Effect of electroacupuncture at "Fenglong" (ST 40) on liver cholesterol metabolism in hyperlipidemia rats based on AMPK/mTOR pathway

JIN Shuwen; LIU Jiabao; LI Dan; ZHANG Xi; LIU Manqi; ZHANG Hongxing; PAN Xiaoli

doi:10.13703/j.0255-2930.20231011-k0002

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Effect of electroacupuncture at "Fenglong" (ST 40) on liver cholesterol metabolism in hyperlipidemia rats based on AMPK/mTOR pathway

Study on Mechanism | 更新时间：2024-10-12

- Effect of electroacupuncture at "Fenglong" (ST 40) on liver cholesterol metabolism in hyperlipidemia rats based on AMPK/mTOR pathway
- Chinese Acupuncture & Moxibustion Vol. 44, Issue 10, Pages: 1155-1164(2024)
- 作者机构：
  
  1.湖北中医药大学针灸骨伤学院，武汉　430061
  2.江汉大学医学部
  3.江汉大学针灸研究所，湖北武汉　430056
  4.湖北时珍实验室，武汉　430061
- 作者简介：
- 基金信息：
- DOI：10.13703/j.0255-2930.20231011-k0002
  CLC：
- Received：11 October 2023，
  
  Online First：28 May 2024，
  
  Published：12 October 2024
- 稿件说明：
移动端阅览
JIN Shuwen, LIU Jiabao, LI Dan, et al. Effect of electroacupuncture at "Fenglong" (ST 40) on liver cholesterol metabolism in hyperlipidemia rats based on AMPK/mTOR pathway[J]. Chinese Acupuncture & Moxibustion, 2024, 44(10): 1155-1164.
DOI：

JIN Shuwen, LIU Jiabao, LI Dan, et al. Effect of electroacupuncture at "Fenglong" (ST 40) on liver cholesterol metabolism in hyperlipidemia rats based on AMPK/mTOR pathway[J]. Chinese Acupuncture & Moxibustion, 2024, 44(10): 1155-1164. DOI： 10.13703/j.0255-2930.20231011-k0002.

摘要

目的：

观察电针“丰隆”穴对高脂血症（HPL）大鼠腺苷酸活化蛋白激酶（AMPK）、哺乳动物雷帕霉素靶蛋白（mTOR）表达水平及下游胆固醇代谢相关分子胆固醇调节元件结合蛋白-2（SREBP-2）、3-羟基3-甲基戊二酰辅酶A还原酶（HMGCR）、三磷酸腺苷结合盒转运体G5/G8（ABCG5/G8）表达的影响，探讨电针干预HPL的可能机制。

方法：

将30只SPF级雄性SD大鼠随机分为空白组、模型组、AMPK激动剂组、电针组和电针+AMPK抑制剂组，每组6只。采用高脂饲料喂养法制备HPL模型。造模完成后，电针组大鼠行电针干预，穴取双侧“丰隆”，予疏密波，频率2 Hz/100 Hz，电流1 mA，每次30 min，每天1次；AMPK激动剂组予以腹腔注射AMPK激动剂A-769662（30 mg/kg），每天2次；电针+AMPK抑制剂组于电针干预前30 min腹腔注射AMPK抑制剂化合物C（Compound C，25 mg/kg），每天1次。3组均每周连续干预5 d，干预4周。全自动生化分析仪检测大鼠血脂相关指标[血清胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）及丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）含量]；HE染色和油红O染色法观察大鼠肝脏组织形态；称重计算肝指数，ELISA法检测大鼠肝脏组织TC、TG含量及粪便TC、胆汁酸含量；Western blot法检测大鼠肝脏组织AMPK、mTOR蛋白磷酸化水平；免疫组化法检测大鼠肝脏组织SREBP-2、HMGCR和ACBG5/G8阳性表达。

结果：

造模后，模型组、AMPK激动剂组、电针组和电针+AMPK抑制剂组大鼠血清TC、TG和LDL-C含量均高于空白组（

0.01）；各组大鼠血清HDL-C含量比较，差异无统计学意义（

0.05）。干预后，与空白组比较，模型组大鼠血清TC、TG、LDL-C、ALT、AST含量，肝指数，肝脏组织TC、TG含量及粪便TC、胆汁酸含量均升高（

0.01）；HE和油红O染色可见肝细胞排列紊乱，细胞内有明显的大泡性脂滴，明显脂质蓄积；肝脏组织磷酸化腺苷酸活化蛋白激酶（p-AMPK）蛋白表达、p-AMPK/AMPK比值降低（

0.01），磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）蛋白表达、p-mTOR/mTOR比值升高（

0.01）；肝脏组织SREBP-2、HMGCR、ABCG5、ABCG8阳性表达升高（

0.01，

0.05）。与模型组比较，AMPK激动剂组和电针组大鼠血清TC、TG、LDL-C、ALT、AST含量，肝指数及肝脏组织TC、TG含量降低（

0.01），粪便TC、胆汁酸含量升高（

0.05，

0.01）；HE染色和油红O染色可见肝细胞排列较规则，脂质蓄积明显减轻；肝脏组织p-AMPK蛋白表达、p-AMPK/AMPK比值升高（

0.01），p-mTOR蛋白表达、p-mTOR/mTOR比值降低（

0.01）；肝脏组织SREBP-2、HMGCR阳性表达降低（

0.01），ABCG5、ABCG8阳性表达升高（

0.05，

0.01）。与电针组比较，电针+AMPK抑制剂组大鼠血清TC、TG、LDL-C、ALT、AST含量，肝指数及肝脏组织TC、TG含量升高（

0.05，

0.01），粪便TC、胆汁酸含量降低（

0.01）；肝脏脂质蓄积加重；肝脏组织p-AMPK蛋白表达、p-AMPK/AMPK比值降低（

0.01，

0.05），p-mTOR蛋白表达、p-mTOR/mTOR比值升高（

0.05，

0.01）；肝脏组织SREBP-2、HMGCR阳性表达升高（

0.01），ABCG5、ABCG8阳性表达降低（

0.01）。

结论：

电针“丰隆”穴可以改善HPL大鼠高血脂水平，其机制可能与调节AMPK/mTOR信号通路，抑制下游胆固醇合成相关分子SREBP-2、HMGCR表达及上调胆固醇外排分子ABCG5、ABCG8表达，从而减轻肝脏胆固醇堆积、增加胆固醇排泄有关。

Abstract

Objective

To observe the effects of electroacupuncture (EA) at "Fenglong" (ST 40) on the expression of adenosine 5ʹ-monophosphate-activated protein kinase (AMPK)

mammalian target of rapamycin (mTOR)

and the expression of the downstream molecules related to cholesterol metabolism i.e. sterol regulatory element binding protein-2 (SREBP-2)

recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR)

and adenosine triphosphate binding cassette transporter G5/G8(ABCG5/G8) in the rats with hyperlipidemia (HPL) so as to explore the possible mechanism of EA in the intervention of HPL.

Methods

Thirty SPF-grade male SD rats were randomly divided into a blank group

a model group

an AMPK agonist group

an EA group

and an EA+AMPK inhibitor group

6 rats in each group. The high-fat feeding method was adopted to establish HPL model. After successfully modeled

the rats in the EA group received EA intervention at bilateral "Fenglong" (ST 40)

with disperse-dense wave

in the frequency of 2 Hz/100 Hz

the intensity of 1 mA. EA was given once daily

for 30 min in one intervention. In the AMPK agonist group

the intraperitoneal injection with AMPK agonist A-769662 was administered

30 mg/kg

twice a day. In the EA+AMPK inhibitor group

the intraperitoneal injection of AMPK inhibitor Compound C was administered

25 mg/kg

once a day

30 min before EA intervention. In the intervention groups

the interventions were delivered continuously for 5 days a week and lasted 4 weeks. Using automated biochemical analyzer

the blood lipid-related indexes (serum total cholesterol [TC]

triglycerides [TG]

low-density lipoprotein cholesterol [LDL-C] and high-density lipoprotein cholesterol [HDL-C] as well as alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were detected in the rats. HE staining and oil red O staining were used to observe the morphology of liver tissue. Liver index was calculated by the weight. Using ELISA

the contents of TC and TG of liver tissue and the contents of of TC and bile acid in feces were detected. The protein phosphorylation levels of AMPK and mTOR in the liver tissue were detected using Western blot; and the positive expression of SREBP-2

HMGCR and ACBG5/G8 was detected using immunohistochemical staining.

Results

After modeling

the levels of serum TC

TG and LDL-C of rats in the model group

the AMPK agonist group

the EA group and the EA+AMPK inhibitor group were all higher than those in the blank group (

0.01); and there was no statistically difference in the levels of serum HDL-C among groups (

0.05). After intervention

compared with the blank group

in the model group

the levels of serum TC

LDL-C

ALT and AST

the liver index

the levels of TC and TG in liver tissue

the levels of TC and the bile acid in feces were increased (

0.01); HE and oil red O staining showed that the hepatocytes were disordered

and there were macrovesicular lipid droplets in the cells and the obvious lipid accumulation; the protein expression of phosphorylated AMPK (p-AMPK) in liver tissue and the ratio of p-AMPK and AMPK were reduced (

0.01)

he protein expression of phosphorylated mTOR (p-mTOR) and the ratio of p-mTOR and mTOR were elevated (

0.01); and the positive expression of SREBP-2

HMGCR

ABCG5 and ABCG8 in liver tissue was increased (

0.01

0.05). Compared with the model group

in the AMPK agonist group and the EA group

the levels of serum TC

LDL-C

ALT and AST

liver indexes

the levels of TC and TG in liver tissue were reduced (

0.01)

while the levels of TC and bile acid in feces were increased (

0.05

0.01); HE staining and oil red O staining showed that the hepatocytes were in order

and lipid accumulation; the protein expression of p-AMPK and the ratio of p-AMPK and AMPK in liver tissue increased (

0.01)

while the protein expression of p-mTOR and the ratio of p-mTOR and mTOR decreased (

0.01); the positive expression of SREBP-2 and HMGCR in liver tissue was reduced (

0.01)

while that of ABCG5 and ABCG8 up-regulated (

0.05

0.01) . Compared with the EA group

in the EA+AMPK inhibitor group

the levels of serum TC

LDL-C

ALT and AST

liver index

the levels of TC and TG in liver tissue were increased (

0.05

0.01)

while the levels of TC and bile acid in feces were reduced (

0.01); lipid accumulation was aggravated; the protein expression of p-AMPK and the ratio of p-AMPK and AMPK in liver tissue were reduced (

0.01

0.05)

while the protein expression of p-mTOR and the ratio of p-mTOR and mTOR elevated (

0.05

0.01); the positive expression of SREBP-2 and HMGCR in liver tissue was increased (

0.01)

while that of ABCG5 and ABCG8 was down-regulated (

0.01).

Conclusion

EA at "Fenglong" (ST 40) can attenuate hyperlipidemia in HPL rats. It may be achieved by regulating the AMPK/mTOR pathway

inhibiting the expression of cholesterol synthesis related molecules

SREBP-2 and HMGCR

and up-regulating the expression of cholesterol excretion molecules

ABCG5 and ABCG8

thereby reducing liver cholesterol accumulation and increasing cholesterol excretion.

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