丝氨酸蛋白酶23在系统性硬化病皮肤纤维化中的作用和机制

doi:10.19723/j.issn.1671-167X.2025.05.014

摘要/Abstract

摘要： 目的: 系统性硬化病(systemic sclerosis, SSc)患者的皮肤成纤维细胞中丝氨酸蛋白酶23(serine protease 23，PRSS23)mRNA水平升高，本研究旨在探索PRSS23表达上调对SSc患者皮肤纤维化的作用及机制。方法: 采用免疫组织化学方法检测SSc患者和健康对照者皮肤组织石蜡切片中PRSS23蛋白的表达水平。分离新鲜皮肤组织的成纤维细胞，采用实时荧光定量PCR(real-time quantitative PCR，RT-qPCR)和蛋白免疫印迹法检测PRSS23在皮肤成纤维细胞中的mRNA和蛋白表达。采用慢病毒感染的方式构建过表达PRSS23的皮肤成纤维细胞BJ细胞系，以400 μmol/L过氧化氢刺激12 h后，采用膜联蛋白V/7-AAD染色检测凋亡成纤维细胞的比例，采用流式细胞仪和蛋白免疫印迹法检测凋亡相关蛋白激活型半胱氨酸-天冬氨酸蛋白酶-3(Caspase-3)的表达。采用RT-qPCR和酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法检测成纤维细胞分泌的白细胞介素-6(interleukin 6，IL-6)和肿瘤坏死因子-α(tumor necrosis factor alpha，TNF-α)水平。结果: 与健康对照组比较，SSc患者皮肤组织中的PRSS23蛋白表达显著升高[4.952 (3.806~5.439) vs. 0.806 (0.395~1.173)，P＜0.001]。PRSS23主要表达在成纤维细胞中，SSc患者分离的皮肤成纤维细胞中PRSS23的mRNA[27.59 (25.02~30.00) vs. 1.00，P＜0.001]和蛋白表达水平[0.675 (0.587~0.837) vs. 0.451 (0.342~0.502)，P=0.029]均显著上调。相较于对照组，过表达PRSS23的皮肤成纤维细胞抗凋亡能力增强，过氧化氢诱导后凋亡细胞的比例显著减少[(5.043±1.097)% vs. (17.480±3.212)%，P=0.022]，凋亡相关蛋白激活型Caspase-3表达水平也显著降低[(0.718±0.022) vs. (1.422±0.105)，P=0.003]，而炎症因子IL-6的mRNA水平[(99.780±1.796) vs. (1.000±0.004)，P＜0.001]和蛋白分泌水平[(211.600±2.431) ng/L vs. (65.930±1.768) ng/L，P＜0.001]显著上调，同时，TNF-α的mRNA水平[(3.555±0.555) vs. (1.000±0.004)，P＜0.001]和蛋白分泌水平[(41.190±0.949) ng/L vs. (31.150±0.360) ng/L，P＜0.001]显著上调。结论: PRSS23在SSc患者皮肤成纤维细胞中的表达增加，可能通过抑制细胞凋亡，并促进炎症因子IL-6和TNF-α的分泌，调控皮肤成纤维细胞向促炎型转化，参与皮肤纤维化的发展。

关键词: 系统性硬化病, 成纤维细胞, 丝氨酸蛋白酶类, 细胞凋亡

Abstract: Objective: It has been reported that the mRNA expression of serine protease 23 (PRSS23) was increased in skin fibroblasts from systemic sclerosis patients (SSc). The purpose of this study is to explore the pathogenetic effect and mechanism of PRSS23 in skin fibrosis of SSc. Methods: The expression of PRSS23 in skin tissues from the SSc patients and healthy controls was detected by immunohisto-chemistry. Fibroblasts isolated from fresh skin tissue were used to detect the expression of PRSS23 by real-time quantitative PCR (RT-qPCR) and Western blot. Overexprssion of PRSS23 in BJ, the fibroblasts cell line of skin, was constructed by lentivirus. After stimulation with 400 μmol/L hydrogen peroxide for 12 h, Annexin V/7-AAD staining was used to detect apoptosis of fibroblasts; flow cytometry and Western blot were used to detect the expression of apoptosis-related protein cleaved Caspase-3. The expression of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in fibroblasts was detected by RT-qPCR and enzyme linked immunosorbent assay (ELISA). Results: Compared with the healthy controls, the expression of PRSS23 in skin tissues of the SSc patients was significantly increased [4.952 (3.806-5.439) vs. 0.806 (0.395-1.173), P < 0.001], and fibroblast was the main cell that expressed PRSS23. The mRNA [27.59 (25.02-30.00) vs. 1.00, P < 0.001] and protein [0.675 (0.587-0.837) vs. 0.451 (0.342-0.502), P=0.029] of PRSS23 in skin fibroblasts isolated from the SSc patients were significantly up-regulated. Compared with the control group, the anti-apoptotic ability of skin fibroblasts overexpressing PRSS23 was enhanced, and the proportion of apoptotic cells was significantly reduced after hydrogen peroxide induction [(5.043±1.097)% vs. (17.480±3.212)%, P=0.022], the expression of apoptosis-related protein cleaved Caspase-3 was also markedly reduced [(0.718±0.022) vs. (1.422±0.105), P=0.003]. In addition, the mRNA [(99.780±1.796) vs. (1.000±0.004), P < 0.001] and protein [(211.600±2.431) ng/L vs. (65.930±1.768) ng/L, P < 0.001] of IL-6 in the fibroblasts overexpressing PRSS23 were significantly up-regulated; the mRNA[(3.555±0.555) vs. (1.000±0.004), P < 0.001] and protein levels [(41.190±0.949) ng/L vs. (31.150±0.360) ng/L, P < 0.001] of TNF-α in the fibroblasts overexpressing PRSS23 were also significantly up-regulated. Conclusion: The expression of PRSS23 is increased in skin fibroblasts of SSc patients. PRSS23 can inhibit cell apoptosis, promote the secretion of inflammatory factors such as IL-6 and TNF-α, and regulate the process that skin fibroblasts transform into pro-inflammatory type. So, PRSS23 is associated with the development of skin fibrosis.

Key words: Systemic sclerosis, Fibroblasts, Serine proteases, Apoptosis

中图分类号:

R593.2

袁显墩, 李照华, 徐丹, 李婷, 方丹, 穆荣. 丝氨酸蛋白酶23在系统性硬化病皮肤纤维化中的作用和机制[J]. 北京大学学报（医学版）, 2025, 57(5): 903-910.

Xiandun YUAN, Zhaohua LI, Dan XU, Ting LI, Dan FANG, Rong MU. Pathogenesis and mechanism of serine protease 23 in skin fibrosis of systemic sclerosis[J]. Journal of Peking University (Health Sciences), 2025, 57(5): 903-910.

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Gene	GenBank ID	Primer sequences (5′ to 3′)	Length/bp
IL-6	NM_000600	Forward: 5′-ACTCACCTCAGAACGAATTG-3′ Reversed: 5′-CCATCTTGGAAGGTTCAGGTTG-3′	149
TNF-α	NM_000594	Forward: 5′-CCTCTAATCAGCCTCTG-3′ Reversed: 5′-GAGGACCTGGAGTAGAG-3′	220
PRSS23	NM_009104	Forward: 5′-TGTGCTGGGCAAGTGAG-3′ Reversed: 5′-AGTTCCCTTATGACTGGGG-3′	174

Items	Sample size (n)	Value, M (P₂₅-P₇₅)
Mean optical density
HC	6	0.806 (0.395-1.173)
SSc	6	4.952 (3.806-5.439)^***
Protein relative expression
HC	4	0.451 (0.342-0.502)
SSc	4	0.675 (0.587-0.837)^*
mRNA relative expression
HC	6	1.000
SSc	6	27.590 (25.020-30.000)^***

Items	Sample size (n)	Value, ${\bar x}$±SE
Protein relative expression (PRSS23)
Mock	3	0.199±0.023
Overexpressed	3	0.730±0.037^***
mRNA relative expression
Mock	4	1.000
Overexpressed	4	28.160±0.507^***

Items	Sample size (n)	Value, ${\bar x}$±SE
Apoptosis ratio
Mock	3	(17.480±3.212)%
Overexpressed	3	(5.043±1.097)%^*
Positive cell ratio
Mock	4	(12.510±1.472)%
Overexpressed	4	(7.460±0.866)%^*
Protein relative expression (cleaved Caspase 3)
Mock	4	1.422±0.105
Overexpressed	4	0.718±0.022^**

Items	Sample size (n)	Value, ${\bar x}$±SE
IL-6 mRNA relative expression
Mock	3	1.000
Overexpressed	3	99.780±1.796^***
TNF-α mRNA relative expression
Mock	4	1.000
Overexpressed	4	3.555±0.555^**
IL-6 protein content (cell supernatant)/(ng/L)
Mock	4	65.930±1.768
Overexpressed	4	211.600±2.431^***
TNF-α protein content (cell supernatant)/(ng/L)
Mock	4	31.150±0.360
Overexpressed	4	41.190±0.949^***