嗜酸乳杆菌调节NO及其氧化介质对动脉粥样硬化模型大鼠的影响
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摘要
目的研究嗜酸乳杆菌对一氧化氮(NO)及其氧化介质表达的影响,探讨嗜酸乳杆菌抗动脉粥样硬化的机制。方法 24只SPF级雄性大鼠随机分为正常饮食组、高脂饮食组和嗜酸乳杆菌组,每组8只。正常饮食组大鼠以普通饲料喂养,高脂饮食组以高脂饲料+腹腔注射维生素D3+免疫损伤法+FeSO_4喂养建立动脉粥样硬化模型,嗜酸乳杆菌组大鼠在高脂饮食组的基础上,每天灌胃0.5 mL嗜酸乳杆菌菌液(1×109CFU/mL),实验第4、8、12周末称量动物体质量。喂养12周后处死动物,检测血清氧化低密度脂蛋白(oxLDL)、NO、精氨酸、精氨酸酶、过氧亚硝基(ONOO-)含量。分离大鼠主动脉血管,HE染色观察主动脉形态学变化,Real-time PCR检测eNOS、iNOS mRNA表达,Western blot检测主动脉NF-κB p65亚基的表达。结果 (1)整个实验过程中,高脂饮食组和嗜酸乳杆菌组大鼠体质量变化差异无统计学意义(P>0.05),但较正常饮食组均有明显增长(P <0.01)。(2)HE染色结果显示,高脂饮食大鼠中主动脉形成广泛的动脉粥样硬化病变,嗜酸乳杆菌组大鼠的主动脉形态得到明显改善,仅内皮细胞增生,没有观察到平滑肌细胞的坏死。(3)与正常饮食组相比,高脂饮食组大鼠血清oxLDL、ONOO-、精氨酸酶、主动脉iNOS mRNA和细胞核NF-κB p65表达水平升高(P<0.01),血清NO和精氨酸含量、主动脉eNOS mRNA及胞质NF-κB p65亚基表达水平降低(P<0.01);而嗜酸乳杆菌恰好能逆转上述改变(P<0.01)。结论嗜酸乳杆菌可能通过调节NOS的表达、增加NO生物利用度、保护内皮功能来发挥抗动脉粥样硬化作用。
Objective To investigate the effect of lactobacillus acidophilus on the expression of nitric oxide(NO) and its oxidative mediators, and to explore the anti-atherosclerosis mechanism of lactobacillus acidophilus. Methods Twentyfour male SPF rats were randomly divided into normal diet group, high-fat diet group and lactobacillus acidophilus group,with 8 rats in each group. The rats in the normal diet group were fed with normal diet, and the high-fat diet group was fed with high-fat diet + intraperitoneal injection of vitamin D3 + immune injury + FeSO_4 to establish an atherosclerosis model.The rats in the lactobacillus acidophilus group were intragastrically administered with 0.5 mL of lactobacillus acidophilus per day(l×10~9 CFU/mL) on the basis of the high-fat diet. Rats were weighed at the 4 ~(th), 8~(th) and 12~(th) weekends. After 12 weeks of feeding, the serum was taken from abdominal aorta to detect the contents of oxidized low density lipoprotein(oxLDL), NO,arginine, arginase, and peroxynitrite(ONOO-). The aorta was isolated to observe the morphological change by HE staining.The expressions of eNOS and iNOS mRNA were detected by Real-time PCR, and the expressions of NF-κB p65 subunits were detected by Western blot assay. Results(1) There was no significant difference in body weight between the high-fat diet group and the lactobacillus acidophilus group during the whole experiment(P > 0.05),but both were significantly increased compared with those of the normal diet group(P < 0.01).(2) HE staining showed that the aorta formed a wide range of atherosclerotic lesions in the high-fat diet rats, and the aortic morphology was significantly improved in the lactobacillus acidophilus group. Only endothelial cell proliferation was found, and no smooth muscle cell necrosis was observed in this group.(3) Compared with normal diet group, the serum expressions of oxLDL, ONOO-, arginase, aortic iNOS mRNA and nuclear NF-κB p65 were increased in high-fat diet group(P < 0.01), and the serum arginine content and aortic eOS mRNA expression levels were decreased(P<0.01). While lactobacillus acidophilus can just reverse the above changes(P < 0.01).Conclusion Lactobacillus acidophilus may exert anti-atherosclerotic effects by regulating the expression of NOS,increasing NO bioavailability, and protecting endothelial function.
Objective To investigate the effect of lactobacillus acidophilus on the expression of nitric oxide(NO) and its oxidative mediators, and to explore the anti-atherosclerosis mechanism of lactobacillus acidophilus. Methods Twentyfour male SPF rats were randomly divided into normal diet group, high-fat diet group and lactobacillus acidophilus group,with 8 rats in each group. The rats in the normal diet group were fed with normal diet, and the high-fat diet group was fed with high-fat diet + intraperitoneal injection of vitamin D3 + immune injury + FeSO_4 to establish an atherosclerosis model.The rats in the lactobacillus acidophilus group were intragastrically administered with 0.5 mL of lactobacillus acidophilus per day(l×10~9 CFU/mL) on the basis of the high-fat diet. Rats were weighed at the 4 ~(th), 8~(th) and 12~(th) weekends. After 12 weeks of feeding, the serum was taken from abdominal aorta to detect the contents of oxidized low density lipoprotein(oxLDL), NO,arginine, arginase, and peroxynitrite(ONOO-). The aorta was isolated to observe the morphological change by HE staining.The expressions of eNOS and iNOS mRNA were detected by Real-time PCR, and the expressions of NF-κB p65 subunits were detected by Western blot assay. Results(1) There was no significant difference in body weight between the high-fat diet group and the lactobacillus acidophilus group during the whole experiment(P > 0.05),but both were significantly increased compared with those of the normal diet group(P < 0.01).(2) HE staining showed that the aorta formed a wide range of atherosclerotic lesions in the high-fat diet rats, and the aortic morphology was significantly improved in the lactobacillus acidophilus group. Only endothelial cell proliferation was found, and no smooth muscle cell necrosis was observed in this group.(3) Compared with normal diet group, the serum expressions of oxLDL, ONOO-, arginase, aortic iNOS mRNA and nuclear NF-κB p65 were increased in high-fat diet group(P < 0.01), and the serum arginine content and aortic eOS mRNA expression levels were decreased(P<0.01). While lactobacillus acidophilus can just reverse the above changes(P < 0.01).Conclusion Lactobacillus acidophilus may exert anti-atherosclerotic effects by regulating the expression of NOS,increasing NO bioavailability, and protecting endothelial function.
引文
[1] Yang X, Li Y, Li Y, et al. Oxidative stress-mediatedatherosclerosis:mechanisms and therapies[J]. Front Physiol,2017,8:600. doi:10.3389/fphys.2017.00600.
[2] Kattoor AJ,Pothineni NVK,Palagiri D,et al. Oxidative stress inatherosclerosis[J]. Curr Atheroscler Rep,2017,19(11):42. doi:10.1007/s11883-017-0678-6.
[3] Kundu P,Blacher E,Elinav E,et al. Our gut microbiome:Theevolving inner self[J]. Cell,2017,171(7):1481-1493. doi:10.1016/j.cell.2017.11.024.
[4] Ascher S,Reinhardt C. The gut microbiota:an emerging risk factor for cardiovascular and cerebrovascular disease[J]. Eur J Immunol,2018,48(4):564-575. doi:10.1002/eji.201646879.
[5] Anjum N,Maqsood S,Masud T,et al. Lactobacillus acidophilus:characterization of the species and application in food production[J]. Crit Rev Food Sci Nutr,2014,54(9):1241-1251.doi:10.1080/10408398.2011.621169.
[6] Yamashita T. Intestinal immunity and gut microbiota inatherogenesis[J]. J Atheroscler Thromb,2017,24(2):110-119.doi:10.5551/jat.38265.
[7] Jie Z,Xia H,Zhong SL,et al. The gut microbiome in atheroscleroticcardiovascular disease[J]. Nat Commun,2017,8(1):845. doi:10.1038/s41467-017-00900-1.
[8] Chen L,Liu W,Li Y,et al. Lactobacillus acidophilus ATCC 4356attenuates the atherosclerotic progression through modulation ofoxidative stress and inflammatory process[J]. IntImmunopharmacol,2013,17(1):108-115. doi:10.1016/j.intimp.2013.05.018.
[9] Amaretti A,di Nunzio M,Pompei A,et al. Antioxidant properties ofpotentially probiotic bacteria:in vitro and in vivo activities[J].Appl Microbiol Biotechnol,2013,97(2):809-817. doi:10.1007/s00253-012-4241-7.
[10]Zhang YH. Nitric oxide signalling and neuronal nitric oxidesynthase in the heart under stress[J]. F1000Res,2017,6:742. doi:10.12688/f1000research.10128.1.
[11]Forstermann U,Xia N,Li H. Roles of vascular oxidative stress andnitric oxide in the pathogenesis of atherosclerosis[J]. Circ Res,2017,120(4):713-735. doi:10.1161/CIRCRESAHA.116.309326.
[12]张安邦,黄昕,李令根,等.复合方法制备SD大鼠动脉粥样硬化模型[J].中国中西医结合外科杂志,2015,21(3):282-285.Zhang AB,Huang X,Li LG,et al. Multi-factor methods to establishatherosclerosis model in SD Rats[J]. Chinese Journal of Surgery ofIntegrated Traditional and Western Medicine,2015,21(3):282-285. doi:10.3969/j.issn.1007-6948.2015.03.021.
[13]Paudel KR, Panth N, Kim DW. Circulating endothelialmicroparticles:a key hallmark of atherosclerosis progression[J].Scientifica(Cairo),2016,2016:8514056. doi:10.1155/2016/8514056.
[14]Napoli C,de Nigris F,Williams-Ignarro S,et al. Nitric oxide andatherosclerosis:an update[J]. Nitric Oxide,2006,15(4):265-279.doi:10.1016/j.niox.2006.03.011
[15]Hare JM,Stamler JS. NO/redox disequilibrium in the failing heartand cardiovascular system[J]. J Clin Invest,2005,115(3):509-517. doi:10.1172/JCI24459.
[16]Lee J,Bae EH,Ma SK,et al. Altered nitric oxide system incardiovascular and renal diseases[J]. Chonnam Med J,2016,52(2):81-90. doi:10.4068/cmj.2016.52.2.81.
[17]Vanhoutte PM,Shimokawa H,Feletou M,et al. Endothelialdysfunction and vascular disease-a 30th anniversary update[J].Acta Physiol(Oxf),2017,219(1):22-96. doi:10.1111/apha.12646.
[18]Bartnicki P,Kowalczyk M,Franczyk-Skora B,et al. Evaluation ofendothelial(dys)function,left ventricular structure and function inpatients with chronic kidney disease[J]. Curr Vasc Pharmacol,2016,14(4):360-367.
[19]Rabelo LA,Ferreira FO,Nunes-Souza V,et al. Arginase as acritical prooxidant mediator in the binomial endothelialdysfunction-atherosclerosis[J]. Oxid Med Cell Longev,2015,2015:924860. doi:10.1155/2015/924860.
[20]Lubrano V,Balzan S. LOX-1 and ROS,inseparable factors in theprocess of endothelial damage[J]. Free Radic Res,2014,48(8):841-848. doi:10.3109/10715762.2014.929122.
[21]Pautz A,Art J,Hahn S,et al. Regulation of the expression ofinducible nitric oxide synthase[J]. Nitric Oxide,2010,23(2):75-93. doi:10.1016/j.niox. 2010.04.007.
[22]Li H,Forstermann U. Uncoupling of endothelial NO synthase inatherosclerosis and vascular disease[J]. Curr Opin Pharmacol,2013,13(2):161-167. doi:10.1016/j.coph.2013.01.006.
[23]Sukhovershin RA,Yepuri G,Ghebremariam YT. Endothelium-derived nitric oxide as an antiatherogenic mechanism:implicationsfor therapy[J]. Methodist Debakey Cardiovasc J,2015,11(3):166-171. doi:10.14797/mdcj-11-3-166.
[24]Degendorfer G,Chuang CY,Hammer A,et al. Peroxynitrous acidinduces structural and functional modifications to basementmembranes and its key component,laminin[J]. Free Radic BiolMed,2015,89:721-733. doi:10.1016/j.freeradbiomed.2015.09.018.
[25]Fordjour PA,Wang Y,Shi Y,et al. Possible mechanisms of C-reactive protein mediated acute myocardial infarction[J]. Eur JPharmacol,2015,760:72-80. doi:10.1016/j.ejphar.2015.04.010.
[26]Ahmad Z,Ng CT,Fong LY,et al. Cryptotanshinone inhibits TNF-alpha-induced early atherogenic events in vitro[J]. J Physiol Sci,2016,66(3):213-220. doi:10.1007/s12576-015-0410-7.
[27]Sies H,Berndt C,Jones DP. Oxidative Stress[J]. Annu RevBiochem,2017,86:715-748. doi:10.1146/annurev-biochem-061516-045037.
[28]Galley HF,Webster NR. Physiology of the endothelium[J]. Br JAnaesth,2004,93(1):105-113. doi:10.1093/bja/aeh163.2018-08-142018-11-20
[2] Kattoor AJ,Pothineni NVK,Palagiri D,et al. Oxidative stress inatherosclerosis[J]. Curr Atheroscler Rep,2017,19(11):42. doi:10.1007/s11883-017-0678-6.
[3] Kundu P,Blacher E,Elinav E,et al. Our gut microbiome:Theevolving inner self[J]. Cell,2017,171(7):1481-1493. doi:10.1016/j.cell.2017.11.024.
[4] Ascher S,Reinhardt C. The gut microbiota:an emerging risk factor for cardiovascular and cerebrovascular disease[J]. Eur J Immunol,2018,48(4):564-575. doi:10.1002/eji.201646879.
[5] Anjum N,Maqsood S,Masud T,et al. Lactobacillus acidophilus:characterization of the species and application in food production[J]. Crit Rev Food Sci Nutr,2014,54(9):1241-1251.doi:10.1080/10408398.2011.621169.
[6] Yamashita T. Intestinal immunity and gut microbiota inatherogenesis[J]. J Atheroscler Thromb,2017,24(2):110-119.doi:10.5551/jat.38265.
[7] Jie Z,Xia H,Zhong SL,et al. The gut microbiome in atheroscleroticcardiovascular disease[J]. Nat Commun,2017,8(1):845. doi:10.1038/s41467-017-00900-1.
[8] Chen L,Liu W,Li Y,et al. Lactobacillus acidophilus ATCC 4356attenuates the atherosclerotic progression through modulation ofoxidative stress and inflammatory process[J]. IntImmunopharmacol,2013,17(1):108-115. doi:10.1016/j.intimp.2013.05.018.
[9] Amaretti A,di Nunzio M,Pompei A,et al. Antioxidant properties ofpotentially probiotic bacteria:in vitro and in vivo activities[J].Appl Microbiol Biotechnol,2013,97(2):809-817. doi:10.1007/s00253-012-4241-7.
[10]Zhang YH. Nitric oxide signalling and neuronal nitric oxidesynthase in the heart under stress[J]. F1000Res,2017,6:742. doi:10.12688/f1000research.10128.1.
[11]Forstermann U,Xia N,Li H. Roles of vascular oxidative stress andnitric oxide in the pathogenesis of atherosclerosis[J]. Circ Res,2017,120(4):713-735. doi:10.1161/CIRCRESAHA.116.309326.
[12]张安邦,黄昕,李令根,等.复合方法制备SD大鼠动脉粥样硬化模型[J].中国中西医结合外科杂志,2015,21(3):282-285.Zhang AB,Huang X,Li LG,et al. Multi-factor methods to establishatherosclerosis model in SD Rats[J]. Chinese Journal of Surgery ofIntegrated Traditional and Western Medicine,2015,21(3):282-285. doi:10.3969/j.issn.1007-6948.2015.03.021.
[13]Paudel KR, Panth N, Kim DW. Circulating endothelialmicroparticles:a key hallmark of atherosclerosis progression[J].Scientifica(Cairo),2016,2016:8514056. doi:10.1155/2016/8514056.
[14]Napoli C,de Nigris F,Williams-Ignarro S,et al. Nitric oxide andatherosclerosis:an update[J]. Nitric Oxide,2006,15(4):265-279.doi:10.1016/j.niox.2006.03.011
[15]Hare JM,Stamler JS. NO/redox disequilibrium in the failing heartand cardiovascular system[J]. J Clin Invest,2005,115(3):509-517. doi:10.1172/JCI24459.
[16]Lee J,Bae EH,Ma SK,et al. Altered nitric oxide system incardiovascular and renal diseases[J]. Chonnam Med J,2016,52(2):81-90. doi:10.4068/cmj.2016.52.2.81.
[17]Vanhoutte PM,Shimokawa H,Feletou M,et al. Endothelialdysfunction and vascular disease-a 30th anniversary update[J].Acta Physiol(Oxf),2017,219(1):22-96. doi:10.1111/apha.12646.
[18]Bartnicki P,Kowalczyk M,Franczyk-Skora B,et al. Evaluation ofendothelial(dys)function,left ventricular structure and function inpatients with chronic kidney disease[J]. Curr Vasc Pharmacol,2016,14(4):360-367.
[19]Rabelo LA,Ferreira FO,Nunes-Souza V,et al. Arginase as acritical prooxidant mediator in the binomial endothelialdysfunction-atherosclerosis[J]. Oxid Med Cell Longev,2015,2015:924860. doi:10.1155/2015/924860.
[20]Lubrano V,Balzan S. LOX-1 and ROS,inseparable factors in theprocess of endothelial damage[J]. Free Radic Res,2014,48(8):841-848. doi:10.3109/10715762.2014.929122.
[21]Pautz A,Art J,Hahn S,et al. Regulation of the expression ofinducible nitric oxide synthase[J]. Nitric Oxide,2010,23(2):75-93. doi:10.1016/j.niox. 2010.04.007.
[22]Li H,Forstermann U. Uncoupling of endothelial NO synthase inatherosclerosis and vascular disease[J]. Curr Opin Pharmacol,2013,13(2):161-167. doi:10.1016/j.coph.2013.01.006.
[23]Sukhovershin RA,Yepuri G,Ghebremariam YT. Endothelium-derived nitric oxide as an antiatherogenic mechanism:implicationsfor therapy[J]. Methodist Debakey Cardiovasc J,2015,11(3):166-171. doi:10.14797/mdcj-11-3-166.
[24]Degendorfer G,Chuang CY,Hammer A,et al. Peroxynitrous acidinduces structural and functional modifications to basementmembranes and its key component,laminin[J]. Free Radic BiolMed,2015,89:721-733. doi:10.1016/j.freeradbiomed.2015.09.018.
[25]Fordjour PA,Wang Y,Shi Y,et al. Possible mechanisms of C-reactive protein mediated acute myocardial infarction[J]. Eur JPharmacol,2015,760:72-80. doi:10.1016/j.ejphar.2015.04.010.
[26]Ahmad Z,Ng CT,Fong LY,et al. Cryptotanshinone inhibits TNF-alpha-induced early atherogenic events in vitro[J]. J Physiol Sci,2016,66(3):213-220. doi:10.1007/s12576-015-0410-7.
[27]Sies H,Berndt C,Jones DP. Oxidative Stress[J]. Annu RevBiochem,2017,86:715-748. doi:10.1146/annurev-biochem-061516-045037.
[28]Galley HF,Webster NR. Physiology of the endothelium[J]. Br JAnaesth,2004,93(1):105-113. doi:10.1093/bja/aeh163.2018-08-142018-11-20