However, its curative effects on ischemic stroke and heart diseases and the underlying mechanisms remain unknown. Taking an integrated approach of RNA-seq and network pharmacology analysis, we compared transcriptome profiles of brain and heart ischemia reperfusion injury in C57BL/6J mice to identify common and differential target genes by SXNI. Models for myocardial ischemia reperfusion injury (MIRI) by ligating left anterior descending coronary artery (LAD) for 30 min ischemia and 24 h reperfusion and cerebral ischemia reperfusion injury (CIRI) by middle cerebral artery occlusion (MCAO) for 90 min ischemia and 24 h reperfusion were employed to identify the common mechanisms of SXNI on both cerebral and myocardial ischemia reperfusion. In the CIRI model, ischemic infarct volume was markedly decreased after pre-treatment with SXNI at 0.5, 2.5, and 12.5 mL/kg. In the MIRI model, pre-treatment with SXNI at 2.5 and 12.5 mL/kg improved cardiac function and coronary blood flow and decreased myocardial infarction area. Besides, SXNI at 2.5 mL/kg also markedly reduced the levels of LDH, AST, CK-MB, and CK in serum. RNA-seq analysis identified 329 differentially expressed genes (DEGs) in brain and 94 DEGs in heart after SXNI treatment in CIRI or MIRI models, respectively. Core analysis by Ingenuity Pathway Analysis (IPA) revealed that atherosclerosis signaling and inflammatory response were top-ranked in the target profiles for both CIRI and MIRI after pre-treatment with SXNI. Specifically, Tnfrsf12a was recognized as an important common target, and was regulated by SXNI in CIRI and MIRI. In conclusion, our study showed that SXNI effectively protects brain and heart from I/R injuries via a common Tnfrsf12a-mediated pathway involving atherosclerosis signaling and inflammatory response. It provides a novel knowledge of active ingredients of Ginkgo biloba on cardio-cerebral vascular diseases in future clinical application.</p

Data_Sheet_1.xls

<p>Shuxuening injection (SXNI) is a widely prescribed herbal medicine of Ginkgo biloba extract (EGB) for cerebral and cardiovascular diseases in China. However, its curative effects on ischemic stroke and heart diseases and the underlying mechanisms remain unknown. Taking an integrated approach of RNA-seq and network pharmacology analysis, we compared transcriptome profiles of brain and heart ischemia reperfusion injury in C57BL/6J mice to identify common and differential target genes by SXNI. Models for myocardial ischemia reperfusion injury (MIRI) by ligating left anterior descending coronary artery (LAD) for 30 min ischemia and 24 h reperfusion and cerebral ischemia reperfusion injury (CIRI) by middle cerebral artery occlusion (MCAO) for 90 min ischemia and 24 h reperfusion were employed to identify the common mechanisms of SXNI on both cerebral and myocardial ischemia reperfusion. In the CIRI model, ischemic infarct volume was markedly decreased after pre-treatment with SXNI at 0.5, 2.5, and 12.5 mL/kg. In the MIRI model, pre-treatment with SXNI at 2.5 and 12.5 mL/kg improved cardiac function and coronary blood flow and decreased myocardial infarction area. Besides, SXNI at 2.5 mL/kg also markedly reduced the levels of LDH, AST, CK-MB, and CK in serum. RNA-seq analysis identified 329 differentially expressed genes (DEGs) in brain and 94 DEGs in heart after SXNI treatment in CIRI or MIRI models, respectively. Core analysis by Ingenuity Pathway Analysis (IPA) revealed that atherosclerosis signaling and inflammatory response were top-ranked in the target profiles for both CIRI and MIRI after pre-treatment with SXNI. Specifically, Tnfrsf12a was recognized as an important common target, and was regulated by SXNI in CIRI and MIRI. In conclusion, our study showed that SXNI effectively protects brain and heart from I/R injuries via a common Tnfrsf12a-mediated pathway involving atherosclerosis signaling and inflammatory response. It provides a novel knowledge of active ingredients of Ginkgo biloba on cardio-cerebral vascular diseases in future clinical application.</p

Video_1_Danhong Injection Reversed Cardiac Abnormality in Brain–Heart Syndrome via Local and Remote β-Adrenergic Receptor Signaling.mp4

<p>Ischemic brain injury impacts cardiac dysfunction depending on the part of the brain affected, with a manifestation of irregular blood pressure, arrhythmia, and heart failure. Generally called brain–heart syndrome in traditional Chinese medicine, few mechanistic understanding and treatment options are available at present. We hypothesize that considering the established efficacy for both ischemic stroke and myocardial infarction (MI), Danhong injection (DHI), a multicomponent Chinese patent medicine, may have a dual pharmacological potential for treating the brain–heart syndrome caused by cerebral ischemic stroke through its multi-targeted mechanisms. We investigated the role of DHI in the setting of brain–heart syndrome and determined the mechanism by which it regulates this process. We induced Ischemia/Reperfusion in Wistar rats and administered intravenous dose of DHI twice daily for 14 days. We assessed the neurological state, infarct volume, CT scan, arterial blood pressure, heart rhythm, and the hemodynamics. We harvested the brain and heart tissues for immunohistochemistry and western blot analyses. Our data show that DHI exerts potent anti-stroke effects (infarct volume reduction: ^∗∗p < 0.01 and ^∗∗∗p < 0.001 vs. vehicle. Neurological deficit correction: ^∗p < 0.05 and ^∗∗∗p < 0.001 vs. vehicle), and effectively reversed the abnormal arterial pressure (^∗p < 0.05 vs. vehicle) and heart rhythm (^∗∗p < 0.01 vs. vehicle). The phenotype of this brain–heart syndrome is strikingly similar to those of MI model. Quantitative assessment of hemodynamic in cardiac functionality revealed a positive uniformity in the PV-loop after administration with DHI and valsartan in the latter. Immunohistochemistry and western blot results showed the inhibitory effect of DHI on the β-adrenergic pathway as well as protein kinase C epsilon (PKCε) (^∗∗p < 0.01 vs. model). Our data showed the underlying mechanisms of the brain–heart interaction and offer the first evidence that DHI targets the adrenergic pathway to modulate cardiac function in the setting of brain–heart syndrome. This study has made a novel discovery for proper application of the multi-target DHI and could serve as a therapeutic option in the setting of brain–heart syndrome.</p

Groups of KKAy mice and WT mice.

<p>Groups of KKAy mice and WT mice.</p

DHI improved perfusion of ischemic limbs in KKAy mice.

<p>(A) Representative images of laser Doppler perfusion analysis for WT control mice, KKAy mice treated with AMD3100, KKAy mice treated with or without DHI before surgery and at different time points after surgery. Low perfusion signals (dark blue) were observed in the ischemic hind limb, whereas high perfusion signals (red) were detected in KKAy mice treated with DHI on postoperative day 8 through 35 and in KKAy mice treated with AMD3100 on postoperative days 8, 21, 28, and 35. (B) Hind-limb perfusion recovery was impaired in untreated KKAy mice. The mean hind-limb blood flow was calculated as the ratio of ischemic (left) side to non-ischemic (right) side. (C) DHI or AMD3100 significantly improved perfusion recovery after HLI surgery. <i>*P<0</i> .<i>05</i>, <i>**P<0</i> .<i>01</i>, <i>***P<0</i> .<i>001 vs</i>. <i>KKAy+vehicle</i>.</p

IPA indicated the overlap between angiogenesis and glucose tolerance networks.

<p>(A) Red colored genes are related to glucose tolerance and green colored genes are related to angiogenesis. The arrows indicate effects of regulated genes on other genes. (B) Orange colored are predicted to be activation and blue colored are predicted to be inhibited.</p

<i>In vivo</i> expression of VEGFR-2 in diabetic VEGFR-2-luc mice.

<p>(A-C) Representative Bioluminescent images of HLI mice were obtained at 0, 7 and 10 days under the same imaging conditions. (D) The dynamic measurement of bioluminescent intensities in non-DM, DM+vehicle and DM+DHI groups. Regions of interest (ROI) from displayed images were identified on the HLI sites and quantified as photons per second (p/s). Data is shown as mean ± SEM. n = 5 in each group. <i>*P<0</i>.<i>05</i>, <i>vs</i>. DM+vehicle.</p

Increase of angiogenic factors in DHI-treated ischemic muscle.

<p>(A) Quantitative PCR showed DHI increased the expression of VEGF-A and Flk-1 in KKAy mice. (B) Quantitative PCR showed DHI increased the expression of VEGF-A and Flk-1 in STZ-induced diabetic mice. Data represent the means ± SEM, C57BL/6J: n = 5, KKAy+vehicle: n = 4, KKAy+DHI: n = 6; Non-DM: n = 3, DM+vehicle: n = 5, DM+DHI: n = 5; <i>vs</i>. <i>vehicle</i>, <i>***P<0</i> .<i>001</i>.</p

DHI decreased incidence of limb necrosis in diabetic mice post HLI.

<p>Representative images of hind limbs showing evidence of tissue necrosis. In DM+vehicle group, six out of seven ischemic limbs showed necrosis (86%), while in DM+DHI group, four out of seven ischemic limbs showed necrosis (57%).</p

DHI increased EPC mobilization in KKAy mice.

<p>EPC (defined as Sca-1⁺/Flk-1⁺cells) mobilization after tissue ischemia was determined by flow cytometry in C57BL/6J mice and KKAy mice after administration of saline, DHI or AMD3100. DHI and positive-control AMD3100 both showed a significantly improved EPC mobilization after HLI surgery. <i>*P<0</i> .<i>05 vs</i>. <i>KKAy+vehicle</i>.</p