| BackgroundThe incidence of coronary heart disease in our country is rising recently and are getting younger and younger. Acute myocardial infarction (AMI) is the most dangerous type of coronary heart disease. Every year, almost a hundred million of patients died of AMI. Therefore, the disease is considered the killer of human health.With maturity of Coronary artery bypass surgery, percutaneous coronary angioplasty and other vascular recanalization, the re-perfusion therapy of AMI is widely carried out. Timely restoration of coronary blood flow is a fundamental measure to save the acute ischemic myocardium, but the rapid restoration of coronary blood flow can lead to myocardial reperfusion injury. How to effectively mitigate such damage has been the biggest challenge in reperfusion therapy for maximum benefit in AMI treatment.Baicalin is extracted from the widely used traditional Chinese medicine Scutellaria baicalensis Georigi, one kind of flavonoid class of compounds. Baicalin is the major bio-active ingredients of Scutellaria baicalensis Georigi. It has multiple pharmacological role, such as antibacterial, sedative, anti-inflammatory, strong antioxidant properties. Thus, recently, it has been widely studied. Baicalin has been widely studied in variety of pathophysiological conditions. A recent study reported that baicalin pretreatment effectively reduce I-R injury of the liver and brain. In some vitro studies, baicalin and its aglycone baicalein was reported to have a role in protection of myocardial injury. Recently, an in vitro study reported:baicalin pretreatment can alleviate cultured neonatal rat cardiac myocyte apoptosis via anti-inflammatory mechanisms. Tu IH also reported that baicalin can reduce hypoxia-reoxygenation-induced myocardial apoptosis. However, the in vivo effects of baicalin in ischemic heart disease was unclear. A recent study by Peng and his colleagues confirmed:pretreatment via oral baicalin is effective in protection of myocardial ischemic injury through antioxidant mechanisms. However, the effects of baicalin on myocardial I-R injury in vivo and its related mechanisms have not been reported.It has been confirmed that excessive oxygen free radicals production and calcium overload are the main factors leading to myocardial I-R injury. Sustained myocardial ischemia and myocardial reperfusion after ischemia can lead to myocardial apoptosis, although reperfusion can reduce the number of apoptotic cardiomyocytes, but apoptosis of the irreversible myocardial cells accelerated. Under oxidative stress conditions, the outbreak of ROS during early reperfusion will cause a large number of mitochondrial permeability transition pore opening. Mitochondrial dysfunction can lead to apoptosis. We put forward the hypothesis:baicalin pretreatment can protect myocardial I-R injury by inhibiting mitochondrial damage-mediated apoptosis.Objectives:1. We intend to establish the model of myocardial I-R injury in mice to explore the effects of baicalin pretreatment on myocardial I-R injury in vivo;2. To explore whether baicalin can reduce myocardial I-R injury by inhibition of mitochondrial damage-mediated apoptosis. Methods1. In vivo studyWild-type mice underwent45min of left anterior descending coronary artery (LAD) occlusion, followed by2h of reperfusion. The mice were randomized into three groups:sham, vehicle+I/R, baicalin (100mg/kg)+I/R. Baicalin or saline was administered intravenously10min before LAD occlusion.2. In vitro studyTo simulate the process of myocardial I-R, we established the model of hypoxia/reoxygenation in neonatal rat cardiomyocytes. Briefly, neonatal rat cardiomyocytes were isolated and cultured:using trypsin/collagenase digestion.. The cells were randomly divided into3groups:control group (normoxic group), the vehicle+hypoxia/reoxygenation group and the Baicalin+hypoxia/reoxygenation group, three wells were set in each group, and the experiment was repeated three times. The myocardial cell beating and morphological changes were observed. The CCK8assay was used to detect myocardial cell viability; TUNEL staining was used to detect cardiomyocyte apoptosis,; LDH and SOD kits was used to detect the expression levels of LDH and SOD.Results:1. In vivo study(1) Baicalin pretreatment reduces infarct size after reperfusionThe ratio of the AAR to LV was almost the same in the vehicle and baicalin groups (Fig.2A, B), suggesting that the ischemic range was similar in these groups in the present study. The ratios of infarct size to the AAR and infarct size to LV were significantly decreased in the baicalin group (P<0.01vs. saline group)(Fig.2A, B). The average ratios of the infarct size to the AAR were46.75±1.94%and21.09± 2.08%in vehicle and baicalin groups, respectively (Fig.2B). In our data, we found that a higher dose of baicalin (200mg/kg) exerted similar effect on infarct size (Fig. SI A, C).(2) LV function can be preserved by baicalin treatmentFig.3A shows representative M-mode images of LV from sham-operated, saline-and baicalin-treated mice1week following I/R. There was no significant LV dilatation between all groups1week after I/R (Fig.3B), whereas baicalin pretreatment reduced LVESD (2.36±0.08mm) and preserved FS (37±1.09%) as compared to saline (LVESD:2.68±0.15mm and FS:28±2.93%, respectively; P<0.05). LVESD and FS at baseline (sham) were1.85±0.05mm and48±2.04%, respectively (Fig.3C, D).(3) Baicalin pretreatment inhibits I/R-induced apoptosis in the AARRepresentative photographs of TUNEL-positive cells in the vehicle-and baicalin-treated groups are shown in Fig.4A. There was a significantly lower apoptotic index in baicalin-pretreated mice (P<0.01vs. saline group, Fig.4B). As shown in Fig.4C, the level of cleaved caspase-3was decreased in baicalin-pretreated hearts (vs. saline-treated hearts). Western blot analysis indicated that caspase-3activity was inhibited by baicalin pretreatment (Fig.4D, P<0.05vs. saline group). A higher dose of baicalin (200mg/kg) exerted a similar effect on apoptosis (Fig. S2A, B). The ratio of anti-apoptotic protein Bcl-2vs. pro-apoptotic protein Bax (Bcl-2/Bax ratio) was also increased in baicalin-treated hearts (P<0.05vs. saline treated hearts; Fig. S3).(4) Baicalin attenuates mitochondrial damage during reperfusionTo determine the underlying mechanisms of the inhibition of apoptosis, the mitochondrial ultrastructure was examined by TEM. Fig.5A shows the change in mitochondrial morphology in the infarct border zone following I/R. The mitochondrial morphology was distinct in ischemic and non-ischemic regions. Myocardial mitochondria from the sham group were regular-shaped with electron-dense matrixes and unbroken cristae (Fig.5Ba). In contrast, mitochondria from the saline-treated group were swollen with electron-lucent matrixes and broken cristae, an effect of I/R injury (Fig.5Bb). However, the ultrastructure of the mitochondria was preserved in the baicalin-pretreated hearts (Fig.5Bc). Moreover, baicalin pretreatment reduced the extent of mitochondrial swelling (Fig.6A, B, P<0.01) and fission (Fig.6C, D, P<0.01). The number of mitochondrial matrix granules (Fig.7A, B, P<0.01) and the number of mitochondria-associated lipid droplets (Fig.7C, D, P<0.01) were also reduced by baicalin treatment. Mitochondrial swelling, fission, perimitochondrial lipids and matrix granules collectively suggest mitochondrial damage during I/R.(5) Baicalin pretreatment exerts anti-oxidant effects at reperfusionAs shown in Fig.8C, I/R resulted in excessive ROS generation in the saline-treated group (P<0.01vs. sham group). Of note, baicalin treatment markedly decreased the elevated level of ROS (P<0.01vs. saline group). As an enzymatic anti-oxidant defence, CAT and SOD can protect against the detrimental effects of ROS. Baicalin pretreatment significantly increased the activity of Mn-SOD and CAT (P<0.05vs. saline group, Fig.8A, B).(6) Baicalin pretreatment inhibits PKB beta2expression in the AARTo explore the potential mechanism of reduction in myocardial ROS by baicalin treatment, the expression of PKC beta2was measured. The level of PKC beta2was significantly increased in the hearts of saline-treated mice (P<0.01vs. sham-operated mice), but not in the hearts of baicalin-treated mice (P<0.01vs. saline-treated mice). The results of immunohistochemistry (Fig.9A) and western blot analysis (Fig.9B) were complimentary. Supplementary results:(S1) Baicalin pretreatment reduces infarct size.(A) Representative photographs of TTC-stained heart cross-sections.(B) Saline or baicalin-treated hearts have similar ratio of the AAR to LV.(C) The infarct size can be significantly limited by different doses of baicalin (Bai,50,100or200mg/kg). Bai (100mg/kg) had a better efficacy than Bai (50mg/kg) group in infarct-limiting effect. Bai (200mg/kg), however, has a similar efficacy in infarct-limiting efficacy as100mg/kg of Bai (n=6per group).(S2) Baicalin pretreatment reduces apoptosis and the level of plasma LDH after myocardial I/R.(A) Representative micrographs of left mid-ventricular sections with TUNEL staining and immunohistochemical staining of cleaved caspase-3.(B) The proportion of TUNEL-positive cells was significantly reduced by different doses of baicalin (n=6per group).(C) Baicalin pretreatment significantly decreased plasma LDH level2h after reperfusion (n=8per group).(S3) Baicalin pretreatment increases the Bcl-2/Bax ratio following2h reperfusion. Immunohistochemical analysis of Bax (A) and Bcl-2(C) expressions in the hearts from saline-or baicalin-pretreated mice (n=6per group). Western blot analysis indicates inhibition of Bax activity (B) and enhancement of Bcl-2activity (D) in baicalin-treated hearts (P<0.05vs. saline-treated hearts, n=3hearts per group).(E) The Bcl-2/Bax ratio was increased in baicalin group (P<0.05vs. vehicle group).(S4) Baicalin pretreatment inhibits cardiac remodeling.(A) Representative images of mid-ventricular sections stained with Masson trichrome to visualize collagen deposition (arrows) in saline-(a, c and e) or baicalin-pretreated (b, d and f) hearts3weeks after1/R.(B) Baicalin markedly decreased cardiac fibrosis in the myocardium (P<0.01vs. saline-treated group, n=7per group).(C) Baicalin reduced the heart/body weight ratio (P<0.01vs. saline-treated group, n=6per group).(D) Representative micrographs of left mid-ventricular sections stained with HE show attenuation of myocardial hypertrophy (arrows indicate myocyte cross surface area) by baicalin treatment.(E) Baicalin decreased myocyte cross surface area (P<0.01vs. saline-treated group, n=4per group).2. In vivo study(1)SD neonatal rat cardiomyocytes were successfully obtained using continuous trypsin/collagenase digestion:12h after the vaccination of cell suspension by Trypsin/collagenase multiple digestion, cell morphology changed to fusiform or irregular flat, after24hours the spontaneous contraction of individual cells was observed, then the cells gradually spreading to extend pseudopodia interwoven into a network, forming irregular star. The cells contact with each other, forming cell clusters adherent contraction in accordance with the same frequency.Assessment of the model of myocardial hypoxia/reoxygenation. In this part of the experiment, the One-Way ANOVA statistical analysis was used. Our resultsreveald that apoptosis rate in control group was0.05±0.005, apoptosis rate in hypoxia group was0.47±0.174, the apoptosis rate in hypoxia/reoxygenation group was0.72±0.292. Than rate of myocardial apoptosis in hypoxia group increased significantly (P<0.01vs. control). And after restoration of the oxygen supply, the rate of apoptosis didn’t fall but rise (P<0.01), suggesting the success of myocardial hypoxia/reoxygenation model. In addition, we assessed the effects of baicalin pretreatment on myocardial apoptosis rate during myocardial hypoxia/reoxygenation, and our results showed that apoptosis rate in baicalin group was0.62±0.160(P <0.01vs. hypoxia/reoxygenation group.(2) The effects of baicalin pretreatment on the viability of cells after myocardial hypoxia/reoxygenation. The viability (41.84±0.63) was significantly decreased after myocardial hypoxia (P<0.01vs. Control group). And after reoxygenation, myocardial viability (64.76±1.51) increased for (P<0.01vs. Hypoxia group). We further examined the effects of baicalin pretreatment on myocardial cell activity, and our results showed that cell activity in baicalin group was70.66±3.75, therefore, baicalin significantly increased myocardial cell activity (P<0.01vs. H/R group).(3)The effects of baicalin pretreatment on the level of SOD after myocardial hypoxia/reoxygenation. The level (105.10±2.24) was significantly decreased after hypoxia/reoxygenation (P<0.01vs. Control group). We further examined the effects of baicalin pretreatment on the level of SOD, and our results showed that the level of SOD in baicalin group was122.60±2.69, therefore, baicalin significantly increased the level of SOD (P<0.01vs. H/R group)(4) The effects of baicalin pretreatment on the level of LDH following myocardial hypoxia/reoxygenation. After hypoxia/reoxygenation, LDH level was significantly increased to26.8±0.36(P<0.01vs. Control group). We further tested the effects of baicalin pretreatment on LDH activity, and our results revealed that LDH activity in baicalin group was22.2±0.75, therefore, baicalin can significantly reduce the level of LDH following hypoxia/reoxygenation (P<0.01vs. H/R group).Conclusions:These results demonstrate that baicalin pretreatment can protect against myocardial I/R injury by inhibiting mitochondrial impairment-mediated apoptosis. Therefore, baicalin could be a promising agent for alleviating I/R injury in patients undergoing reperfusion therapy. |
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