【摘要】：研究背景:膿毒癥是一種由感染引起的全身性免疫應(yīng)答綜合征(SIRS),屬于炎癥反應(yīng)失控導(dǎo)致全身多種器官發(fā)生功能障礙的一種疾病。膿毒癥所引起的心肌功能障礙是嚴(yán)重的并發(fā)癥之一,發(fā)生率達(dá)50%,這一并發(fā)癥也是導(dǎo)致患者死亡或預(yù)后不良的起因。糖皮質(zhì)激素治療能扭轉(zhuǎn)膿毒癥心肌損傷的危急情況,然而,大劑量糖皮質(zhì)激素突擊療法帶來難以防治的并發(fā)癥,如胃腸道應(yīng)激出血,加重感染等。因此急需開發(fā)新型高效低毒的抗內(nèi)毒素藥物以替代糖皮質(zhì)激素。人參是我國的傳統(tǒng)中藥,部分提取物具有優(yōu)良的改善心功能作用,保護(hù)心肌細(xì)胞抵抗損傷和凋亡。人參二醇組皂苷(panaxadiols saponin,PDS)是一種脂溶性分子量小的二醇組提取物,其毒性較小,本課題組前期對PDS的系列研究發(fā)現(xiàn),PDS可以改善失血性休克犬的心肺功能,還能改善膿毒癥誘發(fā)的腎損傷,對臟器和細(xì)胞的功能及結(jié)構(gòu)都有保護(hù)作用,深入研究其抗心肌損傷作用十分必要。研究目的:小鼠腹腔注射LPS模擬膿毒癥時的心肌損傷模型,對比觀察PDS和地塞米松對LPS誘導(dǎo)心肌損傷小鼠心功能的改善作用,探討抗炎、抗凋亡、抗氧化應(yīng)激等機(jī)制在PDS心臟保護(hù)機(jī)制中的作用,為其或可替代地塞米松類激素藥物治療LPS誘導(dǎo)的心肌損傷提供基礎(chǔ)研究數(shù)據(jù)。方法與結(jié)果:1.建立LPS誘導(dǎo)的小鼠急性心肌損傷模型選擇雄性純系C57BL/6小鼠隨機(jī)分為四組(n=8),分別為:對照組,LPS組,LPS+PDS組,LPS+DEX組。對照組小鼠腹腔注射0.5 ml PBS緩沖液,LPS+PDS組小鼠腹腔注射PDS(25 mg/kg),LPS+DEX組小鼠腹腔注射地塞米松(2.5 mg/kg);1小時后,LPS組、LPS+PDS組、LPS+DEX組小鼠腹腔注射LPS(10 mg/kg)。在LPS注射后7小時,麻醉小鼠進(jìn)行超聲心動圖檢測。在LPS注射后9小時處死小鼠,收集血液和心臟組織進(jìn)行檢測。超聲心動圖結(jié)果顯示,與對照組小鼠相比,LPS組小鼠心臟的射血分?jǐn)?shù)(ejection fraction,EF)、縮短分?jǐn)?shù)(fractional shortening,FS)顯著下降(P0.01),左室后壁變薄,左室腔增大,室間隔變薄(P0.01),同時LPS組小鼠的HE染色結(jié)果顯示,心肌細(xì)胞周圍出現(xiàn)大量炎性細(xì)胞浸潤,肌細(xì)胞纖維變形,表明急性心肌損傷模型建立成功。2.PDS具有保護(hù)LPS誘導(dǎo)的急性心肌損傷小鼠心功能的作用與LPS模型組相比,LPS+PDS組小鼠腹腔注射PDS后,超聲心動圖檢測顯示其心功能的多項指標(biāo)得到改善,EF和FS得到恢復(fù),左心室形態(tài)也趨于正常,這一結(jié)果與LPS+DEX組小鼠結(jié)果相似。另一方面,檢測小鼠心肌損傷標(biāo)志物L(fēng)DH和CK發(fā)現(xiàn),LPS誘發(fā)的心肌炎性損傷使LDH和CK水平顯著升高(P0.01),在經(jīng)過PDS和DEX治療后下降。以上結(jié)果說明PDS和DEX可以在一定程度上改善LPS引起的小鼠心肌損傷,達(dá)到治療效果。3.PDS可降低LPS誘導(dǎo)的急性心肌損傷小鼠血清和心肌組織中TNF-α和IL-6水平增高試劑盒檢測小鼠血清中TNF-α、IL-6的水平,結(jié)果顯示LPS組小鼠與對照組相比TNF-α、IL-6水平明顯升高(P0.05),表明LPS引起大量炎性因子入血;LPS+PDS組的TNF-α和IL-6的含量與LPS組相比,顯著降低(P0.05);DEX組相較于LPS組,TNF-a有所下降,但沒有統(tǒng)計學(xué)意義;IL-6的含量顯著降低(P0.05)。檢測小鼠心肌組織中TNF-α、IL-6的的m RNA水平,發(fā)現(xiàn)小鼠心臟組織中IL-6的m RNA水平組間沒有差異;而TNF-α的m RNA水平在LPS組小鼠中顯著增加(P0.05),PDS和DEX治療后顯著下降(P0.05)。提示PDS和DEX可能是通過抑制炎癥反應(yīng)來起到心肌保護(hù)作用。4.PDS可減少LPS誘導(dǎo)的急性心肌損傷小鼠心肌細(xì)胞凋亡使用Western Blot的方法檢測各實(shí)驗(yàn)組凋亡相關(guān)蛋白在心臟中的表達(dá)情況。結(jié)果顯示LPS組的小鼠與對照組小鼠相比凋亡蛋白Cleaved PARP,Cleaved caspase3,Bax的表達(dá)顯著增加(P0.05),細(xì)胞色素C表現(xiàn)出增多的趨勢;抗凋亡蛋白Bcl-2表達(dá)明顯減少(P0.05)。而LPS+PDS組與LPS組相比凋亡蛋白Cleaved PARP,Cleaved caspase3,Bax,Cyto C的表達(dá)明顯減少(P0.05);DEX治療組凋亡蛋白Cleaved PARP,Cleaved caspase3也明顯減少(P0.05),Bax,和Cyto C有減少趨勢,由此可見,PDS和DEX可以影響凋亡相關(guān)蛋白的表達(dá),減少LPS引起的心臟細(xì)胞凋亡,起到保護(hù)心肌組織的作用。5.PDS可抑制LPS誘導(dǎo)的急性心肌損傷小鼠心臟NF-κB信號通路激活Western Blot結(jié)果顯示LPS組小鼠心臟組織中磷酸化IκBα(p-IκBα)水平顯著升高(P0.05),p-IκBα/t-IκBα比值也明顯增加(P0.01)。當(dāng)給予PDS和DEX作用后,可以顯著降低p-IκBα/t-IκBα比值(P0.01)。提取心肌細(xì)胞核蛋白,檢測各組p50和p65的表達(dá)水平,發(fā)現(xiàn),與對照組小鼠相比,LPS組小鼠心臟細(xì)胞核中NF-κBp50和p65蛋白表達(dá)水平顯著升高(P0.05),當(dāng)給予了PDS和DEX治療后,NF-κB p50和p65蛋白表達(dá)水平都有了明顯的下降(P0.05)�？梢�,NF-κB信號通路的活化被PDS和DEX抑制,LPS誘導(dǎo)的p50和p65蛋白入核減少,逆轉(zhuǎn)了LPS誘導(dǎo)的小鼠心肌損傷。6.PDS可減輕LPS誘導(dǎo)的急性心肌損傷小鼠心肌氧化應(yīng)激水平檢測了四組小鼠心肌組織中NAD+/NADH的變化,發(fā)現(xiàn)在LPS組小鼠中這一比值明顯高于對照組(P0.01),提示在LPS引起的小鼠急性心肌損傷中,NAD+/NADH體內(nèi)穩(wěn)態(tài)破壞,細(xì)胞氧化損傷加重,心肌組織中MDA水平也明顯增加(P0.05),SOD的表達(dá)明顯減少(P0.05)。而經(jīng)過PDS和DEX治療后,NAD+/NADH比值出現(xiàn)明顯的下調(diào)(P0.05),MDA水平也出現(xiàn)下調(diào),SOD表達(dá)恢復(fù)(P0.05),說明減少細(xì)胞氧化損傷可能也是PDS抗LPS心肌損傷的機(jī)制之一。結(jié)論:1.PDS具有與地塞米松類似的改善LPS誘導(dǎo)的急性心肌損傷小鼠心臟的收縮功能,抑制炎性因子產(chǎn)生,減少心肌細(xì)胞的凋亡和氧化應(yīng)激損傷的作用。2.PDS改善LPS誘導(dǎo)的急性心肌損傷的作用機(jī)制可能與抑制NF-κB信號通路的活化有關(guān)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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