灵宝护心丹对动脉粥样硬化小鼠肠道黏膜屏障和肠道菌群的影响

Effect of Lingbao Huxin Pill on intestinal mucosal barrier and gut microbiota in mice with atherosclerosis

目的探讨灵宝护心丹对动脉粥样硬化模型小鼠的改善效果及其对肠道菌群结构的调节作用,并比较其与他汀类药物对肠道菌群影响的差异性。方法高脂饮食喂养ApoE^(−/−)小鼠12周诱导动脉粥样硬化模型,随机分为模型组及灵宝护心丹低、中、高剂量(4.68、9.36、18.72 mg/kg)组和阿托伐他汀(2.4 mg/kg)组。造模成功后连续给药8周,C57BL/6小鼠喂养普通饲料作为空白组。通过油红O染色和苏木素-伊红(hematoxylin-eosin,HE)染色观察小鼠主动脉窦的斑块面积和病理结构改变,检测血清总胆固醇(total cholesterol,TC)、三酰甘油(triacylglycerol,TG)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low-density lipoprotein cholestero,LDL-C)水平,采用16S rRNA测序技术检测小鼠粪便微生物群的多样性和丰度,免疫荧光染色和qRT-PCR检测结肠紧密连接蛋白Occludin的表达情况。结果与模型组比较,灵宝护心丹能够有效减少动脉斑块沉积和局部炎症损伤,降低TC、TG、LDL-C水平(P<0.01、0.001),改善肠道微生物的丰富度和多样性,且与他汀类药物治疗后的肠道微生物有显著差异,灵宝护心丹组的差异物种是Parasutterella属、Sutterellaceae科、Burkholderiales目和γ-变形菌纲等,阿托伐他汀组的差异物种是拟杆菌属、拟杆菌科、Butyricimonas属和Marinifilaceae科等。免疫荧光和qRT-PCR结果显示灵宝护心丹显著增加结肠Occludin的表达(P<0.05),提高小鼠肠道黏膜屏障的完整性。结论灵宝护心丹具有改善动脉粥样硬化、调血脂、保护肠黏膜屏障的作用,其机制可能与调节肠道菌群有关。

Objective To explore the improvement effect of Lingbao Huxin Pill(灵宝护心丹)on atherosclerosis model mice and its regulatory effect on the structure of intestinal flora,and compare its effects with statins on intestinal flora.Methods ApoE^(−/−)mice were fed high-fat diet for 12 weeks to induce atherosclerosis model,and were randomly divided into model group,Lingbao Huxin Pill low-,medium-,high-dose(4.68,9.36,18.72 mg/kg)and atorvastatin(2.4 mg/kg)group.After the model was successfully established,C57BL/6 mice were fed with ordinary feed by continuous intragastric administration for eight weeks as a blank group.The plaque area and pathological structural changes of aortic sinus in mice were observed through oil red O staining and hematoxylin-eosin(HE)staining;Total cholesterol(TC),triacylglycerol(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)levels in serum were detected;16S rRNA sequencing technology was used to detect the diversity and abundance of fecal microbiota in mice;Immunofluorescence staining and qRT-PCR were used to detect the expression of tight junction protein Occludin in colon.Results Compared with model group,Lingbao Huxin Pill effectively reduced arterial plaque deposition and local inflammatory damage,reduced TC,TG and LDL-C levels in serum(P<0.01,0.001),improved the richness and diversity of intestinal microorganisms,and there was a significant difference between Lingbao Huxin Pill and statin treatment in intestinal microorganisms.The different species in Lingbao Huxin Pill group were Parasutterella,Sutterellaceae,Burkholderiales andγ-Proteobacteria,etc.The different species in atorvastatin group were Bacteroides,Bacteroidaceae,Butyricimonas and Marinifilaceae,etc.The results of immunofluorescence and qRT-PCR showed that Lingbao Huxin Pill significantly increased the expression of Occludin in colon(P<0.05)and improved the integrity of the intestinal mucosal barrier in mice.Conclusion Lingbao Huxin Pill can improve atherosclerosis,regulate blood lipid,and protect the intestinal mucosal barrier.Its mechanism may be related to the regulation of intestinal flora.