当归补血汤通过miR-27a/TGF-β1/Smad3通路抑制HK-2细胞纤维化作用及机制研究

赵烨; 葛凡; 李子航; 朱景天; 薛梅

doi:10.14148/j.issn.1672-0482.2022.0592

当归补血汤通过miR-27a/TGF-β1/Smad3通路抑制HK-2细胞纤维化作用及机制研究

doi: 10.14148/j.issn.1672-0482.2022.0592

赵烨^1,,
葛凡²,
李子航²,
朱景天²,
薛梅^2, ,

1.
南京中医药大学药学院, 江苏南京 210023
2.
南京中医药大学中医学院·中西医结合学院, 江苏南京 210023

基金项目:

国家自然科学基金青年科学基金项目 81904085

江苏省自然科学基金面上项目 BK20191412

详细信息

作者简介:
赵烨, 女, 硕士研究生, E-mail: 547583769@qq.com

通讯作者:
薛梅, 女, 副教授, 主要从事中药方剂治疗糖尿病及其并发症相关研究, E-mail: 290427@njucm.edu.cn

中图分类号: R285.5
计量
- 文章访问数: 330
- HTML全文浏览量: 71
- PDF下载量: 34
- 被引次数: 0
出版历程
- 收稿日期: 2022-01-19
- 网络出版日期: 2022-07-09
- 发布日期: 2022-07-10

Study on the Effect and Mechanism of Danggui Buxue Decoction Inhibiting HK-2 Cell Fibrosis through miR-27a/TGF-β1/Smad3 Pathway

ZHAO Ye^1
,,
GE Fan²,
LI Zi-hang²,
ZHU Jing-tian²,
XUE Mei^{2
, ,}

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的探讨当归补血汤通过miR-27a调控HK-2细胞纤维化的机制。方法 HK-2细胞分为对照组、高糖组、高糖+空白血清组、高糖+当归补血汤含药血清组。采用MTT法检测各组细胞增殖情况; 采用Western blot法检测纤维化相关因子波形蛋白(Vimentin)、Ⅳ型胶原(COL Ⅳ)、α-平滑肌肌动蛋白(α-SMA)、p-Smad3/Smad3和转化生长因子(TGF)-β1蛋白的表达情况; qPCR法检测miR-27a及纤维化相关因子mRNA的表达情况; 通过转染miR-27a抑制剂及Smad3-siRNA并向培养基中加入TGF-β1, 观察miR-27a的表达和纤维化相关蛋白表达的情况。结果 MTT结果显示, 与对照组相比, 高糖组细胞增殖率明显降低(P < 0.05, P < 0.001), 当归补血汤含药血清作用24、48 h后细胞增殖率明显提高(P < 0.05, P < 0.001)。Western blot结果显示, 当归补血汤含药血清明显抑制高糖诱导的HK-2细胞纤维化相关蛋白COL Ⅳ、α-SMA、Vimentin、TGF-β1、p-Smad3/Smad3的表达(P < 0.05, P < 0.01)。qPCR结果显示, 当归补血汤含药血清明显抑制高糖诱导的HK-2细胞COL Ⅳ、α-SMA、Vimentin、TGF-β1 mRNA和miR-27a的表达(P < 0.05, P < 0.01)。TGF-β1处理细胞后, 细胞纤维化加重, miR-27a的表达升高(P < 0.05, P < 0.01), 当归补血汤含药血清可明显降低TGF-β1诱导的纤维化细胞中miR-27a的表达升高(P < 0.001)。转染miR-27a抑制剂及Smad3-siRNA可明显降低TGF-β1诱导的细胞纤维化蛋白COL Ⅳ、α-SMA、Vimentin、TGF-β1、p-Smad3/Smad3表达的升高(P < 0.05, P < 0.01)。结论当归补血汤含药血清可促进HK-2细胞增殖, 并通过miR-27a/TGF-β1/Smad3信号通路调控HK-2细胞纤维化。
- 当归补血汤 /
- HK-2 /
- miR-27a /
- TGF-β1/Smad3 /
- 纤维化
Abstract: OBJECTIVE To investigate the mechanism of Danggui Buxue Decoction regulating HK-2 cell fibrosis through miR-27a. METHODS HK-2 cells were divided into control group, high glucose group, high glucose+blank serum group, and high glucose+Danggui Buxue Decoction-containing serum group. The proliferation of cells in each group was detected by MTT assay; The expression of the fibrosis-related factors Vimentin, COL Ⅳ, α-SMA, p-Smad3/Smad3 and TGF-β1 protein was measured by Western blot; The qPCR was used to detect the expression of miR-27a and fibrosis-related factors mRNA; The expression of miR-27a and fibrosis-related proteins were observed by transfecting miR-27a inhibitor and Smad3-siRNA, as well as adding TGF-β1 to the culture medium. RESULTS MTT results showed that compared with the control group, the cell proliferation rate in the high glucose group significantly decreased (P < 0.05, P < 0.001), and Danggui Buxue Decoction significantly promoted cell proliferation (P < 0.05, P < 0.001). Western blot showed that Danggui Buxue Decoction significantly inhibited the expression of fibrosis-related proteins COL Ⅳ, α-SMA, Vimentin, TGF-β1 and p-Smad3/Smad3 in HK-2 cells induced by high glucose (P < 0.05, P < 0.01). The qPCR results were consistent with Western blot results. After TGF-β1 treatment of cells, cell fibrosis aggravated, and the expression of miR-27a increased (P < 0.05, P < 0.01), Danggui Buxue Decoction significantly reduced the elevation of miR-27a in TGF-β1-induced cellular fibrosis (P < 0.001). Transfection of miR-27a inhibitor and Smad3-siRNA could significantly reduce the increase of TGF-β1-induced cellular fibrosis protein expression (P < 0.05, P < 0.01). CONCLUSION Danggui Buxue Decoction-containing serum can promote the proliferation of HK-2 cells and regulate HK-2 cell fibrosis through miR-27a/TGF-β1/Smad3 signaling pathway.
- Danggui Buxue Decoction /
- HK-2 /
- miR-27a /
- TGF-β1/Smad3 /
- fibrosis

HTML全文

图 1 当归补血汤含药血清处理HK-2细胞48 h纤维化相关蛋白表达的条带

注: A.对照组；B.高糖组；C.高糖+空白血清组；D.高糖+含药血清组

Figure 1. Electrophoresis bands of fibrosis-related proteins in HK-2 cells treated with DBD containing serum for 48 h

下载: 全尺寸图片幻灯片

图 2 转染Smad3-siRNA及miR-27a抑制剂对TGF-β1处理后HK-2细胞纤维化相关蛋白表达的影响

注: anti-miR-27a.miR-27a抑制剂组; miR-Ctrl.miR-27a抑制剂阴性对照组; Smad3-siRNA.Smad3-siRNA组; Con-siRNA.Smad3-siRNA阴性对照组。组间比较, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001。

Figure 2. Transfection of Smad3-siRNA and miR-27a inhibitor on the expression of fibrosis-related proteins in HK-2 cells after TGF-β1 treatment

下载: 全尺寸图片幻灯片

表 1 qPCR引物序列

Table 1. Primer sequences for qPCR

基因	正向引物	反向引物
TGF-β1	5'-GCGTCTAATGGTGGAAACC-3'	5'-GAGCAACACGGGTTCAGGTA-3'
Vimentin	5'-CAGCATGGACGTTCGTCTG-3'	5'-AACCACGGTITGGTCCTTGG-3'
α-SMA	5'-GTGTGTGTGTCTTGGTAATGG-3'	5'-CCTTGTGTCCTGATCGTTG-3'
COL Ⅳ	5'-GAAGACATCCCACCAATCAC-3'	5'-CACGTCATCGCACAACAC-3'
β-actin	5'-GGGACCTGACTGACTACCTC-3'	5'-TCATACTCCTGCTTGCTGAT-3'
miR-27a	5'-GCGCGTTCACAGTGGCTAAG-3'	5'-AGTGCAGGGTCCGAGGTATT-3'
U6	5'-CTCGCTTCGGCAGCACA-3'	5'-AACGCTTCACGAATTTGCGT-3'

下载: 导出CSV

表 2 当归补血汤含药血清对HK-2细胞增殖率的影响(x±s, %, n=6)

Table 2. Effect of Danggui Buxue Decoction (DBD) containing serum on HK-2 proliferative rate (x±s, %, n=6)

组别	12 h	24 h	48 h
对照组	100.00±21.72	100.00±18.10	100.00±13.19
高糖组	68.99±19.31^*	48.16±18.84^***	52.84±16.66^***
高糖+空白血清组	72.75±14.66^*	62.72±16.42^**	58.57±8.56^***
高糖+含药血清组	85.40±19.99	81.09±15.55^#	91.89±15.48^###
注: 与对照组比较，^P < 0.05, ^P < 0.01, ^**P < 0.001;与高糖+空白血清组比较，^#P < 0.05, ^###P < 0.001。

下载: 导出CSV

表 3 当归补血汤对HK-2细胞纤维化相关蛋白表达的影响(x±s, n=3)

Table 3. Effect of DBD containing serum on the expression of fibrosis-related proteins in HK-2 cells (x±s, n=3)

组别	COL Ⅳ/β-actin	Vimentin/β-actin	α-SMA/β-actin	TGF-β1/β-actin	p-Smad3/Smad3
对照组	1.00±0.06	1.00±0.17	1.00±0.14	1.00±0.09	1.00±0.09
高糖组	2.17±0.17^***	1.99±0.13^**	1.90±0.13^**	2.55±0.08^***	1.59±0.21^*
高糖+空白血清组	1.98±0.14^**	2.30±0.25^**	2.02±0.11^***	2.48±0.09^***	1.58±0.21^*
高糖+含药血清组	1.07±0.19^##	1.50±0.18^#	1.56±0.10^##	1.09±0.28^##	1.21±0.08^#
注: 与对照组比较, ^P < 0.05, ^P < 0.01, ^**P < 0.001;与高糖+空白血清组比较, ^#P < 0.05, ^##P < 0.01。

下载: 导出CSV

表 4 当归补血汤对HK-2细胞纤维化相关mRNA表达的影响(x±s, n=3)

Table 4. Effect of DBD containing serum on the expression of fibrosis-related mRNA in HK-2 cells (x±s, n=3)

组别	COL Ⅳ/β-actin	Vimentin/β-actin	α-SMA/β-actin	TGF-β1/β-actin	miR-27a/U6
对照组	1.00±0.13	1.00±0.10	1.00±0.14	1.00±0.06	1.00±0.10
高糖组	1.94±0.40^*	1.30±0.06^*	1.48±0.12^*	1.58±0.07^***	1.47±0.13^**
高糖+空白血清组	1.83±0.15^**	1.41±0.07^**	1.63±0.21^*	1.80±0.40^*	2.10±0.19^***
高糖+含药血清组	1.44±0.20^##	1.21±0.07^##	1.15±0.14^#	1.09±0.22^#	1.16±0.22^##
注: 与对照组比较，^P < 0.05, ^P < 0.01, ^**P < 0.001;与高糖+空白血清组比较，^#P < 0.05, ^##P < 0.01。

下载: 导出CSV

表 5 TGF-β1处理HK-2细胞对miR-27a表达的影响(x±s, n=3)

Table 5. The effect of TGF-β1 treatment on the expression of miR-27a in HK-2 cells (x±s, n=3)

组别	miR-27a/U6
对照组	1.00±0.04
5 ng·mL^-1 TGF-β1组	2.23±0.69^*
10 ng·mL^-1 TGF-β1组	4.09±0.63^**
注: 与对照组比较, ^P < 0.05, ^*P < 0.01。

下载: 导出CSV

表 6 TGF-β1及当归补血汤含药血清处理HK-2细胞对miR-27a表达的影响(x±s, n=3)

Table 6. The effect of TGF-β1 and DBD containing serum on the expression of miR-27a in HK-2 cells (x±s, n=3)

组别	miR-27a/U6
对照组	1.00±0.01
当归补血汤组	0.33±0.08^***
注: 与对照组比较, ^***P < 0.001。

下载: 导出CSV

参考文献(26)

[1]	AVOGARO A, FADINI GP. Microvascular complications in diabetes: A growing concern for cardiologists[J]. Int J Cardiol, 2019, 291: 29-35. doi: 10.1016/j.ijcard.2019.02.030
[2]	YANG SF, ABDULLA R, LU C, et al. Inhibition of microRNA-376b protects against renal interstitial fibrosis via inducing macrophage autophagy by upregulating Atg5 in mice with chronic kidney disease[J]. Kidney Blood Press Res, 2018, 43(6): 1749-1764. doi: 10.1159/000495394
[3]	TANG FJ, HAO YR, ZHANG X, et al. Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy[J]. Drug Des Devel Ther, 2017, 11: 2813-2826. doi: 10.2147/DDDT.S143805
[4]	WU LN, WANG QZ, GUO F, et al. Involvement of miR-27a-3p in diabetic nephropathy via affecting renal fibrosis, mitochondrial dysfunction, and endoplasmic Reticulum stress[J]. J Cell Physiol, 2021, 236(2): 1454-1468. doi: 10.1002/jcp.29951
[5]	HOU XY, TIAN JW, GENG J, et al. microRNA-27a promotes renal tubulointerstitial fibrosis via suppressing PPARγ pathway in diabetic nephropathy[J]. Oncotarget, 2016, 7(30): 47760-47776. doi: 10.18632/oncotarget.10283
[6]	SHI XQ, YUE SJ, TANG YP, et al. A network pharmacology approach to investigate the blood enriching mechanism of Danggui buxue Decoction[J]. J Ethnopharmacol, 2019, 235:227-242.
[7]	ZHANG R, HAN X, HUANG T, et al. Danggui Buxue Tang inhibited mesangial cell proliferation and extracellular matrix accumulation through GAS5/NF-κB pathway[J]. Biosci Rep, 2019, 39(10): BSR20181740. doi: 10.1042/BSR20181740
[8]	WANG WK, ZHOU Y, FAN L, et al. The antidepressant-like effects of Danggui Buxue Decoction in GK rats by activating CREB/BDNF/TrkB signaling pathway[J]. Phytomedicine, 2021, 89: 153600. doi: 10.1016/j.phymed.2021.153600
[9]	GAO DH, GUO YJ, LI XJ, et al. An aqueous extract of Radix astragali, Angelica sinensis, and Panax notoginseng is effective in preventing diabetic retinopathy[J]. Evid Based Complement Alternat Med, 2013, 2013: 578165.
[10]	薛梅, 卞勇, 周俊杰, 等. 当归补血汤主要吸收成分对GK大鼠肾保护作用研究[J]. 南京中医药大学学报, 2018, 34(2): 190-193. http://xb.njucm.edu.cn/article/id/ZR2018_0219 XUE M, BIAN Y, ZHOU JJ, et al. Renal protective effect of absorbed bioactive compounds of Danggui buxue decoction on GK rats[J]. J Nanjing Univ Tradit Chin Med, 2018, 34(2): 190-193. http://xb.njucm.edu.cn/article/id/ZR2018_0219
[11]	CALLE P, HOTTER G. Macrophage phenotype and fibrosis in diabetic nephropathy[J]. Int J Mol Sci, 2020, 21(8): 2806. doi: 10.3390/ijms21082806
[12]	ZENG LF, XIAO Y, SUN L. A glimpse of the mechanisms related to renal fibrosis in diabetic nephropathy[J]. Adv Exp Med Biol, 2019, 1165: 49-79.
[13]	BROSIUS FC 3rd, ALPERS CE, BOTTINGER EP, et al. Mouse models of diabetic nephropathy[J]. J Am Soc Nephrol, 2009, 20(12), 2503-2512. doi: 10.1681/ASN.2009070721
[14]	WANG JP, FANG CY, WANG SX, et al. Danggui Buxue Tang ameliorates bleomycin-induced pulmonary fibrosis in rats through inhibiting transforming growth factor-β1/Smad3/plasminogen activator inhibitor-1 signaling pathway[J]. J Tradit Chin Med, 2020, 40(2): 236-244.
[15]	CHEN Y, CHEN Q, LU J, et al. Effects of Danggui Buxue Decoction on lipid peroxidation and MMP-2/9 activities of fibrotic liver in rats[J]. Chin J Integr Med, 2009, 15(6): 435-441. doi: 10.1007/s11655-009-0435-y
[16]	WANG LN, MA JW, GUO CX, et al. Danggui Buxue Tang attenuates tubulointerstitial fibrosis via suppressing NLRP3 inflammasome in a rat model of unilateral ureteral obstruction[J]. Biomed Res Int, 2016, 2016: 9368483.
[17]	WANG JY, GAO YB, MA MF, et al. Effect of miR-21 on renal fibrosis by regulating MMP-9 and TIMP1 in kk-ay diabetic nephropathy mice[J]. Cell Biochem Biophys, 2013, 67(2): 537-546. doi: 10.1007/s12013-013-9539-2
[18]	LIU HF, WANG XH, LIU SF, et al. Effects and mechanism of miR-23b on glucose-mediated epithelial-to-mesenchymal transition in diabetic nephropathy[J]. Int J Biochem Cell Biol, 2016, 70: 149-160. doi: 10.1016/j.biocel.2015.11.016
[19]	WU LN, WANG QZ, GUO F, et al. microRNA-27a induces mesangial cell injury by targeting of PPARγ and its in vivo knockdown prevents progression of diabetic nephropathy[J]. Sci Rep, 2016, 6: 26072. doi: 10.1038/srep26072
[20]	LAN HY. Diverse roles of TGF-β/Smads in renal fibrosis and inflammation[J]. Int J Biol Sci, 2011, 7(7): 1056-1067. doi: 10.7150/ijbs.7.1056
[21]	侯晓艳. microRNA-27a在糖尿病肾病肾小管间质纤维化中的作用及机制研究[D]. 广州: 南方医科大学, 2017. HOU XY. A study of the mechanism and effect of microRNA-27a on renal tubulointerstitial fibrosis in diabetic nephropathy[D]. Guangzhou: Southern Medical University, 2017.
[22]	KRUPA A, JENKINS R, LUO DD, et al. Loss of microRNA-192 promotes fibrogenesis in diabetic nephropathy[J]. J Am Soc Nephrol, 2010, 21(3): 438-447. doi: 10.1681/ASN.2009050530
[23]	ZHENG ZJ, GUAN MP, JIA YJ, et al. The coordinated roles of miR-26a and miR-30c in regulating TGFβ1-induced epithelial-to-mesenchymal transition in diabetic nephropathy[J]. Sci Rep, 2016, 6: 37492. doi: 10.1038/srep37492
[24]	SATO M, MURAGAKI Y, SAIKA S, et al. Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction[J]. J Clin Invest, 2003, 112(10): 1486-1494. doi: 10.1172/JCI200319270
[25]	KELLENBERGER T, KRAG S, DANIELSEN CC, et al. Differential effects of Smad3 targeting in a murine model of chronic kidney disease[J]. Physiol Rep, 2013, 1(7): e00181. doi: 10.1002/phy2.181
[26]	ZHONG X, CHUNG ACK, CHEN HY, et al. Smad3-mediated upregulation of miR-21 promotes renal fibrosis[J]. J Am Soc Nephrol, 2011, 22(9): 1668-1681. doi: 10.1681/ASN.2010111168