人参二醇组皂苷对LPS导致的小鼠心肌损伤的保护作用
发布时间：2018-09-08 07:14
【摘要】：研究背景:脓毒症是一种由感染引起的全身性免疫应答综合征(SIRS),属于炎症反应失控导致全身多种器官发生功能障碍的一种疾病。脓毒症所引起的心肌功能障碍是严重的并发症之一,发生率达50%,这一并发症也是导致患者死亡或预后不良的起因。糖皮质激素治疗能扭转脓毒症心肌损伤的危急情况,然而,大剂量糖皮质激素突击疗法带来难以防治的并发症,如胃肠道应激出血,加重感染等。因此急需开发新型高效低毒的抗内毒素药物以替代糖皮质激素。人参是我国的传统中药,部分提取物具有优良的改善心功能作用,保护心肌细胞抵抗损伤和凋亡。人参二醇组皂苷(panaxadiols saponin,PDS)是一种脂溶性分子量小的二醇组提取物,其毒性较小,本课题组前期对PDS的系列研究发现,PDS可以改善失血性休克犬的心肺功能,还能改善脓毒症诱发的肾损伤,对脏器和细胞的功能及结构都有保护作用,深入研究其抗心肌损伤作用十分必要。研究目的:小鼠腹腔注射LPS模拟脓毒症时的心肌损伤模型,对比观察PDS和地塞米松对LPS诱导心肌损伤小鼠心功能的改善作用,探讨抗炎、抗凋亡、抗氧化应激等机制在PDS心脏保护机制中的作用,为其或可替代地塞米松类激素药物治疗LPS诱导的心肌损伤提供基础研究数据。方法与结果:1.建立LPS诱导的小鼠急性心肌损伤模型选择雄性纯系C57BL/6小鼠随机分为四组(n=8),分别为:对照组,LPS组,LPS+PDS组,LPS+DEX组。对照组小鼠腹腔注射0.5 ml PBS缓冲液,LPS+PDS组小鼠腹腔注射PDS(25 mg/kg),LPS+DEX组小鼠腹腔注射地塞米松(2.5 mg/kg);1小时后,LPS组、LPS+PDS组、LPS+DEX组小鼠腹腔注射LPS(10 mg/kg)。在LPS注射后7小时,麻醉小鼠进行超声心动图检测。在LPS注射后9小时处死小鼠,收集血液和心脏组织进行检测。超声心动图结果显示,与对照组小鼠相比,LPS组小鼠心脏的射血分数(ejection fraction,EF)、缩短分数(fractional shortening,FS)显著下降(P0.01),左室后壁变薄,左室腔增大,室间隔变薄(P0.01),同时LPS组小鼠的HE染色结果显示,心肌细胞周围出现大量炎性细胞浸润,肌细胞纤维变形,表明急性心肌损伤模型建立成功。2.PDS具有保护LPS诱导的急性心肌损伤小鼠心功能的作用与LPS模型组相比,LPS+PDS组小鼠腹腔注射PDS后,超声心动图检测显示其心功能的多项指标得到改善,EF和FS得到恢复,左心室形态也趋于正常,这一结果与LPS+DEX组小鼠结果相似。另一方面,检测小鼠心肌损伤标志物LDH和CK发现,LPS诱发的心肌炎性损伤使LDH和CK水平显著升高(P0.01),在经过PDS和DEX治疗后下降。以上结果说明PDS和DEX可以在一定程度上改善LPS引起的小鼠心肌损伤,达到治疗效果。3.PDS可降低LPS诱导的急性心肌损伤小鼠血清和心肌组织中TNF-α和IL-6水平增高试剂盒检测小鼠血清中TNF-α、IL-6的水平,结果显示LPS组小鼠与对照组相比TNF-α、IL-6水平明显升高(P0.05),表明LPS引起大量炎性因子入血;LPS+PDS组的TNF-α和IL-6的含量与LPS组相比,显著降低(P0.05);DEX组相较于LPS组,TNF-a有所下降,但没有统计学意义;IL-6的含量显著降低(P0.05)。检测小鼠心肌组织中TNF-α、IL-6的的m RNA水平,发现小鼠心脏组织中IL-6的m RNA水平组间没有差异;而TNF-α的m RNA水平在LPS组小鼠中显著增加(P0.05),PDS和DEX治疗后显著下降(P0.05)。提示PDS和DEX可能是通过抑制炎症反应来起到心肌保护作用。4.PDS可减少LPS诱导的急性心肌损伤小鼠心肌细胞凋亡使用Western Blot的方法检测各实验组凋亡相关蛋白在心脏中的表达情况。结果显示LPS组的小鼠与对照组小鼠相比凋亡蛋白Cleaved PARP,Cleaved caspase3,Bax的表达显著增加(P0.05),细胞色素C表现出增多的趋势;抗凋亡蛋白Bcl-2表达明显减少(P0.05)。而LPS+PDS组与LPS组相比凋亡蛋白Cleaved PARP,Cleaved caspase3,Bax,Cyto C的表达明显减少(P0.05);DEX治疗组凋亡蛋白Cleaved PARP,Cleaved caspase3也明显减少(P0.05),Bax,和Cyto C有减少趋势,由此可见,PDS和DEX可以影响凋亡相关蛋白的表达,减少LPS引起的心脏细胞凋亡,起到保护心肌组织的作用。5.PDS可抑制LPS诱导的急性心肌损伤小鼠心脏NF-κB信号通路激活Western Blot结果显示LPS组小鼠心脏组织中磷酸化IκBα(p-IκBα)水平显著升高(P0.05),p-IκBα/t-IκBα比值也明显增加(P0.01)。当给予PDS和DEX作用后,可以显著降低p-IκBα/t-IκBα比值(P0.01)。提取心肌细胞核蛋白,检测各组p50和p65的表达水平,发现,与对照组小鼠相比,LPS组小鼠心脏细胞核中NF-κBp50和p65蛋白表达水平显著升高(P0.05),当给予了PDS和DEX治疗后,NF-κB p50和p65蛋白表达水平都有了明显的下降(P0.05)。可见,NF-κB信号通路的活化被PDS和DEX抑制,LPS诱导的p50和p65蛋白入核减少,逆转了LPS诱导的小鼠心肌损伤。6.PDS可减轻LPS诱导的急性心肌损伤小鼠心肌氧化应激水平检测了四组小鼠心肌组织中NAD+/NADH的变化,发现在LPS组小鼠中这一比值明显高于对照组(P0.01),提示在LPS引起的小鼠急性心肌损伤中,NAD+/NADH体内稳态破坏,细胞氧化损伤加重,心肌组织中MDA水平也明显增加(P0.05),SOD的表达明显减少(P0.05)。而经过PDS和DEX治疗后,NAD+/NADH比值出现明显的下调(P0.05),MDA水平也出现下调,SOD表达恢复(P0.05),说明减少细胞氧化损伤可能也是PDS抗LPS心肌损伤的机制之一。结论:1.PDS具有与地塞米松类似的改善LPS诱导的急性心肌损伤小鼠心脏的收缩功能,抑制炎性因子产生,减少心肌细胞的凋亡和氧化应激损伤的作用。2.PDS改善LPS诱导的急性心肌损伤的作用机制可能与抑制NF-κB信号通路的活化有关。
[Abstract]:BACKGROUND: Sepsis is a systemic immune response syndrome (SIRS) caused by infection, which is a disease of multiple organ dysfunction caused by uncontrolled inflammation. Myocardial dysfunction caused by sepsis is one of the serious complications with a incidence of 50%. This complication also leads to death or prognosis of patients. Glucocorticoid therapy can reverse the critical condition of myocardial injury in sepsis. However, high dose of glucocorticoid shock therapy brings difficult prevention and treatment complications, such as gastrointestinal stress bleeding, aggravating infection and so on. Therefore, it is urgent to develop new high-efficiency and low-toxicity anti-endotoxin drugs to replace glucocorticoid. Panaxadiols saponin (PDS) is a kind of glycol extract with low fat-soluble molecular weight, and its toxicity is small. Our previous study on PDS showed that PDS can improve hemorrhagic shock dogs. Cardiopulmonary function can also improve the kidney injury induced by sepsis. It has protective effect on the function and structure of organs and cells. It is necessary to study the anti-myocardial injury effect of PDS and dexamethasone. To explore the role of anti-inflammatory, anti-apoptotic and anti-oxidative stress mechanisms in cardioprotective mechanism of PDS, and to provide basic research data for its or alternative dexamethasone hormones in the treatment of LPS-induced myocardial injury. Mice were randomly divided into four groups: control group, LPS group, LPS + PDS group, LPS + DEX group. Control group mice were injected with 0.5 ml PBS buffer, LPS + PDS group mice were injected with PDS (25 mg/kg), LPS + DEX group mice were injected with dexamethasone (2.5 mg/kg); one hour later, LPS group, LPS + PDS group, LPS + DEX group mice were injected with LPS (10 mg/kg). The anesthetized mice were sacrificed 9 hours after LPS injection and the blood and heart tissues were collected for examination. Left ventricular posterior wall became thinner, left ventricular cavity enlarged and interventricular septum thinned (P 0.01). Meanwhile, the results of HE staining in LPS group showed that a large number of inflammatory cells infiltrated around myocardial cells and myocyte fibers deformed, indicating that the establishment of acute myocardial injury model was successful. 2. PDS has protective effect on heart function in LPS-induced acute myocardial injury mice and LPS model. Compared with the LPS + PDS group, echocardiographic examination showed that many indexes of cardiac function were improved, EF and FS were restored, and left ventricular morphology was normal in LPS + PDS group. This result was similar to that in LPS + DEX group. On the other hand, detection of LDH and CK markers of myocardial injury in mice showed that LPS induced myocarditis injury. These results suggest that PDS and DEX can improve LPS-induced myocardial injury in mice to a certain extent and achieve therapeutic effect. 3. PDS can reduce LPS-induced acute myocardial injury in mice serum and myocardial tissue TNF-a and IL-6 levels increased kit detection mice. The levels of TNF-a and IL-6 in serum were significantly higher in LPS group than those in control group (P 0.05), indicating that LPS caused a large number of inflammatory factors to enter the blood; the levels of TNF-a and IL-6 in LPS + PDS group were significantly lower than those in LPS group (P 0.05); the levels of TNF-a in DEX group were lower than those in LPS group, but there was no statistical significance. The levels of TNF-alpha and IL-6 in myocardium of mice were detected, and there was no significant difference between the two groups. However, the levels of TNF-alpha m RNA increased significantly in LPS group (P 0.05), and decreased significantly after PDS and DEX treatment (P 0.05). Protective effect of PDS on myocardium. 4. PDS can reduce the apoptosis of cardiomyocytes induced by LPS in mice with acute myocardial injury. Western Blot method was used to detect the expression of apoptosis-related proteins in the heart of experimental groups. Compared with LPS group, the expression of apoptotic proteins Cleaved PARP, Cleaved caspase 3, Bax and C YTO C decreased significantly in LPS + PDS group (P 0.05). The expression of apoptotic proteins Cleaved PARP, Cleaved caspase 3, Bax and C YTO C also decreased significantly in DEX treatment group (P 0.05). Thus, PDS and DEX can affect the expression of apoptosis-related proteins, reduce LPS-induced cardiac apoptosis, and play a protective role in myocardial tissue. 5. PDS can inhibit LPS-induced acute myocardial injury in mice heart NF-kappa B signal pathway activation Western Blot results show that LPS-induced mice heart tissue phosphorylated I-kappa B alpha (p-I-kappa B alpha) level is significant. The ratio of p-I kappa B alpha to t-I kappa B alpha was significantly increased (P 0.05), and the ratio of p-I kappa B alpha to t-I kappa B alpha was significantly decreased (P 0.01) after treatment with PDS and DEX. The expression levels of NF-kappa B P50 and p65 protein decreased significantly after treatment with PDS and DEX (P 0.05). The activation of NF-kappa B signaling pathway was inhibited by PDS and DEX. LPS-induced decrease of P50 and p65 protein entry into the nucleus reversed LPS-induced acute myocardial injury in mice. The changes of NAD+/NADH in myocardial tissue of mice in four groups were detected by oxidative stress. The results showed that the ratio of NAD+/NADH in LPS group was significantly higher than that in control group (P After PDS and DEX treatment, the ratio of NAD+/NADH decreased significantly (P 0.05), the level of MDA decreased, and the expression of SOD recovered (P 0.05), indicating that reducing cell oxidative damage may also be one of the mechanisms of PDS against LPS-induced myocardial injury. Conclusion: 1. PDS can improve LPS-induced acute myocardial injury similar to dexamethasone. The mechanism of PDS improving LPS-induced acute myocardial injury may be related to the inhibition of activation of NF-kappa B signaling pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
本文编号：2229769
[Abstract]:BACKGROUND: Sepsis is a systemic immune response syndrome (SIRS) caused by infection, which is a disease of multiple organ dysfunction caused by uncontrolled inflammation. Myocardial dysfunction caused by sepsis is one of the serious complications with a incidence of 50%. This complication also leads to death or prognosis of patients. Glucocorticoid therapy can reverse the critical condition of myocardial injury in sepsis. However, high dose of glucocorticoid shock therapy brings difficult prevention and treatment complications, such as gastrointestinal stress bleeding, aggravating infection and so on. Therefore, it is urgent to develop new high-efficiency and low-toxicity anti-endotoxin drugs to replace glucocorticoid. Panaxadiols saponin (PDS) is a kind of glycol extract with low fat-soluble molecular weight, and its toxicity is small. Our previous study on PDS showed that PDS can improve hemorrhagic shock dogs. Cardiopulmonary function can also improve the kidney injury induced by sepsis. It has protective effect on the function and structure of organs and cells. It is necessary to study the anti-myocardial injury effect of PDS and dexamethasone. To explore the role of anti-inflammatory, anti-apoptotic and anti-oxidative stress mechanisms in cardioprotective mechanism of PDS, and to provide basic research data for its or alternative dexamethasone hormones in the treatment of LPS-induced myocardial injury. Mice were randomly divided into four groups: control group, LPS group, LPS + PDS group, LPS + DEX group. Control group mice were injected with 0.5 ml PBS buffer, LPS + PDS group mice were injected with PDS (25 mg/kg), LPS + DEX group mice were injected with dexamethasone (2.5 mg/kg); one hour later, LPS group, LPS + PDS group, LPS + DEX group mice were injected with LPS (10 mg/kg). The anesthetized mice were sacrificed 9 hours after LPS injection and the blood and heart tissues were collected for examination. Left ventricular posterior wall became thinner, left ventricular cavity enlarged and interventricular septum thinned (P 0.01). Meanwhile, the results of HE staining in LPS group showed that a large number of inflammatory cells infiltrated around myocardial cells and myocyte fibers deformed, indicating that the establishment of acute myocardial injury model was successful. 2. PDS has protective effect on heart function in LPS-induced acute myocardial injury mice and LPS model. Compared with the LPS + PDS group, echocardiographic examination showed that many indexes of cardiac function were improved, EF and FS were restored, and left ventricular morphology was normal in LPS + PDS group. This result was similar to that in LPS + DEX group. On the other hand, detection of LDH and CK markers of myocardial injury in mice showed that LPS induced myocarditis injury. These results suggest that PDS and DEX can improve LPS-induced myocardial injury in mice to a certain extent and achieve therapeutic effect. 3. PDS can reduce LPS-induced acute myocardial injury in mice serum and myocardial tissue TNF-a and IL-6 levels increased kit detection mice. The levels of TNF-a and IL-6 in serum were significantly higher in LPS group than those in control group (P 0.05), indicating that LPS caused a large number of inflammatory factors to enter the blood; the levels of TNF-a and IL-6 in LPS + PDS group were significantly lower than those in LPS group (P 0.05); the levels of TNF-a in DEX group were lower than those in LPS group, but there was no statistical significance. The levels of TNF-alpha and IL-6 in myocardium of mice were detected, and there was no significant difference between the two groups. However, the levels of TNF-alpha m RNA increased significantly in LPS group (P 0.05), and decreased significantly after PDS and DEX treatment (P 0.05). Protective effect of PDS on myocardium. 4. PDS can reduce the apoptosis of cardiomyocytes induced by LPS in mice with acute myocardial injury. Western Blot method was used to detect the expression of apoptosis-related proteins in the heart of experimental groups. Compared with LPS group, the expression of apoptotic proteins Cleaved PARP, Cleaved caspase 3, Bax and C YTO C decreased significantly in LPS + PDS group (P 0.05). The expression of apoptotic proteins Cleaved PARP, Cleaved caspase 3, Bax and C YTO C also decreased significantly in DEX treatment group (P 0.05). Thus, PDS and DEX can affect the expression of apoptosis-related proteins, reduce LPS-induced cardiac apoptosis, and play a protective role in myocardial tissue. 5. PDS can inhibit LPS-induced acute myocardial injury in mice heart NF-kappa B signal pathway activation Western Blot results show that LPS-induced mice heart tissue phosphorylated I-kappa B alpha (p-I-kappa B alpha) level is significant. The ratio of p-I kappa B alpha to t-I kappa B alpha was significantly increased (P 0.05), and the ratio of p-I kappa B alpha to t-I kappa B alpha was significantly decreased (P 0.01) after treatment with PDS and DEX. The expression levels of NF-kappa B P50 and p65 protein decreased significantly after treatment with PDS and DEX (P 0.05). The activation of NF-kappa B signaling pathway was inhibited by PDS and DEX. LPS-induced decrease of P50 and p65 protein entry into the nucleus reversed LPS-induced acute myocardial injury in mice. The changes of NAD+/NADH in myocardial tissue of mice in four groups were detected by oxidative stress. The results showed that the ratio of NAD+/NADH in LPS group was significantly higher than that in control group (P After PDS and DEX treatment, the ratio of NAD+/NADH decreased significantly (P 0.05), the level of MDA decreased, and the expression of SOD recovered (P 0.05), indicating that reducing cell oxidative damage may also be one of the mechanisms of PDS against LPS-induced myocardial injury. Conclusion: 1. PDS can improve LPS-induced acute myocardial injury similar to dexamethasone. The mechanism of PDS improving LPS-induced acute myocardial injury may be related to the inhibition of activation of NF-kappa B signaling pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
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