香砂六君子汤调控CircRNA-0067835对脾虚高脂血症大鼠胆固醇外排的影响

孟嘉伟; 宋囡; 王莹; 陈丝; 王群; 赵秋宇; 贾连群

doi:10.14148/j.issn.1672-0482.2022.0147

香砂六君子汤调控CircRNA-0067835对脾虚高脂血症大鼠胆固醇外排的影响

doi: 10.14148/j.issn.1672-0482.2022.0147

辽宁中医药大学中医脏象理论及应用教育部重点实验室, 辽宁沈阳 110847

基金项目:

国家自然科学基金面上项目 82074145

辽宁省“兴辽英才计划”项目 XLYC1902100

辽宁省科学技术计划项目 2019-ZD-0436

详细信息

作者简介:
孟嘉伟, 女, 硕士研究生, E-mail: 458973701@qq.com

通讯作者:
贾连群, 女, 教授, 博士生导师, 主要从事中西医结合防治心血管疾病研究, E-mail: jlq-8@163.com

中图分类号: R285.5
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- 被引次数: 0
出版历程
- 收稿日期: 2021-08-23
- 网络出版日期: 2022-03-01
- 发布日期: 2022-02-10

Effect of Xiangsha Liujunzi Decoction Regulating CircRNA-0067835 on Cholesterol Efflux in Hyperlipidemia Rats with Spleen Deficiency

Ministry of Education Key Laboratory of Visceral Phenomenon Theory and Application of Traditional Chinese Medicine, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China

摘要

摘要: 目的探讨香砂六君子汤对脾虚高脂血症大鼠脂质沉积的影响及分子生物学机制。方法 30只SD大鼠随机分成空白对照组、高脂组、脾虚高脂组、香砂六君子汤高剂量组及香砂六君子汤正常剂量组。使用不节饮食加游泳直至力竭的复合方法15 d, 建立脾虚模型。随后饲喂高脂饲料10周, 检测大鼠血脂水平, 确定高脂血症模型复制成功后, 香砂六君子汤高剂量组及正常剂量组分别给予香砂六君子汤11.34、5.67 g·kg^-1·d^-1灌胃, 其余3组给予相应体积的生理盐水灌胃, 4周后取材。检测5组大鼠血脂水平。HE染色观察肝脏病理变化。qPCR法检测CircRNA-0067835、miR-155基因水平。qPCR及Western blot法检测PPARγ、LXR、ABCA1、ABCG1 mRNA及蛋白表达水平。结果与空白对照组比较, 高脂组和脾虚高脂组HE染色可见明显肿胀的肝脏细胞以及清晰的脂肪空泡, 血清TC、TG、LDL-C水平以及miR-155基因表达升高(P < 0.01), HDL-C水平, CircRNA-0067835基因表达，PPARγ、LXR、ABCA1、ABCG1 mRNA和蛋白表达均降低(P < 0.05，P < 0.01)。相较于脾虚高脂组, 香砂六君子汤高剂量组及正常剂量组TC、TG和LDL-C水平显著降低(P < 0.01), HDL-C水平显著升高(P < 0.01), 肿胀的肝脏细胞得到明显改善、脂肪空泡减少, miR-155表达降低(P < 0.01), CircRNA-0067835表达升高(P < 0.01), PPARγ、LXR、ABCA1、ABCG1 mRNA及蛋白表达均升高(P < 0.05，P < 0.01)。香砂六君子汤高剂量组与正常剂量组之间部分存在统计学意义(P < 0.05, P < 0.01)。结论香砂六君子汤可能通过影响CircRNA-0067835调节PPARγ介导的胆固醇外排过程改善高脂血症大鼠胆固醇代谢异常, 进而起到防治血脂异常的作用。
- 高脂血症 /
- 脾虚 /
- 香砂六君子汤 /
- 胆固醇代谢 /
- CircRNA-0067835 /
- miR-155 /
- PPARγ
Abstract: OBJECTIVE To investigate the effect of Xiangsha Liujunzi Decoction on lipid deposition in rats with spleen deficiency and hyperlipidemia and its molecular biological mechanism. METHODS Thirty SD rats were randomly divided into 5 groups: blank control group, high-fat group, spleen-deficiency and high-fat group, high-dose Xiangsha Liujunzi Decoction group, and normal-dose Xiangsha Liujunzi Decoction group. A compound method of unregulated diet and swimming until exhaustion was used for 15 days to establish a model of spleen deficiency. After feeding the high-fat diet for 10 weeks, the blood lipid levels of rats were tested to confirm that the hyperlipidemia model was successfully replicated. Xiangsha Liujunzi Decoction high-dose group and normal-dose group were given Xiangsha Liujunzi Decoction of 11.34, 5.67 g·kg^-1·d^-1 intragastrically, the other 3 groups were given corresponding volume of normal saline intragastrically, and the samples were taken 4 weeks later. The blood lipid levels of the 5 groups of rats were detected. HE staining was used to observe the pathological changes of the liver. qPCR method was used to detect CircRNA-0067835 and miR-155 gene levels. The expression levels of PPARγ, LXR, ABCA1, ABCG1 mRNA and protein were detected by qPCR and Western blot. RESULTS Compared with the blank control group, HE staining of high-fat group and spleen-deficiency and high-fat group showed obvious swollen liver cells and clear fatty vacuoles. The serum contents of TC, TG, LDL-C and the expression of miR-155 increased (P < 0.01), HDL-C level, the expression of CircRNA-0067835, the mRNA and protein expressions of PPARγ, LXR, ABCA1, ABCG1 all decreased (P < 0.05, P < 0.01). Compared with the spleen-deficiency and high-fat group, the TC, TG and LDL-C levels of Xiangsha Liujunzi Decoction high-dose group and normal-dose group significantly reduced (P < 0.01), and the HDL-C level significantly increased (P < 0.01), swollen liver cells were significantly improved, fatty vacuoles reduced, miR-155 expression decreased (P < 0.01), CircRNA-0067835 expression increased (P < 0.01), PPARγ, LXR, ABCA1, ABCG1 mRNA and protein expressions all increased (P < 0.05, P < 0.01). There was statistical significance between Xiangsha Liujunzi Decoction high-dose group and the normal-dose group (P < 0.05, P < 0.01). CONCLUSION Xiangsha Liujunzi Decoction may improve cholesterol metabolism in hyperlipidemia rats by influencing CircRNA-0067835 to regulate the cholesterol efflux process mediated by PPARγ, thereby preventing and treating dyslipidemia.
- hyperlipidemia /
- spleen deficiency /
- Xiangsha Liujunzi Decoction /
- cholesterol metabolism /
- CircRNA-0067835 /
- miR-155 /
- PPARγ

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图 1 各组大鼠肝脏组织病理形态比较(HE, ×200)

Figure 1. Comparison of pathological morphology of liver tissues in each group(HE, ×200)

下载: 全尺寸图片幻灯片

图 2 各组大鼠肝脏组织脂质沉积情况比较(油红O, ×200)

Figure 2. Comparison of lipid deposition of liver tissues in each group (Oil red O, ×200)

下载: 全尺寸图片幻灯片

图 3 各组大鼠肝脏CircRNA-0067835、miR-155与PPARγ、LXR、ABCA1、ABCG1 mRNA表达的比较

注: 与空白对照组比较, ^**P < 0.01;与脾虚高脂组比较, ^▲▲P < 0.01;与香砂六君子汤正常剂量组比较, ^△△P < 0.01。x±s, n=3。

Figure 3. The expressions of CircRNA-0067835, miR-155 and PPARγ, LXR, ABCA1, ABCG1 mRNA in each group

下载: 全尺寸图片幻灯片

图 4 各组大鼠肝脏PPARγ、LXR、ABCA1、ABCG1蛋白表达的比较

与空白对照组比较，^*P < 0.05, ^**P < 0.01;与脾虚高脂组比较，^▲▲P < 0.01。x±s, n=3。

Figure 4. The expressions of PPARγ, LXR, ABCA1, ABCG1 in each group

下载: 全尺寸图片幻灯片

表 1 qPCR引物序列

Table 1. The qPCR primer sequence

基因	引物序列	长度/ bp
CircRNA-	上游: GTTTCATACTTTACGCTTGT	734
0067835	下游: GCCTGGGTTTGTTTCT
PPARγ	上游: ACCACTCGCATTCCTTTGAC	156
	下游: TGATCGCACTTTGGTATTCTTG
LXR	上游: AGACCAGGGAGGCAACACTT	157
	下游: CTTTTGTGGACGAAGCTCTG
ABCA1	上游: GAGCAAAGCCAAGCATCTTC	172
	下游: TAGAACGGGCAGGTTGGTAG
ABCG1	上游: CTGTGCGTTTTGTGCTGTTC	185
	下游: GTAGGCTGGGATGGTGTCAA
GAPDH	上游GTGTTTCCTCGTCCCGTAGA	202
	下游CCTTGACTGTGCCGTTGAAT
miR-155	GGCCTCCTACCTGTTAGCATTAA	-
U6	上游: CTCGCTTCGGCAGCACATATACT	-
	下游: ACGCTTCACGAATTTGCGTGTC

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表 2 各组大鼠血脂水平比较(x±s, mmol·L^-1, n=6)

Table 2. The comparison of serum lipid levels in each group(x±s, mmol·L^-1, n=6)

	TC	TG	LDL-C	HDL-C
空白对照组	1.28±0.02	0.41±0.02	0.19±0.2	1.24±0.03
高脂组	1.65±0.02^**	0.65±0.02^**	0.33±0.03^**	0.93±0.03^**
脾虚高脂组	1.75±0.03^**	0.73±0.02^**	0.38±0.02^**	0.80±0.06^**
香砂六君子汤高剂量组	1.48±0.04^▲▲△△	0.51±0.02^▲▲	0.23±0.02^▲▲△	1.13±0.03^▲▲△
香砂六君子汤正常剂量组	1.57±0.02^▲▲	0.52±0.01^▲▲	0.27±0.02^▲▲	1.05±0.03^▲▲
注: 与空白对照组比较, ^**P < 0.01;与脾虚高脂组比较, ^▲▲P < 0.01;与香砂六君子汤正常剂量组比较, ^△P < 0.05, ^△△P < 0.01。

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