天山雪莲对老年脂质代谢异常(气虚血瘀型)小鼠的改善作用及机制研究

Study on improving effects and mechanism of Saussureae Involucratae Herba in aged mice with abnormal lipid metabolism(qi deficiency and blood stasis syndrome)

目的 结合体内外实验研究天山雪莲Saussureae Involucratae Herba对老年脂质代谢异常(气虚血瘀型)小鼠的影响与作用机制。方法 采用高脂喂食配合力竭游泳建立拟临床老年脂质代谢异常(气虚血瘀型)小鼠模型,设置对照组、模型组、罗格列酮组及天山雪莲低、中、高剂量(0.3、0.9、1.8 g/kg)组,各给药组连续给药5周。检测各组小鼠葡萄糖耐量、血脂等指标;HE染色检测脂肪组织形态;定量反转录聚合酶链式反应(quantitative reverse transcriptase polymerase chain reaction,q RTPCR)法检测脂肪组织解偶联蛋白-1(uncoupling protein 1,Ucp1)、DNA聚合酶γ1(DNA polymerase γ1,Polg1)基因表达;免疫组化法检测脂肪组织UCP1的蛋白表达。体外实验进一步研究作用机制,将棕色脂肪细胞分为对照组、罗格列酮组及天山雪莲低、中、高剂量(0.025、0.05、0.1 mg/mL)组。q RT-PCR法检测棕色脂肪细胞中Ucp1、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(peroxisome proliferator activated receptor γ coacti-vator-1α,Pgc-1α)、核呼吸因子1(nuclear respiratory factor1,Nrf1)、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ,Pparγ)、PR结构域蛋白16(positive regulatory domain-containing 16,Prdm16)、线粒体转录因子A(mitochondrial transcription factor A,Tfam)、葡萄糖转运蛋白4(glucose transporter 4,Glut 4)的基因表达;免疫荧光法和Mito Tracker染色检测棕色脂肪细胞Ucp1蛋白表达水平及线粒体数量。结果 与对照组比较,模型小鼠表现对抗性减弱、舌质出现紫瘀等气虚血瘀的表征,空腹血糖、曲线下面积(area under curve,AUC)、腹股沟白色脂肪组织(inguinal white adipose tissue,i WAT)指数及总胆固醇(total cholesterol,TC)、三酰甘油(triglyceride,TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)含量显著升高(P<0.01),棕色脂肪组织(brown adipose tissue,BAT)指数显著降低(P<0.01),BAT和iWAT白色化特征明显且Ucp1阳性细胞表达显著减少(P<0.01)。与模型组对比,天山雪莲给药组小鼠血脂指标、糖代谢指标、iWAT指数显著改善(P<0.01),BAT指数显著升高(P<0.05),iWAT和BAT形态棕色化特征明显,BAT中Polg1、Ucp1等基因表达水平显著升高(P<0.05、0.01),脂肪组织中Ucp1蛋白表达水平显著升高(P<0.05、0.01)。体外实验中,与对照组相比,天山雪莲干预后棕色脂肪细胞Prdm16、Pparγ、Glut 4、Tfam基因表达水平及线粒体数量和Ucp1蛋白表达显著升高(P<0.05、0.01)。结论 天山雪莲通过调控脂肪可塑性,促进白色脂肪棕色化以及棕色脂肪活化,进而提高线粒体功能改善老年小鼠气虚血瘀型脂质代谢异常。

Objective To study the effects and mechanisms of Tianshan Xuelian(Saussureae Involucratae Herba, SI) on aged mice with abnormal lipid metabolism(qi deficiency and blood stasis syndrome) combined in vivo and in vitro experiments. Methods The aged mice model of abnormal lipid metabolism(qi deficiency and blood stasis syndrome) were established by vigorous swimming and high-fat diet. The mice were divided into control group, model group, rosiglitazone group and low, medium and high dose(0.3, 0.9, 1.8 g/kg) of SI groups, and each administrated group was orally given drugs or solvent respectively for consecutive 5 weeks. The parameters of glucose tolerance and blood lipids of the mice in each group were measured by biochemical method. And adipose tissues morphological changes were detected by HE staining. The genes expression of uncoupling protein 1(Ucp1), DNA polymerase γ1(Polg1) in adipose tissue were detected using quantitative reverse transcriptase polymerase chain reaction(qRT-PCR). The protein expression of Ucp1 in adipose tissue was detected by immunohistochemistry. In vitro, brown adipocytes were divided into five groups, including control group, low, medium and high dose(0.025, 0.05, 0.1 mg/mL) of SI groups, and rosiglitazone group, further mechanism of action. The genes expression of Ucp1,peroxisome proliferator-activated receptor γ(Pparγ), positive regulatory domain-containing 16(Prdm16), mitochondrial transcription factor A(Tfam), glucose transporter 4(Glut4) were detected by qRT-PCR. The protein expression of Ucp1 expression was detected by immunofluorescence. Mitochondrial Mito tracker staining was used to detect mitochondrial number changes in brown adipocytes. Results Compared with the control group, the model mice showed decreased resistance, purple blood stasis and other characteristics of qi deficiency and blood stasis in the tongue, fasting blood glucose, area under curve(AUC), inguinal white adipose tissue(iWAT) index, total cholesterol(TC), triglyceride(TG) and low-density lipoprotein cholesterol(LDL-C) content was significantly increased(P < 0.01), and the brown adipose tissue(BAT) index was significantly decreased(P < 0.01), the white characteristics of BAT and iWAT and the expression of Ucp1positive cells were significantly decreased(P < 0.01). Compared with model group, the blood lipid index, glucose metabolism index and i WAT index were significantly improved(P < 0.01), BAT index was significantly increased(P < 0.05), iWAT and BAT morphologic browning characteristics were obvious, and the expression levels of Polg1 and Ucp1 genes in BAT were significantly increased(P < 0.05,0.01), the expression of Ucp1 protein in adipose tissue significantly increased(P < 0.05, 0.01) of mice in SI administration group. In vitro experiments, compared with the control group, the expression levels of Prdm16, Pparγ, Glut4, Tfam genes, mitochondrial number and Ucp1 protein expression in brown adipocytes after SI intervention were significantly increased(P < 0.05, 0.01). Conclusion SI could improve abnormal lipid metabolism in aged mouse models with qi deficiency and blood stasis by regulating fat plasticity, and ameliorating mitochondrial dysfunction.