摘要
目的探讨TLR4基因敲除小鼠动脉粥样硬化模型Wnt5a表达的变化及中药护心康的干预作用。方法SPF级TLR4基因敲除小鼠共68只,随机取10只为对照组,予以普通饲料喂养;其余58只予以高脂饲料建立动脉粥样硬化模型,喂养13周后从造模动物中随机选取5只解剖观察其胸主动脉,光镜下发现粥样斑块为造模成功。造模成功动物随机分为模型组、护心康组、阿托伐他汀组,Wnt抑制剂WIF组,每组12只,分别予以相应处理。正常组不做处理,模型组予以生理盐水灌胃并腹腔注射,其余各组均予相应药物灌胃和腹腔注射处理,灌胃容积均为0. 5 ml、腹腔注射容积均为0. 3 ml,每日1次。治疗8周后处死小鼠,心脏采血,酶比色法检测血脂;取胸主动脉,光镜下观察动脉粥样硬化形成情况;免疫组化法检测斑块中Wnt5a蛋白的表达。结果 (1)血脂检测:造模后动物胆固醇、甘油三酯、低密度脂蛋白和高密度脂蛋白水平比正常组明显升高(P <0. 05);阿托伐他汀和护心康可以降低胆固醇和低密度脂蛋白水平(P<0. 05),WIF对二者的降低作用不明显;三种药物对甘油三酯和高密度脂蛋白的影响相对较小。(2)光镜观察:正常组无斑块形成,模型组可见内膜增厚,大量斑块明显形成,血管腔明显减小;各药物干预组斑块病变相对较轻。(3)Wnt5a蛋白表达:模型组中Wnt5a蛋白表达明显高于正常组(P <0. 01);与模型组相比,三种药物均能降低Wnt5a,Wnt抑制剂WIF降低作用较强(P <0. 01),护心康组和阿托伐他汀组也有明显的降低作用(P <0. 05); WIF降低Wnt5a的作用较阿托伐他汀强(P <0. 05)。结论 TLR4基因敲除小鼠动脉粥样硬化时Wnt5a表达明显升高,中药护心康能降低血脂和Wnt5a在动脉粥样硬化中的表达,从而可以减轻动脉粥样硬化的形成。
Objective To investigate Huxinkang intervention in atherosclerosis and changes of Wnt5 a. Methods 68 SPF TLR4 gene knockout mice were fed. 10 of them were randomly selected as control( normal) group and fed with normal diet. The remaining 58 mice were modeled with high-fat diet for 13 weeks,then,5 mice were observed regarding thoracic aorta. Atheroma plaque was found under light microscopy,indicating successful modeling. The successful modeled mice were randomized into model group,Huxinkang group,atorvastatin group,and Wnt inhibitor WIF group,12 in each group. The normal group was not treated. The model group was given normal saline by intraperitoneal injection.The other groups were given corresponding drugs by intragastric administration and intraperitoneal injection. The intragastric volume was 0. 5 ml,and the intraperitoneal injection volume was 0. 3 ml. 8 weeks after treatment,the mice were sacrificed for their thoracic aortae which were observed under light microscope to evaluate atherosclerosis. The expression of Wnt5 a proteins in plaques was detected by immunohistochemistry. Results(1)Blood lipid test: The levels of cholesterol,triglyceride,LDL and HDL in modeled animals were significantly higher than in normal group( P <0. 05). Atorvastatin and Huxinkang groups had decreased cholesterol and LDL levels( P < 0. 05),while WIF had no significant effect on the two; effects of the three drugs on triglyceride and HDL were not significant.(2)Light microscopy: no plaques were formed in control group. A large amount of plaques was formed in model group,featuring thickened intima and reduced vascular lumen. plaque lesions in each drug intervention group were relatively light.(3)For Wnt5 a protein expression: model group was significantly higher than in the normal group( P < 0. 01). Compared with model group,the three drugs groups showed decreased Wnt5 a,WIF being the most significant( P < 0. 01). Huxinkang group and atorvastatin group also showed Wnt5 a decrease( P < 0. 05) but not as much. Conclusion Wnt5 a expression significantly increased in TLR4 gene knockout mice with atherosclerosis. Huxinkang can reduce blood lipids and Wnt5 a against atherosclerosis.
引文
[1]Scheen AJ.From atherosclerosis to atherothrombosis:from a silent chronic pathology to an acute critical event[J].Rev Med Liege,2018,3(5):224-228.
[2]Huang X,Zhong L,Hendriks J,et al.The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells[J].Int J Mol Sci,2018,19(2):561.
[3]文川,徐浩,黄启福,等.活血中药对ApoE基因缺陷小鼠血脂及动脉粥样硬化斑块炎症反应的影响[J].中国中西医结合杂志,2005,25(4):345-348.
[4]Wang F,Liu Z,Park SH,et al.Myeloid Wnt5a Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice[J].Arterioscler Thromb Vasc Biol,2018,38(7):1468-1478.
[5]Speranza FJ,Mahankali M,Gomez-Cambronero J.Macrophage migration arrest due to a winning balance of Rac2/Sp1 repression over Wnt5a-induced PLD expression[J].J Leukoc Biol,2013,94(5):953-962.
[6]Kerr GE,Young JC,Horvay K,et al.Regulated Wnt/beta-catenin signaling sustains adult spermatogenesis in mice[J].Biol Reprod,2014,90(1):3.
[7]Halleskog C,Mulder J,Dahlstrom J,et al.WNT signaling in activated microglia is proinflammatory[J].Glia,2011,59(1):119-31.
[8]Gustafson B,Hammarstedt A,Andersson CX,et al.Inflamed aipose tissue:a culprit underlying the metabolic syndrome and atherosclerosis[J].Arterioscler Thromb Vasc Biol,2007,27(11):2276-22839.
[9]王国倩,喻敏成,喻正科,等.护心康片治疗冠心病不稳定型心绞痛临床疗效及对肿瘤坏死因子-α和N端脑钠肽的影响[J].中国中医药信息杂志,2016,23(3):16-19.
[10]王国倩,喻正科,陈志红,等.护心康片治疗不稳定型心绞痛气虚痰瘀互阻证的临床研究[J].中医药导报,2015,21(14):21-24.
[11]刘叶辉,毛以林,李庆,等.护心康对痰瘀互结证冠心病患者血清高敏C反应蛋白的影响[J].按摩与康复医学,2012,3(6):27-29.
[12]屈波,蔡光先,刘柏炎,等.护心康对动脉粥样硬化兔模型血脂的干预研究[J].实用预防医学,2007,14(3):686-687.
[13]文礼湘,屈波,蔡光先.护心康对动脉粥样硬化兔TLR4的干预作用[J].实用预防医学,2013,20(2):222-223.
[14]屈波,蔡光先,董晓斐,等.护心康对动脉粥样硬化兔NF-κB的干预作用[J].实用预防医学,2015,22(5):613-614.
