原花青素经肠道微生态途径对脂质代谢的调节

Procyanidin regulates lipid metabolism through gut microbiota

目的研究原花青素(Proanthocyanidin,PC)经肠道微生态途径调节脂质代谢。方法取成年雄性SD大鼠和长爪沙鼠,经基础饲料适应性喂养1周后,采集尾静脉血,收集血清,检测血清总胆固醇(Total cholesterol,TC)水平,并按TC及体重分为基础对照组、模型对照组、低剂量组(25 mg/kg PC)、中剂量组(100 mg/kg PC)、高剂量组(150 mg/kg PC)、阳性对照组(非诺倍特80 mg/kg),基础对照组饲以普通基础饲料,其余各组均饲以高脂饲料,每天灌胃1次,大鼠连续灌胃8周,沙鼠连续灌胃2周。大鼠8周末、沙鼠2周末,经股动脉采血,分离血清,处死前3 d收集72 h粪便。采用全自动生化仪检测TC、甘油三酯(Triglyceride,TG)、高密度脂蛋白胆固醇(High-density lipopro-tein cholesterol,HDL-C)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol,LDL-C)、血总胆汁酸(Total bileacid,TBA)水平;循环酶法试剂盒测定粪TBA排出量水平;双抗体两步夹心ELISA法测定卵磷脂胆固醇酰基转移酶(Lecithin-cholesterol acyltransferase,LCAT)活性。解剖处死的大鼠和沙鼠,进行病理组织学分析。提取大鼠、沙鼠小肠、盲肠细菌基因组DNA,采用PCR-变性梯度凝胶电泳(Denatured gradient gel electrophoresis,DGGE)进行动物模型肠道微生态菌群多样性变化分析。结果模型对照组大鼠和沙鼠TC、TG水平明显高于基础对照组,差异有统计学意义(P<0.05),表明高脂模型建模成功。与模型对照组相比,各剂量组和阳性对照组大鼠、沙鼠TC、TG、LDL-C、血TBA水平均降低,LCAT活性、粪TBA排出量均明显升高,差异有统计学意义(P<0.05);各剂量组大鼠肝脏损伤、肝细胞肿胀、变性等病变程度明显减轻,肝组织病理变化明显改善。DGGE检测及图像分析显示,大鼠各剂量组肠道优势菌群多样性明显增加,随着PC干预剂量加大,中、高剂量组肠道菌群多样性明显减少;沙鼠低剂量组与模型对照组的肠道菌群结构基本相似,而中、高剂量组肠道菌群多样性明显减少,肠道优势菌群结构明显恢复。结论 100、150 mg/kg PC干预的实验动物模型中,肠道菌群结构多样性明显恢复,提示PC可通过肠道菌群这一靶标,进行脂质代谢的调节。

Objective Objectives To investigate the possible mechanism of procyanidin （PC） in regulating lipid metabolism through gut microbiota. Methods Male adult SD rats and Meriones unguiculatus were fed with basal forage for one week, of which the serum samples were collected and determined for total cholesterol （TC） level. These animals were divided into six groups according to their TC levels and bodyweights. The animals in normal control group were fed with common forage, while those in the other five groups with high lipid forage. The animals in three test groups were treated with PC by oral route at low （25 mg/kg）, moderate （100 mg/kg） and high （150 mg/kg） dosages respectively, while those in positive control group with Fenofibrate at a dosage of 80 mg/kg, once a day for 8 （rats） and 2 （M. unguicu- latus） weeks. The rats were killed at end of 8 weeks, while M. unguiculatus at end of 2 weeks, of which serum samples were collected. However, the fecal samples of animals within 72 h before being killed were collected. The TC, triglyceride （TG）, high density lipoproteinprotein cholesterol （HDL-C）, low-density lipoprotein cholesterol （LDL-C） and total bile acid （TBA） levels in sera were determined by full-automatic biochemical analyzer, while the TBA level in feces by enzymatic cycling assay kit, and the lecithin cholesterol acyl transferase （LCAT） activity by two-step double antibody sandwich ELISA. The histopathology of killed animals was observed. The genomic DNAs were extracted from bacteria in small intestines and intestinum caecum of the animals, and analyzed for the changes of predominant bacteria of gut microbiota by PCR-denaturing gradient gel electrophoresis （DGGE）. Results The TC and TG levels of animals in model control group were significantly higher than those in normal control group （P 〈 0. 05）, indicating successful establishment of animal model of hyperlipidemia. As compared with those in model control group, the serum TC, TG, LDL-C and TBA levels in three test groups decressed significantly, while LCAT activity and fecal TBA level increased significantly（each P 〈 0. 05）. The liver lesion, hepatic cell swelling and degeneration in three test groups significantly improved. DGGE and imaging showed that the diversity of predominant bacteria from gut microbiota in three test groups increased significantly, while decreased significantly with the increasing dosage of TC. The bacterial profile of M. unguiculatus treated with 25 mg / kg PC was similar to that in model control group. However, the diversity of gut microbiota decreased significantly, and the predominant bacteria showed an obvious tendency of restoration, in 100 and 150 mg/ kg group. Conclusion The predominant bacterial profile of gut microbiota of experimental animal model showed an obviouse tendency of restoration after intervention by PC and dosages of 100 and 150 mg/kg, indicating the PC might regulate the lipid metabolism via gut microbiota pathway.