二十二碳六烯酸抑制炎癥及內(nèi)質(zhì)網(wǎng)應(yīng)激改善肺血管重塑
發(fā)布時(shí)間：2018-08-23 13:38
【摘要】：目的:肺高壓(PH)是肺血管阻力持續(xù)升高,右心室負(fù)荷不斷增加,進(jìn)而導(dǎo)致進(jìn)行性右心衰竭甚至死亡的常見(jiàn)嚴(yán)重并發(fā)癥。近年來(lái)研究認(rèn)為肺血管炎癥及血管細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)在PH的發(fā)生發(fā)展中起重要作用。二十二碳六烯酸(DHA)是n-3多不飽和脂肪酸(n-3PUFA)的主要成分,具有調(diào)節(jié)內(nèi)質(zhì)網(wǎng)應(yīng)激及抗炎等效應(yīng)。我們前期研究發(fā)現(xiàn)DHA能改善低氧性肺動(dòng)脈高壓。本研究擬采用野百合堿(MCT)誘導(dǎo)的大鼠肺高壓模型,進(jìn)一步探討DHA的防治肺高壓作用及其機(jī)制。方法:1.實(shí)驗(yàn)動(dòng)物分組:SD (200-250 g)大鼠40只,每組10只,隨機(jī)分為4組:對(duì)照組(生理鹽水注射),MCT造模組(腹腔單次注射MCT,4周),MCT造模組+DHA灌胃預(yù)防組(腹腔注射MCT當(dāng)天即給予DHA灌胃,持續(xù)4周),MCT造模+DHA治療組(腹腔注射MCT兩周后給予DHA每天灌胃,持續(xù)2周)。(1)右心導(dǎo)管法檢測(cè)各組大鼠肺動(dòng)脈平均壓;沿室間隔分離右心室(RV),左室+室間隔(LV+S)分別稱取質(zhì)量,計(jì)算右心室肥厚指數(shù):RV/ (LV+S) ; (2)實(shí)時(shí)定量PCR檢測(cè)肺組織細(xì)胞因子IL6,TNF-α,MCP-1及IL1β基因表達(dá);(3)免疫組化法檢測(cè)①肺動(dòng)脈中膜厚度②肺組織及阻力性肺動(dòng)脈周圍CD3+T細(xì)胞及CD68+巨噬細(xì)胞計(jì)數(shù);(4)免疫印跡法檢測(cè)肺組織內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)水平;(5)免疫熒光雙染檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白在肺組織中膜的表達(dá)差異。2.組織塊貼壁法培養(yǎng)大鼠肺動(dòng)脈平滑肌細(xì)胞(PASMC)并進(jìn)行免疫熒光細(xì)胞鑒定。(1)免疫印跡及細(xì)胞免疫熒光檢測(cè)PASMC內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白表達(dá)水平;(2)實(shí)時(shí)定量PCR檢測(cè)PASMC的NFATcl-4基因表達(dá)水平;(3)免疫印跡檢測(cè)NFATcl的總蛋白水平及核轉(zhuǎn)移情況;(4)CCK8及BrdU攝入檢測(cè)PASMC增殖;(5)流式細(xì)胞術(shù)檢測(cè)PASMC細(xì)胞周期;(6)免疫印跡檢測(cè)細(xì)胞周期相關(guān)蛋白表達(dá)水平。結(jié)果:(1)與對(duì)照組大鼠相比,MCT組大鼠4周后平均肺動(dòng)脈壓顯著升高(MCT: 48.2±3.1 mmHg vs control: 20.5±1.3 mmHg;p0.05) ; DHA預(yù)防及治療可顯著降低MCT誘導(dǎo)的PH大鼠平均肺動(dòng)脈壓(MCT+DHA預(yù)防組:25.3±2.1mmHg; MCT+DHA 治療組:31.5±3.4mmHg;p0.05 vs MCT組)及右心指數(shù)(MCT+DHA預(yù)防組:0.37±0.02;]MCT+DHA治療組:0.42±0.01; p0.05vsMCT組);并顯著抑制MCT誘導(dǎo)的大鼠肺動(dòng)脈中膜增厚。(2) DHA預(yù)防及治療可顯著減少M(fèi)CT誘導(dǎo)的大鼠肺組織IL1β,TNFα,IL-6和MCP-1基因表達(dá)(p值均0.05);并抑制MCT誘導(dǎo)的大鼠肺組織及肺血管周圍CD3+T細(xì)胞及CD68+巨噬細(xì)胞的聚集。(3) DHA預(yù)防及治療可顯著減少M(fèi)CT誘導(dǎo)的大鼠肺組織內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白p-IRE1,PDI和GRP78蛋白表達(dá)(p值均0.05);免疫熒光雙染表明DHA主要減少肺動(dòng)脈平滑肌層PDI的表達(dá)。(4)在培養(yǎng)的PASMC,PDGF-BB刺激能顯著增加內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白p-IRE1,PDI和GRP78蛋白表達(dá)(p值均0.05)而DHA預(yù)孵能抑制PDGF-BB誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白的表達(dá);免疫熒光表明PDGF-BB刺激PASMC后PDI熒光信號(hào)增強(qiáng),而預(yù)孵DHA能減弱熒光信號(hào)。(5)PDGF-BB 刺激能誘導(dǎo) PASMC 的 NFATc1,NFATc2 及 NFATc3基因表達(dá)增加(p0.05 vs對(duì)照組)而對(duì)NFATc4無(wú)顯著影響;預(yù)孵DHA能逆轉(zhuǎn)PDGF-BB誘導(dǎo)的NFATc1及NFATc2的基因表達(dá),而對(duì)NFATc3無(wú)顯著影響。(6) PDGF-BB刺激能誘導(dǎo)PASMC的NFATc1總蛋白表達(dá)增加,蛋白核轉(zhuǎn)移增多,而預(yù)孵DHA及內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-PBA能產(chǎn)生類似的抑制PDGF-BB誘導(dǎo)性NFATc1總蛋白表達(dá)增加及活性增強(qiáng)。(7) PDGF-BB刺激PASMC能顯著促進(jìn)細(xì)胞周期蛋白cyclin D1表達(dá),抑制G1期相關(guān)負(fù)性蛋白p21和p27表達(dá),處于細(xì)胞周期S期百分比增多,細(xì)胞增殖增加;而DHA預(yù)孵或NFATc1 siRNA干擾能產(chǎn)生類似的抑制PDGF-BB誘導(dǎo)的cyclinD1表達(dá)增加,恢復(fù)p21和p27的蛋白水平,將細(xì)胞阻滯于G1期,抑制PDGF-BB誘導(dǎo)的PASMC的增殖。結(jié)論:(1) DHA抑制MCT誘導(dǎo)的PH的肺組織及肺血管周圍炎癥反應(yīng)。(2) DHA抑制PDGF-BB誘導(dǎo)的PASMC內(nèi)質(zhì)網(wǎng)應(yīng)激,減少PASMC增殖,改善肺血管重塑,降低MCT誘導(dǎo)的大鼠PH平均肺動(dòng)脈壓。
[Abstract]:AIM: Pulmonary hypertension (PH) is a common and serious complication of progressive right heart failure (RHF) and death caused by increased pulmonary vascular resistance (PVR) and right ventricular load. Recent studies have shown that pulmonary vascular inflammation and vascular endoplasmic reticulum stress play an important role in the development of PH. Saturated fatty acids (n-3PUFA) are the main components of endoplasmic reticulum (ER) stress and anti-inflammatory effects. Our previous study found that DHA can improve hypoxic pulmonary hypertension. In this study, monocrotaline (MCT)-induced pulmonary hypertension in rats was used to further explore the preventive and therapeutic effects of DHA on pulmonary hypertension and its mechanism. (200-250 g) rats were randomly divided into four groups: control group (saline injection), MCT modeling group (single injection of MCT, 4 weeks), MCT modeling group + DHA gastric lavage prevention group (intraperitoneal injection of MCT on the day of DHA gastric lavage for 4 weeks, continuous 4 weeks), MCT modeling + DHA treatment group (intraperitoneal injection of MCT for two weeks after DHA daily gastric lavage for 2 weeks). The mean pulmonary artery pressure (PAP) was measured by catheterization, the right ventricle (RV) was separated along the interventricular septum, and the left ventricular + septum (LV + S) was weighed and the right ventricular hypertrophy index (RV / LV + S) was calculated. CD3 + T cells and CD68 + macrophages were counted around tissues and pulmonary artery resistance; (4) The expression of ER stress related protein in lung tissues was detected by Western blotting; (5) The expression of ER stress related protein in lung tissues was detected by immunofluorescence double staining. 2. PASMC was cultured by tissue block adherence method and progressed. Immunofluorescent cell identification was performed. (1) Immunoblotting and cytoimmunofluorescence were used to detect the expression of ER stress protein in PASMC; (2) Real-time quantitative PCR was used to detect the expression of NFATcl-4 gene in PASMC; (3) Immunoblotting was used to detect the total protein level and nuclear transfer of NFATcl; (4) CCK8 and BrdU uptake was used to detect the proliferation of PASMC; (5) Flow cytometry was used to detect the expression of PASMC. Results: (1) Compared with the control group, the mean pulmonary artery pressure in MCT group increased significantly after 4 weeks (MCT: 48.2 (+ 3.1) mmHg vs control: 20.5 (+ 1.3) mmHg; p0.05); DHA prevention and treatment could significantly reduce the mean pulmonary artery pressure (MCT + DHA) in MCT-induced PH rats. Group B: 25.3 (+ 2.1 mmHg); MCT + DHA group: 31.5 (+ 3.4 mmHg); P0.05 vs MCT group) and right heart index (MCT + DHA prevention group: 0.37 (+ 0.02); MCT + DHA treatment group: 0.42 (+ 0.01); P0.05 vs MCT group); and significantly inhibited MCT-induced pulmonary artery media thickening in rats. (2) DHA prevention and treatment can significantly reduce MCT-induced pulmonary tissue IL-1 beta, IL-6 and IL-1 and MMC-1. Gene expression (p value was 0.05), and inhibited the accumulation of CD3 + T cells and CD68 + macrophages in lung tissue and perivascular tissues of rats induced by MCT. (3) DHA could significantly reduce the expression of stress-related proteins p-IRE1, PDI and GRP78 (p value was 0.05). Immunofluorescence double staining showed that DHA mainly reduced the expression of lung stress-related proteins p-IRE1, PDI and GRP78 in lung tissue of rats induced by MCT. PDI expression in arterial smooth muscle layer. (4) PDGF-BB stimulation significantly increased the expression of ER stress-related proteins p-IRE1, PDI and GRP78 in cultured PASMC (p 0.05), while DHA pre-incubation inhibited the expression of ER stress-related proteins induced by PDGF-BB. Immunofluorescence showed that PDI fluorescence signal was enhanced after PDGF-BB stimulation of PASMC, while DHA pre-incubation could inhibit the expression of ER stress-related proteins induced by PDGF-BB. (5) PDGF-BB stimulation could induce the expression of NFATc1, NFATc2 and NFATc3 genes in PASMC, but had no significant effect on the expression of NFATc4. Preincubation DHA could reverse the expression of NFATc1 and NFATc2 genes induced by PDGF-BB, but had no significant effect on the expression of NFATc1 protein in PASMC. (6) PDGF-BB stimulation could induce the expression of NFATc1 protein in PASMC. Plus, nuclear transfer increased, while pre-incubation DHA and ER stress inhibitor 4-PBA produced similar inhibition of PDGF-BB-induced total NFATc1 protein expression and activity increase. (7) PDGF-BB stimulated PASMC could significantly promote cyclin D1 expression, inhibit G1-related negative protein p21 and p27 expression, in the S-phase percentage of cell cycle. DHA pre-incubation or NFATc1 siRNA interference could similarly inhibit the increase of cyclin D1 expression induced by PDGF-BB, restore the protein levels of p21 and p27, block the cells in G1 phase and inhibit the proliferation of PASMC induced by PDGF-BB. Inhibiting PDGF-BB-induced endoplasmic reticulum stress, reducing PASMC proliferation, improving pulmonary vascular remodeling, and reducing MCT-induced PH mean pulmonary artery pressure in rats.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R544.1
本文編號(hào)：2199235
[Abstract]:AIM: Pulmonary hypertension (PH) is a common and serious complication of progressive right heart failure (RHF) and death caused by increased pulmonary vascular resistance (PVR) and right ventricular load. Recent studies have shown that pulmonary vascular inflammation and vascular endoplasmic reticulum stress play an important role in the development of PH. Saturated fatty acids (n-3PUFA) are the main components of endoplasmic reticulum (ER) stress and anti-inflammatory effects. Our previous study found that DHA can improve hypoxic pulmonary hypertension. In this study, monocrotaline (MCT)-induced pulmonary hypertension in rats was used to further explore the preventive and therapeutic effects of DHA on pulmonary hypertension and its mechanism. (200-250 g) rats were randomly divided into four groups: control group (saline injection), MCT modeling group (single injection of MCT, 4 weeks), MCT modeling group + DHA gastric lavage prevention group (intraperitoneal injection of MCT on the day of DHA gastric lavage for 4 weeks, continuous 4 weeks), MCT modeling + DHA treatment group (intraperitoneal injection of MCT for two weeks after DHA daily gastric lavage for 2 weeks). The mean pulmonary artery pressure (PAP) was measured by catheterization, the right ventricle (RV) was separated along the interventricular septum, and the left ventricular + septum (LV + S) was weighed and the right ventricular hypertrophy index (RV / LV + S) was calculated. CD3 + T cells and CD68 + macrophages were counted around tissues and pulmonary artery resistance; (4) The expression of ER stress related protein in lung tissues was detected by Western blotting; (5) The expression of ER stress related protein in lung tissues was detected by immunofluorescence double staining. 2. PASMC was cultured by tissue block adherence method and progressed. Immunofluorescent cell identification was performed. (1) Immunoblotting and cytoimmunofluorescence were used to detect the expression of ER stress protein in PASMC; (2) Real-time quantitative PCR was used to detect the expression of NFATcl-4 gene in PASMC; (3) Immunoblotting was used to detect the total protein level and nuclear transfer of NFATcl; (4) CCK8 and BrdU uptake was used to detect the proliferation of PASMC; (5) Flow cytometry was used to detect the expression of PASMC. Results: (1) Compared with the control group, the mean pulmonary artery pressure in MCT group increased significantly after 4 weeks (MCT: 48.2 (+ 3.1) mmHg vs control: 20.5 (+ 1.3) mmHg; p0.05); DHA prevention and treatment could significantly reduce the mean pulmonary artery pressure (MCT + DHA) in MCT-induced PH rats. Group B: 25.3 (+ 2.1 mmHg); MCT + DHA group: 31.5 (+ 3.4 mmHg); P0.05 vs MCT group) and right heart index (MCT + DHA prevention group: 0.37 (+ 0.02); MCT + DHA treatment group: 0.42 (+ 0.01); P0.05 vs MCT group); and significantly inhibited MCT-induced pulmonary artery media thickening in rats. (2) DHA prevention and treatment can significantly reduce MCT-induced pulmonary tissue IL-1 beta, IL-6 and IL-1 and MMC-1. Gene expression (p value was 0.05), and inhibited the accumulation of CD3 + T cells and CD68 + macrophages in lung tissue and perivascular tissues of rats induced by MCT. (3) DHA could significantly reduce the expression of stress-related proteins p-IRE1, PDI and GRP78 (p value was 0.05). Immunofluorescence double staining showed that DHA mainly reduced the expression of lung stress-related proteins p-IRE1, PDI and GRP78 in lung tissue of rats induced by MCT. PDI expression in arterial smooth muscle layer. (4) PDGF-BB stimulation significantly increased the expression of ER stress-related proteins p-IRE1, PDI and GRP78 in cultured PASMC (p 0.05), while DHA pre-incubation inhibited the expression of ER stress-related proteins induced by PDGF-BB. Immunofluorescence showed that PDI fluorescence signal was enhanced after PDGF-BB stimulation of PASMC, while DHA pre-incubation could inhibit the expression of ER stress-related proteins induced by PDGF-BB. (5) PDGF-BB stimulation could induce the expression of NFATc1, NFATc2 and NFATc3 genes in PASMC, but had no significant effect on the expression of NFATc4. Preincubation DHA could reverse the expression of NFATc1 and NFATc2 genes induced by PDGF-BB, but had no significant effect on the expression of NFATc1 protein in PASMC. (6) PDGF-BB stimulation could induce the expression of NFATc1 protein in PASMC. Plus, nuclear transfer increased, while pre-incubation DHA and ER stress inhibitor 4-PBA produced similar inhibition of PDGF-BB-induced total NFATc1 protein expression and activity increase. (7) PDGF-BB stimulated PASMC could significantly promote cyclin D1 expression, inhibit G1-related negative protein p21 and p27 expression, in the S-phase percentage of cell cycle. DHA pre-incubation or NFATc1 siRNA interference could similarly inhibit the increase of cyclin D1 expression induced by PDGF-BB, restore the protein levels of p21 and p27, block the cells in G1 phase and inhibit the proliferation of PASMC induced by PDGF-BB. Inhibiting PDGF-BB-induced endoplasmic reticulum stress, reducing PASMC proliferation, improving pulmonary vascular remodeling, and reducing MCT-induced PH mean pulmonary artery pressure in rats.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R544.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 盧志強(qiáng);張艷軍;莊朋偉;張金保;徐利滿;孫鳳嬌;;肺動(dòng)脈壓的檢測(cè)方法優(yōu)化研究[J];中國(guó)藥理學(xué)通報(bào);2015年07期
2 王靜;戴愛(ài)國(guó);;原代大鼠肺動(dòng)脈平滑肌細(xì)胞的提取和鑒定以及缺氧對(duì)其增殖的影響[J];中國(guó)呼吸與危重監(jiān)護(hù)雜志;2012年02期
，本文編號(hào)：2199235
本文鏈接：http://sikaile.net/yixuelunwen/xxg/2199235.html
最近更新
教材專著
