基于胆固醇代谢途径相关蛋白探讨丹荷颗粒对高胆固醇血症大鼠的作用

Effect of Danhe Granules on Hypercholesterolemia Rats Based on Cholesterol Metabolism Pathway-related Proteins

摘要:

目的 观察丹荷颗粒干预下高胆固醇血症大鼠的药效指标及胆固醇代谢途径相关蛋白水平的变化，研究丹荷颗粒对高胆固醇血症大鼠的治疗作用及机制。方法 将SD大鼠按体质量随机分为空白组和模型组，空白组以维持饲料喂养，模型组持续给予高脂饲料喂养7周。造模1周后检测模型大鼠血清总胆固醇（TC）含量，按TC值将模型组随机分为模型组、普伐他汀钠片组（4.02 mg·kg^-1）、血脂康胶囊组（0.12 g·kg^-1）、丹荷颗粒高、中、低剂量组（4.536、2.268、1.134 g·kg^-1）；模型组分组后，各给药组连续灌胃给药6周，期间每周称体质量1次，每周称取1次饲料加入量与饲料剩余量的差值为摄食量，6周后检测血清TC、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）水平，采用苏木素-伊红（HE）染色与油红O染色，观察肝脏病理状态和脂滴分布情况，进行评分和计算。采集大鼠肝脏，运用蛋白免疫印迹法（Western blot）和免疫组化法（IHC）检测高胆固醇血症大鼠胆固醇代谢相关蛋白磷酸化的腺苷酸活化蛋白激酶（p-AMPK）、腺苷酸活化蛋白激酶（AMPK）、3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMGCR）、低密度脂蛋白受体（LDLR）、胆固醇7α-羟化酶（CYP7A1）、乙酰辅酶A乙酰转移酶2（ACAT2）、载脂蛋白B（ApoB）的表达水平。结果 与空白组比较，模型组血清TC水平显著升高（P<0.01），TG水平变化差异无统计学意义，HDL-C水平明显降低（P<0.05），肝脏指数、脂肪变性评分、病理状态总得分及油红O染色的阳性面积比率显著升高（P<0.01），p-AMPK、p-AMPK/AMPK、LDLR、CYP7A1蛋白表达水平明显降低（P<0.05，P<0.01），AMPK、HMGCR、ACAT2蛋白表达水平明显升高（P<0.05，P<0.01）。与模型组比较，丹荷颗粒各剂量组TC水平明显降低（P<0.05），普伐他汀钠片组和丹荷颗粒中剂量组油红O染色的阳性面积比率明显降低（P<0.05）；各给药组p-AMPK、p-AMPK/AMPK蛋白表达水平明显升高（P<0.05，P<0.01），HMGCR、ACAT2水平显著降低（P<0.01），ApoB水平有下降的趋势；普伐他汀钠片组和丹荷颗粒各剂量组CYP7A1水平明显升高（P<0.05，P<0.01），普伐他汀钠片组、血脂康胶囊组和丹荷颗粒高、中剂量组LDLR水平明显升高（P<0.05，P<0.01）。结论 丹荷颗粒可降低血清TC水平，改善肝脏脂肪变性。可能通过激活AMPK，下调HMGCR的表达，抑制胆固醇的合成；上调LDLR和CYP7A1表达，促进胆固醇的摄取与排泄；下调ACAT2和ApoB的表达，减少胆固醇的吸收和LDL等脂蛋白的装配；发挥治疗高胆固醇血症的作用。

Abstract:

Objective To explore the therapeutic effect and mechanism of the Danhe granules on hypercholesterolemia rats by observing the changes in the efficacy indicators and the levels of proteins related to the cholesterol metabolism pathway in the rats under the intervention of Danhe granules.Method SD rats were randomly assigned to either the blank group or the model group based on their body weight. The blank group had normal chow diets， while the model group was fed high-fat diets for seven weeks. One week after the establishment of the model， the content of the serum total cholesterol （TC） in the model rats was detected. According to the TC value， the model group was further randomly divided into a control group， pravastatin sodium tablet group（4.02 mg·kg^-1）， Xuezhikang capsule group（0.12 g·kg^-1）， high-dose， middle-dose， and low-dose groups of Danhe granules（4.536， 2.268， 1.134 g·kg^-1）. After grouping the model groups， each treatment group received continuous oral gavage for six weeks， with weekly measurements of body weight and food intake （the difference between feed intake and feed surplus）. Six weeks later， the levels of serum TC， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） were measured. The liver pathology and lipid droplet distribution were evaluated by hematoxylin-eosin （HE） staining and oil red O staining， with scoring and calculation conducted. Rat liver tissue was collected， and western blot and immunohistochemistry （IHC） were used to detect the expression levels of cholesterol metabolism-related proteins namely phosphorylated adenosine 5'-monophosphate （AMP）-activated protein kinase （p-AMPK）， AMPK， 3-hydroxy-3-methyl glutaryl coenzyme A reductase （HMGCR）， low-density lipoprotein receptor （LDLR）， cholesterol 7α-hydroxylase （CYP7A1）， Acyl-coenzyme A： cholesterol acyltransferase 2 （ACAT2）， and apolipoprotein B （ApoB） in hypercholesterolemia rats.Result Compared with the blank group， the model group showed a significantly higher level of serum TC （P<0.01）. The TG level had no significant change， and the HDL-C level was significantly decreased （P<0.05）. The liver index， steatosis score， total score of pathological state， and the positive area ratio of oil red O staining were significantly increased （P<0.01）， and the protein expression levels of p-AMPK， p-AMPK/AMPK， LDLR， and CYP7A1 were significantly decreased （P<0.05， P<0.01）， while the protein expression levels of AMPK， HMGCR， and ACAT2 were significantly increased （P<0.05， P<0.01）. Compared with the model group， the TC level in each dose group of Danhe granules was significantly decreased （P<0.05）， and the positive area ratio of oil red O staining in the pravastatin sodium tablet group and medium-dose group of Danhe granules was significantly decreased （P<0.05）. In each administration group， the protein expression levels of p-AMPK and p-AMPK/AMPK were significantly increased （P<0.05， P<0.01）， and the levels of HMGCR and ACAT2 were significantly decreased （P<0.01）. The ApoB level showed a downward trend. The CYP7A1 level in the pravastatin sodium tablet group and each dose group of Danhe granules was significantly increased （P<0.05， P<0.01）， and the LDLR level in the pravastatin sodium tablet group， Xuezhikang capsule group， and high-dose and medium-dose groups of Danhe granules was significantly increased （P<0.05， P<0.01）.Conclusion Danhe granules can reduce serum TC levels and improve hepatic steatosis. It may activate AMPK， down-regulate the expression of HMGCR， and inhibit cholesterol synthesis. It can also up-regulate the expression of LDLR and CYP7A1， promote cholesterol uptake and excretion， down-regulate the expression of ACAT2 and ApoB， reduce cholesterol absorption and assembly of LDL and other lipoproteins， and thus play a role in the treatment of hypercholesterolemia.