马尾松针提取物调控Nrf2-ARE通路治疗雄激素性脱发的研究

朱红柳; 魏跃钢; 闵仲生; 高以红; 羊剑秋

doi:10.14148/j.issn.1672-0482.2022.0129

马尾松针提取物调控Nrf2-ARE通路治疗雄激素性脱发的研究

doi: 10.14148/j.issn.1672-0482.2022.0129

1.
南京中医药大学江阴附属医院, 江苏江阴 214400
2.
南京中医药大学附属医院, 江苏南京 210029

基金项目:

国家自然科学基金青年科学基金项目 81804100

详细信息

通讯作者:
朱红柳, 女, 副主任中医师, 博士研究生, 主要从事雄激素性脱发研究, E-mail: 17768318386@163.com

中图分类号: R285.5
计量
- 文章访问数: 241
- HTML全文浏览量: 101
- PDF下载量: 21
- 被引次数: 0
出版历程
- 收稿日期: 2021-03-16
- 网络出版日期: 2022-03-01
- 发布日期: 2022-02-10

Effects of Pine Massoniana Needle Extract on Androgenetic Alopecia Model Rats via Nrf2-ARE Mediated Signaling Pathway

1.
Jiangyin Hospital Affiliated to Nanjing University of Chinese Medicine, Jiangyin 214400, China
2.
The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China

摘要

摘要: 目的观察马尾松针提取物对双氢睾酮(Dihydrotestosterone, DHT)诱导的雄激素性脱发(Androgenetic alopecia, AGA)模型小鼠的干预效应, 并通过核因子E2相关因子2(Nrf2)-抗氧化反应元件(ARE)信号通路探讨马尾松针提取物促毛发生长的作用机制。方法采用DHT皮下注射建立AGA小鼠模型, 48只雄性C57BL/6小鼠随机分为空白组, DHT组, 原花青素组, 马尾松针提取物(松针)低、中、高剂量组, 每组8只。空白组不予任何处理, DHT组仅皮下注射DHT 30 mg·d^-1, 原花青素组皮下注射DHT并灌服原花青素B2 5 mg·kg^-1, 松针低、中、高剂量组皮下注射DHT, 同时分别灌服马尾松针提取物4、8、12 mg·kg^-1。每组均给药4周, 每周拍照并记录毛发生长情况。d28后取其背部圆形皮片, 刮取毛发称质量, 行HE染色。计算生长期、休止期毛囊数量并计算二者比值。取部分皮肤组织, 检测ROS和MDA含量。qPCR和Western blot法检测Nrf2、Keap1、NQO1、HO-1、TGF-β1 mRNA与蛋白的表达水平。结果马尾松针提取物呈剂量依赖性地促进AGA小鼠的毛发生长, 松针高剂量组与原花青素组毛发质量以及生长期/休止期毛囊比值均无显著性差异。马尾松针a提取物能够降低组织内ROS和MDA含量, 呈剂量依赖性地促进Nrf2、NQO1、HO-1 mRNA与蛋白的表达, 抑制Keap1、TGF-β1 mRNA与蛋白的表达。结论马尾松针提取物可能通过激活Nrf2-ARE信号通路, 改善氧化应激水平, 从而促进AGA小鼠毛发的生长。
- 马尾松针提取物 /
- 雄激素性脱发 /
- 氧化应激 /
- Nrf2-ARE通路
Abstract: OBJECTIVE To investigate the hair growth-promoting activity and the possible mechanism of pine massoniana needle extract (PMNE) on mice by using the dihydrotestosterone (DHT) induced androgenetic alopecia (AGA) model. METHODS 48 C57BL/6 mice were randomly divided into 6 groups: control group without any treatment, DHT group (30 mg ·d^-1 DHT only), procyanidin group (DHT+procyanidin B2 5 mg ·kg^-1), PMNE low dose group (DHT+PMNE 4 mg ·kg^-1), PMNE medium dose group (DHT+PMNE 8 mg ·kg^-1) and PMNE high dose group (DHT+PMNE 12 mg ·kg^-1). PMNE and procyanidin B2 were given orally every day. The hair growth of mice was observed and evaluated at d7, d14, d21 and d28 after administration. The circle skins of all mice were harvested at the same depilated aera after 28 days. The hair from these skins was weighed. Some skin tissue section was obtained for HE staining, and the number of hair follicles were calculated. Some skin tissues were obtained to detect the contents of ROS and MDA and the mRNA and protein levels of Keap1, Nrf2, NQO1, HO-1 and TGF-β1 by qPCR and Western blot. RESULTS PMNE promoted the hair growth of AGA mice and showed a dose-dependent effect. PMNE of 12 mg ·kg^-1 could alleviate the DHT-induced AGA in mice and achieve a similar effect to procyanidin B2. PMNE reduced the contents of ROS and MDA, promoted the mRNA and protein expressions of Nrf2, NQO1, HO-1 and reduced the mRNA and protein expressions of Keap1 and TGF-β1. CONCLUSIONS PMNE can promote the hair growth of AGA mice by regulating the Nrf2-ARE signaling pathway.
- pine massoniana needle extract /
- androgenetic alopecia /
- oxidative stress /
- Nrf2-ARE signaling pathway

HTML全文

图 1 各组小鼠毛发生长情况的比较

Figure 1. Comparison of hair growth in each group

下载: 全尺寸图片幻灯片

图 2 各组小鼠背部毛发生长情况评分比较

Figure 2. Comparison of hair growth scores in each group

下载: 全尺寸图片幻灯片

图 3 各组小鼠背部毛发质量比较

注: 与空白组比较, ^***P < 0.001;与DHT组比较, ^###P < 0.001。x±s, n=8。

Figure 3. Comparison of hair weight in each group

下载: 全尺寸图片幻灯片

图 4 各组小鼠背部圆形皮片HE染色(×100)

Figure 4. HE staining of the circle skin in each group (×100)

下载: 全尺寸图片幻灯片

图 5 流式细胞术检测各组小鼠背部皮肤组织中ROS含量

注: 与空白组比较, ^**P < 0.01;与DHT组比较, ^#P < 0.05。x±s, n=6。

Figure 5. Comparison of ROS content in each group by flow cytometry

下载: 全尺寸图片幻灯片

图 6 各组小鼠背部皮肤组织中MDA含量比较

注: 与空白组比较, ^**P < 0.01;与DHT组比较, ^##P < 0.01。x±s, n=6。

Figure 6. Comparison of MDA content in each group

下载: 全尺寸图片幻灯片

图 7 qPCR检测各组小鼠皮肤组织中Nrf2、NQO1、HO-1、Keap1及TGF-β1 mRNA的表达

注: 与空白组比较, ^*P < 0.05, ^**P < 0.01;与DHT组比较, ^#P < 0.05, ^##P < 0.01。x±s, n=6。

Figure 7. Expressions of Nrf2, NQO1, HO-1, Keap1, TGF-β1 mRNA in each group by qPCR

下载: 全尺寸图片幻灯片

图 8 Western blot检测各组小鼠皮肤组织中Nrf2、NQO1、HO-1、Keap1及TGF-β1蛋白表达

注: 与空白组比较, ^*P < 0.05;与DHT组比较, ^#P < 0.05, ^##P < 0.01。x±s, n=6。

Figure 8. Expressions of Nrf2, NQO1, HO-1, Keap1, TGF-β1 protein in each group by Western blot

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. The sequence of primer

基因	正向引物(5'→3')	反向引物(5'→3')	长度/bp
Nrf2	CTGAACTCCTGGACGGGACTA	CGGTGGGTCTCCGTAAATGG	20
Keap1	TCGAAGGCATCCACCCTAAG	CTCGAACCACGCTGTCAATCT	21
NQO1	TACGACAACGGTCCTTTCCAG	ACAGAAACGCAGGATGCCACT	21
HO-1	AAGCCGAGAATGCTGAGTTCA	GCCGTGTAGATATGGTACAAGGA	23
TGF-β1	CTCCCGTGGCTTCTAGTGC	GCCTTAGTTTGGACAGGATCTG	22
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT	22

下载: 导出CSV

表 2 各组小鼠背部皮肤毛囊数量比较(x±s, n=8)

Table 2. Comparison of the number of hair follicles in each group (x±s, n=8)

组别	生长期	休止期	生长期/休止期
空白组	74.13±31.00	9.63±2.39	7.25±2.81
DHT组	12.38±3.38^**	7.88±4.22	2.13±1.25^*
原花青素组	64.25±19.91^##	7.50±2.14	9.88±5.46^#
松针低剂量组	27.63±15.21	7.00±4.99	6.88±9.05
松针中剂量组	27.63±13.10	5.38±3.16^*	6.63±3.46
松针高剂量组	62.25±28.40^##	5.88±3.44^*	14.13±11.03^##
注: 与空白组比较, ^P < 0.05, ^*P < 0.01;与DHT组比较, ^#P < 0.05, ^##P < 0.01。

下载: 导出CSV

参考文献(13)

[1]	JAIN R, DE-EKNAMKUL W. Potential targets in the discovery of new hair growth promoters for androgenic alopecia[J]. Expert Opin Ther Targets, 2014, 18(7): 787-806. doi: 10.1517/14728222.2014.922956
[2]	WANG TL, ZHOU C, SHEN YW, et al. Prevalence of androgenetic alopecia in China: A community-based study in six cities[J]. Br J Dermatol, 2010, 162(4): 843-847. doi: 10.1111/j.1365-2133.2010.09640.x
[3]	HASLAM IS, JADKAUSKAITE L, SZABO IL, et al. Oxidative damage control in a human (mini-) organ: Nrf2 activation protects against oxidative stress-induced hair growth inhibition[J]. J Invest Dermatol, 2017, 137(2): 295-304. doi: 10.1016/j.jid.2016.08.035
[4]	UPTON JH, HANNEN RF, BAHTA AW, et al. Oxidative stress-associated senescence in dermal papilla cells of men with androgenetic alopecia[J]. J Invest Dermatol, 2015, 135(5): 1244-1252. doi: 10.1038/jid.2015.28
[5]	邱冰, 胡惠静. 松针在医药中的研究进展[J]. 中医药信息, 2017, 34(6): 130-133. doi: 10.3969/j.issn.1002-2406.2017.06.037 QIU BING, HU HJ. Medical research progress of pine needles[J]. Inform Tradit Chin Med, 2017, 34(6): 130-133. doi: 10.3969/j.issn.1002-2406.2017.06.037
[6]	WU DC, LI S, YANG DQ, et al. Effects of Pinus massoniana bark extract on the adhesion and migration capabilities of HeLa cells[J]. Fitoterapia, 2011, 82(8): 1202-1205. doi: 10.1016/j.fitote.2011.08.008
[7]	KAMIMURA A, TAKAHASHI T, MOROHASHI M, et al. Procyanidin oligomers counteract TGF-β₁- and TGF-β₂- induced apoptosis in hair epithelial cells: An insight into their mechanisms[J]. Skin Pharmacol Physiol, 2006, 19(5): 259-265. doi: 10.1159/000093981
[8]	张桂芳, 杜珊珊, 史蕊, 等. 处理方法和提取溶剂对松针提取物抗氧化活性的影响[J]. 中国医院药学杂志, 2017, 37(16): 1602-1606. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGYZ201716013.htm ZHANG GF, DU SS, SHI R, et al. Effect of treatment methods and extraction solvents on the antioxidant activity of pine needle extract[J]. Chin Hosp Pharm J, 2017, 37(16): 1602-1606. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGYZ201716013.htm
[9]	SLOMINSKI A, PAUS R. Melanogenesis is coupled to murine anagen: Toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth[J]. J Invest Dermatol, 1993, 101(S1): 90-97. doi: 10.1111/1523-1747.ep12362991
[10]	LOLLI F, PALLOTTI F, ROSSI A, et al. Androgenetic alopecia: A review[J]. Endocrine, 2017, 57(1): 9-17. doi: 10.1007/s12020-017-1280-y
[11]	MAURYA AK, VINAYAK M. Anticarcinogenic action of quercetin by downregulation of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) via induction of p53 in hepatocellular carcinoma (HepG2) cell line[J]. Mol Biol Rep, 2015, 42(9): 1419-1429. doi: 10.1007/s11033-015-3921-7
[12]	KLOEPPER JE, BARIS OR, REUTER K, et al. Mitochondrial function in murine skin epithelium is crucial for hair follicle morphogenesis and epithelial-mesenchymal interactions[J]. J Invest Dermatol, 2015, 135(3): 679-689. doi: 10.1038/jid.2014.475
[13]	坚哲. Nrf2-ARE信号通路在白癜风氧化应激发病中的作用和机制研究[D]. 西安: 第四军医大学, 2013. JIAN Z. The role of Nrf2-ARE pathway in the oxidative stress pathogenesis of vitiligo and its mechanism[D]. Xi'an: The Fourth Military Medical University, 2013.