川芎嗪对银屑病小鼠皮损内TNF-α、IL-17、VEGF表达的影响

doi:10.11958/20221433

天津医药 ›› 2023, Vol. 51 ›› Issue (6): 590-595.doi: 10.11958/20221433

川芎嗪对银屑病小鼠皮损内TNF-α、IL-17、VEGF表达的影响

朱玉婷¹(), 晏文¹^,^△, 应理晟¹, 王进京²

1 遵义医科大学附属医院皮肤科（邮编563003）
2 遵义医科大学附属医院病理科（邮编563003）

收稿日期:2022-09-13 修回日期:2022-11-02 出版日期:2023-06-15 发布日期:2023-06-20
通讯作者: ^△E-mail：zyyanwen@163.com
作者简介:朱玉婷（1994），女，住院医师，主要从事银屑病发病机制及治疗方面研究。E-mail：1647307775@qq.com
基金资助:
贵州省自然科学基金项目(黔科合基础-ZK（2023）一般578);贵州省科技合作计划项目(黔科合LH字（2015）7473号);遵义市科技计划项目(遵市科合HZ字（2020）217号)

Effects of tetramethylpyrazine on expression levels of TNF-α, IL-17 and VEGF in skin lesions of psoriatic mice

ZHU Yuting¹(), YAN Wen¹^,^△, YING Lisheng¹, WANG Jinjing²

1 Department of Dermatology, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China
2 Department of Pathology, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China

Received:2022-09-13 Revised:2022-11-02 Published:2023-06-15 Online:2023-06-20
Contact: ^△E-mail：zyyanwen@163.com

朱玉婷, 晏文, 应理晟, 王进京. 川芎嗪对银屑病小鼠皮损内TNF-α、IL-17、VEGF表达的影响[J]. 天津医药, 2023, 51(6): 590-595.

ZHU Yuting, YAN Wen, YING Lisheng, WANG Jinjing. Effects of tetramethylpyrazine on expression levels of TNF-α, IL-17 and VEGF in skin lesions of psoriatic mice[J]. Tianjin Medical Journal, 2023, 51(6): 590-595.

摘要/Abstract

摘要：

目的通过研究川芎嗪（TMP）对银屑病小鼠皮损内肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-17及血管内皮生长因子（VEGF）表达的影响及其可能的作用机制。方法 30只BALB/c小鼠剔除背部约4 cm×2 cm大小区域毛发后随机分为正常组，模型组，低、中、高TMP组，每组6只。正常组涂抹凡士林62.5 mg/d，模型组及TMP各组涂抹5%咪喹莫特乳膏62.5 mg/d，连续7 d；于第4天起，低、中、高TMP组分别予腹腔注射20、40、100 mg/kg盐酸川芎嗪注射液，正常组及模型组注入等量生理盐水，1次/d，连续9 d。观察各组小鼠皮损变化，干预结束后取小鼠背部皮损，实时荧光定量PCR（qRT-PCR）法检测各组皮损中TNF-α、IL-17及VEGF的mRNA表达水平；组织切片HE染色及免疫组织化学染色，观察各组皮损的病理变化及检测TNF-α、IL-17、VEGF蛋白表达量。结果皮损组织病理：模型组小鼠呈现为典型的银屑病样病理改变，TMP组皮损的银屑病样病理表现则较模型组依次减轻。qRT-PCR结果显示：与正常组相比，模型组TNF-α、IL-17、VEGF mRNA相对表达量均升高（P<0.05）；与模型组、低TMP组相比，中、高TMP组TNF-α mRNA的表达量下降（P<0.05）；与模型组相比，低、中、高剂量TMP组的IL-17 mRNA表达量依次降低（P<0.05）；与模型组、低、中TMP组相比，高TMP组VEGF mRNA表达量降低（P<0.05）。免疫组化结果显示：TNF-α、IL-17、VEGF均主要表达于表皮细胞的胞浆内，且与正常组相比，模型组的各蛋白表达量均升高（均P<0.05）；与模型组相比，低、中、高TMP组IL-17、VEGF表达量及中TMP组TNF-α表达量降低（均P<0.05）；与低TMP组相比，高TMP组TNF-α、IL-17、VEGF表达量及中TMP组IL-17表达量降低（均P<0.05）；与中TMP组相比，高TMP组VEGF表达量降低（均P<0.05）。结论川芎嗪可通过降低小鼠银屑病样皮损内的TNF-α、IL-17、VEGF的表达水平，改善小鼠银屑病样皮损的炎症表现。

关键词: 银屑病, 肿瘤坏死因子α, 白细胞介素17, 血管内皮生长因子类, 川芎嗪

Abstract:

Objective To investigate the therapeutic effect of tetramethylpyrazine (TMP) on expression levels of tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17) and vascular endothelial growth factor (VEGF) in skin lesions of psoriatic mice and its possible mechanism. Methods Thirty BALB/c mice, half male and half female, were randomly divided into the normal group, the model group and the low, medium and high TMP groups after shaving hair in back area of about 2 cm×4 cm, with 6 mice in each group. Mice in the normal group was smeared with vaseline 62.5 mg/d. Mice in the model group and the TMP group were smeared with imiquimod cream 62.5 mg/d for continuous 7 days. From the 4th day, the low, medium and high TMP groups were respectively injected with ligustrazine hydrochloride 20 mg/kg, 40 mg/kg and 100 mg/kg, and the normal group and the model group were injected with the same amount of normal saline once a day for 9 consecutive days. Changes of skin lesions were observed in each group. After the intervention, back skin lesions of mice were taken. The expression levels of TNF-α, IL-17 and VEGF mRNA in skin lesions were detected by qRT-PCR. The pathological changes of skin lesions and TNF-α, IL-17 and VEGF protein expression levels in skin lesions of each group were detected by HE staining and immunohistochemical staining. Results Mice in the model group showed typical psoriasis-like pathological changes, while the psoriasis-like pathological changes of skin lesions of the TMP group were successively lighter than those of the model group. Compared with the normal group, the expression levels of TNF-α, IL-17 and VEGF mRNA were significantly higher in the model group (all P<0.05). Compared with the model group and the low TMP group, the expression levels of TNF-α mRNA decreased significantly in the medium TMP group and the high TMP group (P<0.05). Compared with the model group, expression levels of IL-17 mRNA were decreased gradually in the three TMP groups (P<0.05). Compared with the model group, the low TMP group and the medium TMP group, the expression level of VEGF mRNA was decreased significantly in the high TMP group (P<0.05). Immunohistochemical results showed that TNF-α, IL-17 and VEGF were mainly expressed in cytoplasm of epidermal cells, and expression levels of above proteins were significantly higher in the model group than those of the normal group (P<0.05). Compared with the model group, expression levels of IL-17 and VEGF were significantly decreased in the low, medium and high TMP groups, and TNF-α was significantly decreased in the medium and high TMP groups (P<0.05). Compared with the low TMP group, expression levels of TNF-α, IL-17 and VEGF were significantly decreased in the high TMP group and IL-17 was also significantly decreased in the medium TMP group (P<0.05). Compared with the medium TMP group, the expression level of VEGF decreased significantly in the high TMP group (P<0.05). Conclusion Tetramethylpyrazine can improve the inflammatory manifestations of psoriasis-like skin lesions in model mice by reducing expression levels of TNF-α, IL-17 and VEGF in psoriasis-like skin lesions of mice.

Key words: psoriasis, tumor necrosis factor-alpha, interleukin-17, vascular endothelial growth factors, tetramethylpyrazine

中图分类号:

R758.63

图/表 7

表1 qRT-PCR引物序列

Tab.1 The primer sequences for qRT-PCR

基因名称	引物序列（5'→3'）	产物大小（bp）
β-actin	上游：CAGCTCAGTAACAGTCCGCC	240
	下游：CCACCATGTACCCAGGCATT	240
TNF-α	上游：GTGAGGAGCACGTAGTCGG	112
	下游：ACGGCATGGATCTCAAAGACA	112
IL-17	上游：TCATTGTGGAGGGCAGACAA	128
	下游：TGACCCCTAAGAAACCCCCA	128
VEGF	上游：AGGAAGGGTAAGCCACTCAC	89
	下游：CCAGGAGGACCTTGTGTGAT	89

图1 各组小鼠皮损大体观

Fig.1 General view of skin lesions in four groups of mice

图2 各组小鼠背部皮损PASI评分比较 3、6、9 d组间比较的F值分别为137.875、102.250及80.921，均P<0.01；a与正常组比较，b与模型组比较，c与低TMP组比较，d与中TMP组比较，P<0.05。

Fig.2 Comparison of PASI scores of skin lesions between the five groups of mice

表2 各组皮损内TNF-α、IL-17、VEGF mRNA相对表达水平比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of relative expression levels of TNF-α, IL-17 and VEGF mRNA in skin lesions between the five groups of mice

组别	TNF-α	IL-17	VEGF
正常组	-0.039 9±0.180 3	-0.050 6±0.209 7	-0.026 6±0.151 8
模型组	1.037 9±0.292 0^a	0.666 4±0.235 0^a	1.278 9±0.254 7^a
低TMP组	0.852 6±0.159 9^a	-0.280 4±0.074 6^ab	1.137 3±0.165 9^a
中TMP组	0.428 8±0.152 9^abc	-0.547 7±0.143 4^abc	0.801 0±0.139 8^a
高TMP组	0.367 7±0.105 0^abc	-0.778 9±0.158 9^abcd	0.327 0±0.058 7^abcd
F	25.437^＊＊	51.276^＊＊	54.334^＊＊

图3 各组皮损HE染色结果（×200）

Fig.3 HE staining results of skin lesions in five groups（×200）

图4 各组皮损内TNF-α、IL-17、VEGF免疫组化结果（×200）

Fig.4 Immunohistochemical results of TNF-α, IL-17 and VEGF in skin lesions of different groups (×200)

表3 各组皮损内TNF-α、IL-17、VEGF蛋白相对表达水平比较（n=6，IOD，$\bar{x}±s$）

Tab.3 Comparison of relative expression levels of TNF-α, IL-17 and VEGF proteins in skin lesions between the five groups of mice

组别	TNF-α	IL-17	VEGF
正常组	0.004±0.002	0.002±0.001	0.001±0.001
模型组	0.019±0.003^a	0.022±0.001^a	0.021±0.003^a
低TMP组	0.017±0.003^a	0.015±0.001^ab	0.019±0.001^ab
中TMP组	0.014±0.001^ab	0.009±0.001^abc	0.012±0.001^ab
高TMP组	0.013±0.001^abc	0.008±0.001^abc	0.008±0.001^abcd
F	98.959^＊＊	718.474^＊＊	367.192^＊＊

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川芎嗪对银屑病小鼠皮损内TNF-α、IL-17、VEGF表达的影响

Effects of tetramethylpyrazine on expression levels of TNF-α, IL-17 and VEGF in skin lesions of psoriatic mice

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