| ObjectiveMyocardial ischemia-reperfusion(MIR) injury is a major contributor to the morbidity and mortality associated with coronary artery disease. MIR is characterized by a rapid increase in cytokines, chemokines and leukocyte influx into the vulnerable parts of the danger zone of the infarct. This is not only the cause in the acute phase inflammatory response leading to myocardial apoptosis, but also the cause leading to adverse cardiac remodeling and further damage to the heart. Atherosclerosis(AS) is a chronic inflammatory disease in the arterial wall, and it is the leading cause to the incidence and mortality of ischemic heart disease. Its characteristics are circulating lipid deposition in the damaged part of the vessel wall especially in the subendothelial space, and cause the complex interactions between the modified lipoproteins and intrinsic vascular cells and the immune system, and ultimately lead to the atherosclerotic plaque rupture and the formation of ischemic heart disease based on its clinical complications.Chinese herbal medicine, especially the ones used to activate circulation to resolve stasis as well as the combined herbal formulations, has been widely used in Traditional Chinese Medicine(TCM) for the treatment of myocardial infarction for hundreds of years. While the efficacy of Chinese herbal medicine is well documented, the underlying molecular mechanisms remain elusive. Therefore we got into this study, in order to bridge the gap between experimental research and clinical applications in medicine. MethodsFirst of all, we highlighted recent studies from literature which was focused on elucidating the cellular and molecular mechanisms using extracted compounds, single herbs or herbal formulations totally about100kinds in experimental settings. Based on the fact that the Myocardial ischemia-reperfusion injury and atherosclerosis shares many pathological processes features, including production of cytokines, chemokines and inflammatory cell infiltration, cell death of cardiomyocytes and intrinsic arterial wall cells such as vascular smooth muscle cells, and Toll-like receptor(TLR)2and4participate in both of these two processes. Hence, herein we investigated the ability of Sparstolonin B(SsnB),a new Chinese herb-derived selective TLR2/4antagonist, to inhibit the TLR2/4-mediated inflammatory responses during cardiomyocyte hypoxia-reoxygenation injury, this injury in rat heart tissue, and atherosclerotic pathological conditions affecting on the aortic vascular smooth muscle cells(VSMC), as well as the underlying responsible mechanisms.Related cellular and molecular biochemistry and immunohistochemical methods were used in this study:(1) Lactate dehydrogenase(LDH) assay was used to evaluate the toxic effects of the Chinese herb-derived compound to the cell activity,(2) Direct cell counting and [3H] thymidine incorporation methods were used for the determination of cell proliferation,(3) Wound healing assay and Transwell chamber assay were used to evaluate the ability of cell migration,(4) Quantitative real-time PCR assay was used for the evaluation of the target gene mRNA expression levels,(5) Enzyme-linked immunosorbent assay(ELISA) was used for the detection of the low-level expression of proteins extracelluarly,(6) Immunoblot assay(Western blot) was used to detect the expression of cell proteins and cell signal transduction molecules intracelluarly,(7) Cells cholesterol kit was used for the detection of intracellular cholesterol levels,(8) Terminal transferase-mediated dUTP nick end labeling(TUNEL) staining was used for the detection of apoptosis cells,(9) periodate acid-Schiff(pAS) staining was used to detect the level of the early occurrence of necrotic cells,(10) Oil Red0staining was used for evaluating the level of intracellular lipid deposition. ResultsFirstly, on the one hand, the results of the literature review from myocardial ischemia and reperfusion injury showed that there were7mechanisms mainly involved in the effects of TCM on MIR, including(1) anti-oxidation,(2) anti-inflammation,(3) anti-apoptosis,(4) protecting mitochondrial function,(5) increasing BMSCs migration,(6) promoting angiogenesis,(7) up-regulating kATP channel subunits and ATPase, and inhibiting calcium overload. On the other hand, the results of the literature review from atherosclerosis showed that there were also7mechanisms mainly involved in the effects of TCM on atherosclerosis, including:(1) protecting the vascular endothelium,(2) reducing the deposition of low-density lipoprotein,(3) inhibition of LDL oxidation,(4) inhibition of monocyte migration and activation,(5) inhibition of vascular smooth muscle cell migration and proliferation,(6) reducing the formation of foam cells,(7) inhibition of chronic inflammation.Secondly, the results of the cardiomyocyte experiments indicated that(1)LDH assay showed that there was not cytotoxicity of SsnB at the proper concentrations on H9c2cardiomyocytes. Therefore,10u M of SsnB was used in the subsequent experiments.(2) Quantitative real-time PCR confirmed that TLR2and TLR4expression was elevated during hypoxia-reoxygenation, and that their up-regulation in cardiomyocytes was significantly inhibited by SsnB (P<0.05).(3) Both the mRNA levels which were detected by qPCR and protein levels which were detected by ELISA of monocyte chemotactic protein-1(MPCl)and high mobility group box1(HMGB1)were up-regulated during hypoxia-reoxygenation, and were significantly attenuated by SsnB (P<0.05).(4) Next we found that ERK1/2and JNK signaling pathways were activated during hypoxia-reoxygenation and SsnB significantly inhibited their activation (P<0.05).(5)Moreover, Transwell cell migration assays revealed that the migration of mouse macrophages to hypoxia-reoxygenation injured cardiomyocytes was significantly reduced by SsnB(P<0.05).Thirdly, the results of the rat heart tissue of left ventricle(LV) experiments indicated that(1) LDH levels were measured and reflected there was not cytotoxicity and cell injury with SsnB at proper concentrations. Therefore,15u M and30μM of SsnB were used in the subsequent experiments.(2)SsnB significantly reduced LDH release from the hypoxic LV tissue slices in a dose-dependent manner.(3)TdT-mediated dUTP-biotin nick end labeling(TUNEL)staining and periodic Acid-Schiff(pAS)staining showed that SsnB protected the cultured LV tissue slices against hypoxia-induced apoptosis(P<0.05)and necrosis(P<0.05), respectively.(4)Quantitative real-time PCR confirmed that the mRNA expressions of MPC1and IL-6were elevated during hypoxia, while their up-regulations in the cultured LV tissue slices were significantly inhibited by SsnB in a dose-dependent manner(P<0.05).Last but not least, the results of the rat aotic artery vascular smooth cells experiments indicated that(1) LDH assay was performed to measure the cytotoxicity of SsnB on VSMC. There was not cytotoxicity of SsnB at the proper concentrations on VSMC cardiomyocytes. Therefore,3μ M,10μM and30μM of SsnB were used in the subsequent experiments.(2) Dirct cell counting, LDH assay and [3H] thymidine incorporation were performed to measure the inhibition of SsnB to VSMC proliferation, and there were significant differences(P<0.05),(3) Wound-healing assay and Transwell assay were used to measure the inhibition of SsnB to VSMC migration, and there were significant differences(P<0.05).(4) Quantitative real-time PCR confirmed that relative inflammatory molecules such as MPC1, IL-6and TNF-α were all decreased by SsnB flowing being elevated by LPS/PDGF stimulation, and there were significant differences (P<0.05).(5) It was observed that ERK1/2and Akt signaling pathways were both activated by LPS/PDGF stimulation, while both of them were significantly inhibited by SsnB(P<0.05).(6) Interestingly, we found that the elevated intracellular cholesterol level in VSMC was also significantly suppressed by SsnB after the cells took in acetylated LDL(ac-LDL), and there were significant differences(P<0.05).(7) Oil Red0staining was performed to confirm the attenuation of SsnB to the intracellular lipid droplets accumulation.(8) Meanwhile, Western blot of ATP-binding membrane cassette transporter A1(ABCA1) indicated that SsnB significantly promoted the intracellular cholesterol efflux, and there was significant difference(P<0.05); qRT-PCR showed that SsnB significantly decreased the CD36mRNA level after being significantly up-regulated by the acLDL plus LPS; while ABCA1mRNA level was significantly restored by SsnB after being significantly down-regulated by the acLDL plus LPS(P<0.05).ConclusionIn conclusion, our data indicate that the new selective TLR2and TLR4antagonist, SsnB,(1) can substantially attenuate hypoxia-reoxygenation induced inflammation of cardiomyocytes, inhibit TLR-mediated downstream inflammatory reactions, reduce the synthesis and release of inflammatory factors, inhibit the recruitment of macrophages and other inflammatory cells to the ischemic necrosis region via inhibiting ERK1/2and JNK signaling pathways.(2)SsnB can also substantially protect cultured rat LV tissue slices against hypoxia induced injury by inhibiting the inflammatory response of myocardial cells independent of inflammatory cell infiltration from the circulation, reducing the expression of inflammatory factors, and reducing hypoxia-induced necrosis and apoptosis of myocardial cells.(3) Meanwhile, SsnB can significantly inhibit the extracellular signal-activated kinase ERK1/2and Akt signaling pathway, reducing the atherosclerosis formation and progression by inhibiting vascular smooth muscle cell migration, proliferation, inflammation and lipid deposition.(4) Accordingly, SsnB has the potential to serve as a therapeutic agent for the prevention of myocardial ischemia-reperfusion injury and atherosclerosis, and these studies represent recent efforts to bridge the gap between the enigma of ancient Chinese herbal medicine and the concepts of modern cell and molecular biology in the treatment of ischemic cardiovascular diseases. |
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