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

袁显墩; 李照华; 徐丹; 李婷; 方丹; 穆荣

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

北京大学学报（医学版） >

2025 , Vol. 57 >Issue 5: 903 - 910

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

论著

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

袁显墩 ,
李照华 ,
徐丹 ,
李婷 ,
方丹 ,
穆荣 ^,*

展开

北京大学第三医院风湿免疫科，北京 100191

收稿日期: 2023-02-28

网络出版日期: 2024-04-10

基金资助

国家自然科学基金(81771706)

版权

收起

Pathogenesis and mechanism of serine protease 23 in skin fibrosis of systemic sclerosis

Xiandun YUAN ,
Zhaohua LI ,
Dan XU ,
Ting LI ,
Dan FANG ,
Rong MU ^,*

Expand

Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191, China

MU Rong, e-mail, murongster@163.com

Received date: 2023-02-28

Online published: 2024-04-10

Supported by

the National Natural Science Foundation of China(81771706)

Copyright

Fold

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

本文引用格式

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

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

系统性硬化病(systemic sclerosis, SSc)又称硬皮病，是一种复杂的自身免疫性疾病，其显著特征是皮肤和内脏纤维化，该病极大地影响了患者的生活质量^[1]。然而，目前缺乏相应的药物治疗皮肤纤维化，对SSc皮肤纤维化发病机制的深入研究有助于开发治疗SSc纤维化的新药物，具有重要的意义。纤维化是异常的组织修复，正常情况下，完成组织修复后的成纤维细胞进入凋亡，完成自我清除，纤维化疾病中，成纤维细胞逃避了凋亡而持续存在，分泌大量的炎症因子，引起慢性炎症，最终导致病理性瘢痕，即纤维化^[2-3]。

最近，有研究报道丝氨酸蛋白酶23(serine protease 23，PRSS23)的mRNA在SSc患者皮肤组织成纤维细胞中表达上调^[4-5]。蛋白酶因与纤维化过程中细胞外基质(extracellular matrix, ECM)的分解和组织重塑中生长因子的激活有关^[6-7]，逐渐受到关注。丝氨酸蛋白酶作为一个庞大的蛋白酶家族，在哺乳动物体内广泛存在，在消化酶、凝血和补体激活方面发挥重要作用^[8-10]。然而，PRSS23在SSc中的作用及机制尚未见报道。本文通过检测皮肤组织、皮肤成纤维细胞中PRSS23的表达水平，明确PRSS23在SSc患者和健康对照中的表达差异。进一步构建过表达PRSS23的皮肤成纤维细胞系，探究PRSS23在SSc皮肤纤维化中的作用及机制。

1 资料与方法

1.1 组织标本来源和免疫组织化学检测

收集2021年1月至2022年10月期间北京大学第三医院患者的前臂皮肤活检样本，其中确诊为SSc的男性和女性患者各3例，年龄25~49岁，平均(37±12)岁，病程5个月至3年，平均(21±16)个月；患者的皮肤表现包括1例肿胀和5例硬化；活检前使用或既往使用过糖皮质激素者2例，服用过免疫抑制剂者3例，未用过前两种药物者1例；未发现皮肤表现、激素及免疫抑制剂对PRRS23的表达有影响。所有患者均符合2013年欧洲抗风湿病联盟(European League Against Rheumatism，EULAR)的SSc诊断分类标准。另选择6例性别、年龄匹配的健康人作为健康对照组。本研究通过北京大学第三医院医学科学研究伦理委员会批准[批件号：(2022)医伦审第(142-03)号]，并获得患者和健康受试者的知情同意书。

6例SSc患者和6例健康对照者的前臂皮肤活检标本经4%(体积分数)多聚甲醛溶液固定24 h以上，石蜡包埋，组织切片厚度为4 μm，常规脱蜡复水，使用抗原修复液(AR0026，博士德生物工程有限公司，中国)室温修复10 min，根据免疫组织化学二步法检测试剂盒(小鼠/兔增强聚合物法检测系统，PV-9000，中山金桥生物技术有限公司，美国)说明书进行操作，使用抗PRSS23(ab201182，Abcam，体积比1 ∶ 500)作为一抗，DAB显色1 min，苏木精复染20 s，返蓝4 min，乙醇梯度脱水，二甲苯透明，中性树胶封片，在偏光显微镜下进行拍摄。

1.2 人皮肤原代成纤维细胞分离

无菌条件下采集SSc患者和健康对照者的前臂皮肤组织，将每例样本置于含1%(质量分数)青霉素-链霉素的PBS缓冲液中反复漂洗5遍，在1.5 mL EP管中用眼科剪将皮肤剪成小碎块，加入0.2%(体积分数) Ⅰ型胶原酶(17101015，Invitrogen，美国) 5 mL，37 ℃消化2 h，1 500 r/min离心3 min，弃去上清液，加1 mL完全培养基重悬，接种于12孔板的1个孔中。

1.3 细胞培养

培养人原代皮肤成纤维细胞和人皮肤成纤维细胞系BJ(FH0191，上海富衡生物科技有限公司，中国)，培养使用的完全培养基为10%(体积分数)胎牛血清(C0235，Gibco，美国)+89%(体积分数)DMEM培养液(11995040，Gibco，美国)+1%(质量分数)青霉素-链霉素-谷氨酰胺(1037 8016，Gibco，美国)，在37 ℃、饱和湿度、5%(体积分数)CO₂的培养箱内培养，将处于对数生长期的细胞用作实验。

1.4 细胞转染和分组

取2×10⁴个对数生长期的BJ细胞接种到6孔板中，每组4个复孔。当6孔板中BJ细胞生长融合度为50%时，将BJ细胞分为PRSS23过表达转染组(转染pCDH-CMV-MCS-EF1-PRSS23-Puro质粒)、空载体对照组(转染pCDH-CMV-MCS-EF1-Puro空质粒)，转染质粒后的细胞在嘌呤霉素存在的条件下培养1周。

1.5 实时荧光定量PCR

实时荧光定量PCR(real-time quantitative PCR，RT-qPCR)检测的样本主要包括两部分，第一部分是1.1小节所述6例SSc患者和6例健康对照者前臂皮肤组织中分离的成纤维细胞，作为临床来源样本；第二部分为过表达PRSS23和转入空载体的人皮肤成纤维细胞系(BJ细胞系)，每组4个复孔，作为实验来源样本。

使用Trizol(DP424，天根生化科技有限公司，中国)提取总RNA后测定RNA浓度，取1 μg总RNA后用高效PCR逆转录试剂盒(FSQ-201，东洋纺生物科技有限公司，日本)合成cDNA，最后使用PCR荧光定量试剂盒(QPK-201，东洋纺生物科技有限公司，日本)进行RT-qPCR分析。RT-qPCR的反应程序为：95 ℃预变性15 s，60 ℃ 15 s，72 ℃ 45 s，循环40次。使用相对标准曲线法(2^-ΔΔCt)确定相对mRNA表达，使用人18S小亚基单位核糖体核酸基因作为参考，引物由北京睿博兴科公司合成，序列见表 1。

表1 IL-6、TNF-α、PRSS23引物序列

Table 1 Primer sequences for IL-6, TNF-α and PRSS23

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

PRSS23, serine protease 23; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; ID, identification.

1.6 蛋白免疫印迹法检测PRSS23和激活型Caspase-3表达

蛋白免疫印迹法检测的样本主要包括两部分，与1.5小节相同。提取总蛋白后用BCA蛋白检测试剂盒(P0012，碧云天生物技术有限公司，中国)测定蛋白浓度，蛋白浓度配平后加入上样缓冲液(P0015L，碧云天生物技术有限公司，中国)，使用SDS-PAGE凝胶电泳分离蛋白，转膜后，用脱脂奶粉室温封闭1 h，加入抗-PRSS23(ab201182，Abcam，体积比1 ∶ 500)和抗激活型半胱氨酸-天冬氨酸蛋白酶-3(Caspase-3)(A11021，ABclonal，体积比1 ∶ 1 000)后，4 ℃孵育过夜，之后加入辣根过氧化物酶(horseradish peroxidase，HRP)耦联的二抗(A0208/A0216，天根生化科技有限公司，中国，体积比1 ∶ 1 000)共孵育1 h，用ECL化学发光底物(170-5061，伯乐科技有限公司，美国)可视化目的蛋白，使用化学发光成像系统采集图像。

1.7 流式细胞检测

检测凋亡时用0.25%(体积分数)胰酶-EDTA将BJ细胞系温和消化成单个细胞。按照膜联蛋白V-FITC/7-AAD荧光双染细胞凋亡检测试剂盒(P-CA-202，武汉普诺赛生命科技公司，中国)的说明进行操作。检测激活型Caspase-3时，按照FITC标记的抗激活型Caspase-3(560901，BD，美国)说明书进行操作，先用0.25%胰酶-EDTA将细胞温和消化成单个细胞，PBS洗1遍，通透固定液室温固定20 min，缓冲液洗2遍后重悬，使每100 μL细胞悬液含有的细胞数为1×10⁶个，每100 μL加20 μL抗体，室温避光孵育30 min，缓冲液洗2遍，最后用200 μL缓冲液重悬细胞。悬液通过40 μm细胞过滤器，在CytoFLEX(Beckman Coulter公司)上对样品进行分析，使用CytExpert软件进行数据分析。

1.8 酶联免疫吸附试验

吸取培养基上清液，在4 ℃以1 800 r/min离心10 min，弃其底部细胞沉淀，得到细胞上清液，用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法测定白细胞介素-6(interleukin 6，IL-6)和肿瘤坏死因子-α(tumor necrosis factor alpha，TNF-α)的蛋白浓度。按照人IL-6/TNF-α预包被酶联免疫吸附试剂盒(1110602/1117202，达优，中国)进行操作，具体步骤为：(1)样本或配好的标准品，设置对照，100 μL/孔；(2)生物素化抗体工作液，50 μL/孔，混匀，室温孵育1 h，洗板3次；(3)链霉亲和素-HRP工作液，100 μL/孔，室温孵育20 min，洗板3次；(4)TMB显色，100 μL/孔，室温避光显色5~30 min；(5)终止液，100 μL/孔，终止反应后，在λ＝450 nm处读取光密度值(D)。用空白培养基作为阴性对照，最终D值=实验孔D值-空白孔D值，根据最终D值以及标准浓度曲线计算蛋白浓度。

1.9 统计学分析

实验数据为至少3个平行试验的平均值，图像数据通过Image J 8.0软件对成像图片进行定量分析。采用GraphPad Prism 8.0软件对试验数据进行统计学处理，主要运用成组设计资料的t检验；临床样本数据选用秩和检验进行比较，用Mann-Whitney U检验计算P值，置信区间为95%，采用双尾检验；其余数据为非临床样本，采用参数检验，用Shapiro-Wilk检验检测数据的正态分布，方差齐性用比较方差的F检验，均以P>0.1为标准，正态分布并且方差齐的数据采用非配对t检验，非正态分布数据采用Mann-Whitney检验，正态分布但方差不齐的数据则使用Welch’s校正，并计算相应P值。P＜0.05为差异有统计学意义。

2 结果

2.1 SSc患者皮肤成纤维细胞中PRSS23的表达

免疫组织化学检测结果显示，SSc患者皮肤组织中PRSS23蛋白表达[4.952 (3.806~5.439)]相较于健康对照组[0.806 (0.395~1.173)]显著增加(Mann-Whitney U检验: U=0, P＜0.001)，且阳性细胞呈长梭型(图 1A)。为验证PRSS23阳性细胞是否为成纤维细胞，分离SSc患者和健康对照者前臂皮肤组织成纤维细胞，以RT-qPCR检测PRSS23 mRNA的表达，SSc患者皮肤成纤维细胞PRSS23的mRNA表达显著高于健康对照者[27.59 (25.02~30.00) vs. 1.00；Mann-Whitney U检验: U=0, P＜0.001；图 1B]。蛋白免疫印记法检测皮肤成纤维细胞中PRSS23蛋白表达水平，与mRNA结果一致，SSc患者皮肤成纤维细胞中PRSS23蛋白表达水平较健康对照上调[0.675 (0.587~0.837) vs. 0.451 (0.342~0.502)；Mann-Whitney U检验: U=0, P=0.029]，约为健康对照者的1.6倍(图 1C)，数据统计结果见表 2。

显示原图|下载原图ZIP|生成PPT

图1 SSc患者皮肤组织成纤维细胞中PRSS23的表达

Figure 1 Expression of PRSS23 in fibroblasts from SSc patients' skin tissue

A, immunohistochemical staining was performed to compare the expression of PRSS23 in skin tissue slices (the magnification is 20× for the left image and 40× for the right image, and the right image shows an enlarged view of the red box in the left image). Compared with the healthy controls, PRSS23 positive expression in skin tissue slices from SSc patients was significantly increased (n=6, P < 0.001). B, protein immunoblotting was used to quantify the protein expression of PRSS23 in human skin fibroblasts. Compared with the healthy controls, PRSS23 protein expression in skin tissue from SSc patients was significantly increased (n=6, P=0.029). C, RT-qPCR was used to quantify the mRNA expression of PRSS23 in human skin fibroblasts. Compared with the healthy controls, PRSS23 mRNA expression in skin tissue from SSc patients was significantly increased (n=6, P < 0.001), and the statistical method used was the Mann-Whitney U test. * P < 0.05, *** P < 0.001. HC, healthy control; SSc, systemic sclerosis; PRSS23, serine protease 23; RT-qPCR, quantitative real-time PCR.

表2 PRSS23在SSc患者皮肤组织成纤维细胞中的表达

Table 2 Expression of PRSS23 in fibroblasts of skin tissue of SSc patients

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)^***

*P＜0.05, ***P＜0.001, HC vs. SSc. PRSS23, serine protease 23; HC, healthy control; SSc, systemic sclerosis.

2.2 PRSS23抑制皮肤成纤维细胞凋亡

进一步探究PRSS23上调对SSc皮肤纤维化的作用及机制。采用慢病毒感染的方式，在皮肤成纤维细胞系BJ中过表达PRSS23。蛋白免疫印迹实验结果显示，转染PRSS23质粒的BJ细胞PRSS23蛋白表达水平相较于空载体对照组显著升高[(0.730±0.037) vs. (0.199±0.023)；Unpaired t检验: t=12.29, P＜0.001；图 2A]。RT-qPCR与蛋白免疫印迹法结果一致，转染PRSS23质粒的成纤维细胞PRSS23 mRNA的表达较空载体对照组明显升高[(28.160±0.507) vs. (1.000)；Unpaired t检验: t=53.57, P＜0.001；图 2B]。数据统计结果见表 3。

显示原图|下载原图ZIP|生成PPT

图2 转染PRSS23质粒的皮肤成纤维细胞过表达PRSS23

Figure 2 Overexpression of PRSS23 in skin fibroblasts transfected with PRSS23 plasmid

A, quantification of PRSS23 protein expression levels in fibroblasts transfected with PRSS23 plasmid or empty vector by Western blot. The PRSS23 protein expression was significantly increased in the overexpression group compared to the empty vector control group (n=4, P < 0.001). B, quantification of PRSS23 mRNA expression levels in two types of cells by RT-qPCR. The mRNA expression of PRSS23 was significantly higher in the overexpression group than in the empty vector control group (n=4, P < 0.001), indicating a successful transfection. The statistical method used was unpaired t test. *** P < 0.001. Mock, empty vector control group; Overexpressed, PRSS23 overexpression group; PRSS23, serine protease 23.

表3 PRSS23在转染PRSS23质粒的皮肤成纤维细胞中的表达

Table 3 Expression of PRSS23 in skin fibroblasts transfected with PRSS23 plasmid

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^***

*** P＜0.001, mock vs. overexpressed. Mock, empty vector control group; Overexpressed, PRSS23 overexpression group; PRSS23, serine protease 23; SE, standard error.

随后，在400 μmol/L过氧化氢处理细胞12 h后，使用膜联蛋白V-FITC/7-AAD细胞凋亡检测试剂盒对细胞染色，以流式细胞仪检测膜联蛋白V和7-AAD双阳细胞的比例。结果显示，与空载体对照组相比，过表达PRSS23的BJ细胞凋亡细胞比例减少[(17.480±3.212)% vs. (5.043±1.097)%；Unpaired t检验: t=3.664, P=0.022；图 3A]。流式细胞术和蛋白免疫印迹法进一步检测激活型Caspase-3，过表达PRSS23的BJ细胞在过氧化氢处理后，流式细胞检测显示，过表达组激活型Caspase-3阳性细胞比例较空载体对照组显著下调[(7.460± 0.866)% vs. (12.510±1.472)%；Unpaired t检验: t=2.957, P=0.026]；蛋白免疫印迹法检测激活型Caspase-3表达显示，过表达组较空载体对照组也显著下调[(0.718±0.022) vs. (1.422±0.105)；Unpaired t检验: t=6.554, P=0.003；图 3B、C]。数据统计结果见表 4。

显示原图|下载原图ZIP|生成PPT

图3 过表达PRSS23抑制皮肤成纤维细胞凋亡

Figure 3 Overexpression of PRSS23 inhibits apoptosis of skin fibroblasts

A, BJ cells transfected with PRSS23 plasmid or empty vector were treated with 400 μmol/L hydrogen peroxide for 12 h to induce apoptosis. After staining with the Annexin V-FITC/7-AAD apoptosis detection kit, cells were analyzed by flow cytometry. Left panel shows the flow cytometry results, and the right panel shows the statistical analysis of apoptosis rate. Compared to the control group, the overexpression of PRSS23 decreased the apoptosis rate (n=4, P=0.022). B, after hydrogen peroxide treatment, cells were stained with anti-cleaved Caspase-3 antibody and analyzed by flow cytometry. Left panel shows the flow cytometry results, and the right panel shows the statistical analysis of cleaved Caspase-3 positive cells. Compared to the control group, the overexpression of PRSS23 reduced the ratio of cleaved Caspase-3 positive cells (n=4, P=0.026). C, the expression level of cleaved Caspase-3 in the two cell groups was quantified by Western blot. Top panel shows the Western blotting results, and the bottom panel shows the relative expression levels of cleaved Caspase-3 (n=4, P=0.003). The statistical analysis was performed using the unpaired t test. * P < 0.05, ** P < 0.01. Mock, empty vector control group; Overexpressed, PRSS23 overexpression group; PRSS23, serine protease 23; FITC, fluorescein isothiocyanate.

表4 PRSS23抑制皮肤成纤维细胞凋亡率和相关凋亡蛋白的测定

Table 4 Determination of the inhibition of apoptosis rate and related apoptotic proteins by PRSS23 in skin fibroblasts

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^**

* P＜0.05, ** P＜0.01, mock vs. overexpressed. Mock, empty vector control group; Overexpressed, PRSS23 overexpression group; PRSS23, serine protease 23; SE, standard error.

2.3 PRSS23促进皮肤成纤维细胞分泌炎症因子

使用RT-qPCR检测过表达PRSS23和空载体对照组BJ细胞中IL-6和TNF-α mRNA的表达，结果显示PRSS23过表达组IL-6的mRNA水平相较于空载体对照组明显上调[(99.780±1.796) vs. (1.000)；Unpaired t检验: t=54.99, P＜0.001]，PRSS23过表达组TNF-α的mRNA水平相较于空载体对照组也明显上调[(3.555±0.555) vs. (1.000)；Unpaired t检验: t=4.604, P＜0.001；图 4A、B]。

显示原图|下载原图ZIP|生成PPT

图4 过表达PRSS23促进皮肤成纤维细胞分泌促炎因子IL-6和TNF-α

Figure 4 Overexpression of PRSS23 promotes the secretion of pro-inflammatory cytokines IL-6 and TNF-α in skin fibroblasts

A, quantification of IL-6 mRNA expression in cells using RT-qPCR. Compared to the control group with empty vector, the PRSS23 overexpression group showed a significant increase in IL-6 mRNA expression (n=4, P < 0.001). B, quantification of TNF-α mRNA expression in cells using RT-qPCR. Compared to the control group with empty vector, the PRSS23 overexpression group showed a significant increase in TNF-α mRNA expression (n=4, P=0.004). C, quantification of IL-6 protein concentration in cell supernatants using ELISA. Compared to the control group with empty vector, the PRSS23 overexpression group showed a significant increase in IL-6 protein concentration (n=4, P < 0.001). D, quantification of TNF-α protein concentration in cell supernatants using ELISA. Compared to the control group with empty vector, the PRSS23 overexpression group showed a significant increase in TNF-α protein concentration (n=4, P < 0.001). Statistical analysis was performed using unpaired t test. ** P < 0.01, *** P < 0.001. Mock, empty vector control group; Overexpressed, PRSS23 overexpression group; PRSS23, serine protease 23; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; RT-qPCR, quantitative real-time PCR; ELISA, enzyme linked immunosorbent assay.

使用ELISA检测细胞培养上清液中蛋白浓度水平，结果显示过表达PRSS23的BJ细胞分泌的IL-6较空载体对照组增多[(211.600±2.431) ng/L vs. (65.930±1.768) ng/L；Unpaired t检验: t=48.47, P＜0.001]，过表达PRSS23的BJ细胞分泌的TNF-α也较空载体对照组增多[(41.190±0.949) ng/L vs. (31.150±0.360) ng/L；Unpaired t检验: t=11.13，P＜0.001；图 4C、D]。数据统计结果见表 5。

表5 PRSS23对皮肤成纤维细胞炎症因子分泌的影响

Table 5 Effect of PRSS23 on the secretion of inflammatory cytokines in skin fibroblasts

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^***

** P＜0.01, *** P＜0.001, mock vs. overexpressed. Mock, empty vector control group; Overexpressed, PRSS23 overexpression group; PRSS23, serine protease 23; SE, standard error; IL-6, interleukin 6; TNF-α, tumor necrosis factor alpha.

3 讨论

本研究通过体外实验证明了SSc患者的皮肤成纤维细胞中PRSS23表达显著上调，这一结论与以往的研究结果一致，2019年Xu等^[4]运用生物信息学技术对SSc和健康皮肤样本进行筛选，发现PRSS23在SSc患者皮肤组织中显著上调，在后续的通路富集中发现，PRSS23可能与凋亡的负向调控及细胞因子活性相关。单细胞测序研究也发现，皮肤瘢痕组织中成纤维细胞PRSS23基因表达上调^{[5, 11]}。在SSc患者的皮肤成纤维细胞中，PRSS23的表达水平与改良Rodnan皮肤评分(modified Rodnan skin score，mRSS)呈正相关^[5]，提示PRSS23与皮肤纤维化密切相关，PRSS23阳性的成纤维细胞具有致纤维化作用。

我们的研究进一步发现，过表达PRSS23的皮肤成纤维细胞通过下调激活型Caspase-3抑制过氧化氢诱导的皮肤成纤维细胞凋亡，这一结果与此前生物信息分析的推论一致^[4]。此外，在胃癌的研究中也发现，PRSS23能促进癌细胞抵抗凋亡，敲减PRSS23后，胃癌细胞中激活型Caspase 3表达上调并观察到发生早期凋亡的细胞比例增加^[9]。替拉那韦诱导胃癌干细胞凋亡的机制也是通过靶向PRSS23，从PRSS23和丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)激酶3(MAPK kinase 3，MKK3)的复合物中释放出MKK3，MKK3能激活p38丝裂原活化蛋白激酶，进而激活IL-24介导的线粒体凋亡通路^[12]。表明PRSS23参与了细胞凋亡过程，在纤维化疾病中，PRSS23是成纤维细胞逃避凋亡的关键基因。

过表达PRSS23可促进成纤维细胞分泌IL-6和TNF-α，其中对IL-6的影响最显著。在纤维化疾病中，经历凋亡逃避的成纤维细胞持续存在，并可能分泌促炎因子^{[2, 13]}。急性炎症是正常伤口愈合的一部分，如果炎症无法消退或转变为慢性炎症，可导致组织修复异常并促进纤维化的发生和发展^[14-16]。人们在多种具有纤维化特征的慢性炎症疾病中发现了IL-6^[17]。关于SSc的研究证明，IL-6通过酪氨酸蛋白激酶2、信号转导及转录激活因子3和细胞外信号调节激酶(extracellular signal-regulated kinase，ERK)通路促进皮肤纤维化^[18]。此外，IL-6是SSc较好的治疗靶点，Khanna等^[19]进行的2期临床试验初步证明了托珠单抗治疗SSc的有效性。TNF-α也在多种纤维化疾病中发挥了重要作用，包括肝、肺和皮肤纤维化^[20]。瘢痕疙瘩中TNF-α的表达高于正常组织^[21]。低浓度TNF-α刺激可导致瘢痕疙瘩中成纤维细胞的增殖^[22]。表明PRSS23是SSc皮肤纤维化的关键致病基因，其可通过促进IL-6和TNF-α的分泌导致纤维化。

根据功能的差异，成纤维细胞可分为不同的亚群^[23-25]。以往的研究主要关注成纤维细胞分泌ECM的能力，而忽略了促炎成纤维细胞的作用。促炎成纤维细胞释放的炎性细胞因子导致慢性炎症，阻碍了组织的正常修复，是纤维化过程中的一个重要环节^{[24, 26]}。本研究结果表明，PRSS23阳性的成纤维细胞具有促炎成纤维细胞的表型，PRSS23是纤维化过程中导致炎症持续存在的一个关键因素。

综上所述，本研究发现SSc患者皮肤组织中的PRSS23表达显著升高，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 mRNA和蛋白表达水平显著上调。皮肤成纤维细胞在过表达PRSS23后，其抗凋亡能力增强，过氧化氢诱导后凋亡细胞的比例显著减少，凋亡相关蛋白激活型Caspase-3的表达水平也显著降低。此外，PRSS23过表达的皮肤成纤维细胞中IL-6和TNF-α的mRNA水平和蛋白分泌水平显著上调。SSc患者皮肤成纤维细胞中PRSS23参与了皮肤纤维化的发生和发展，PRSS23通过抑制细胞凋亡、促进炎症因子分泌使皮肤成纤维细胞持续存在并分泌大量的炎症因子导致慢性炎症，因此，PRSS23可能是治疗SSc有希望的靶点，值得进一步探索。

利益冲突 所有作者均声明不存在利益冲突

作者贡献声明 袁显墩：研究设计，数据收集、分析，文章撰写；李照华：研究设计，数据分析，文章撰写；徐丹、李婷、方丹、穆荣：研究设计，文章撰写及修改。所有作者均对最终文稿进行审读并确认。

参考文献

原文顺序 | 文献年度倒序 | 文中引用次数倒序

1	Domsic RT, Rodriguez-Reyna T, Lucas M, et al. Skin thickness progression rate: A predictor of mortality and early internal organ involvement in diffuse scleroderma[J]. Ann Rheum Dis, 2011, 70(1): 104- 109. DOI

2	Hinz B, Lagares D. Evasion of apoptosis by myofibroblasts: A hallmark of fibrotic diseases[J]. Nat Rev Rheumatol, 2020, 16(1): 11- 31. DOI

3	Dodi AE, Ajayi IO, Chang C, et al. Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli[J]. Respir Res, 2018, 19(1): 91. DOI

4	Xu C, Meng LB, Duan YC, et al. Screening and identification of biomarkers for systemic sclerosis via microarray technology[J]. Int J Mol Med, 2019, 44(5): 1753- 1770.

5	Tabib T, Huang M, Morse N, et al. Myofibroblast transcriptome indicates SFRP2(hi) fibroblast progenitors in systemic sclerosis skin[J]. Nat Commun, 2021, 12(1): 4384. DOI

6	Kanno Y. The role of fibrinolytic regulators in vascular dysfunction of systemic sclerosis[J]. Int J Mol Sci, 2019, 20(3): 619. DOI

7	Menou A, Duitman J, Crestani B. The impaired proteases and anti-proteases balance in idiopathic pulmonary fibrosis[J]. Matrix Biol, 2018(68/69): 382- 403.

8	Page MJ, Di Cera E. Serine peptidases: Classification, structure and function[J]. Cell Mol Life Sci, 2008, 65(7/8): 1220- 1236.

9	Han B, Yang Y, Chen J, et al. PRSS23 knockdown inhibits gastric tumorigenesis through EIF2 signaling[J]. Pharmacol Res, 2019, 142, 50- 57. DOI

10	Rawlings ND, Barrett AJ, Bateman A. MEROPS: The peptidase database[J]. Nucleic Acids Res, 2010, 38(Database issue): D227- D233.

11	Deng CC, Hu YF, Zhu DH, et al. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases[J]. Nat Commun, 2021, 12(1): 3709. DOI

12	Xiong J, Li Y, Tan X, et al. Targeting PRSS23 with tipranavir induces gastric cancer stem cell apoptosis and inhibits growth of gastric cancer via the MKK3/p38 MAPK-IL24 pathway[J]. Acta Pharmacol Sin, 2024, 45(2): 405- 421. DOI

13	Fan L, Wang Q, Liu R, et al. Citrullinated fibronectin inhibits apoptosis and promotes the secretion of pro-inflammatory cytokines in fibroblast-like synoviocytes in rheumatoid arthritis[J]. Arthritis Res Ther, 2012, 14(6): R266. DOI

14	Chaudhuri V, Zhou L, Karasek M. Inflammatory cytokines induce the transformation of human dermal microvascular endothelial cells into myofibroblasts: A potential role in skin fibrogenesis[J]. J Cutan Pathol, 2007, 34(2): 146- 153. DOI

15	Johnson BZ, Stevenson AW, Prêle CM, et al. The role of IL-6 in skin fibrosis and cutaneous wound healing[J]. Biomedicines, 2020, 8(5): 101. DOI

16	Borthwick LA, Wynn TA, Fisher AJ. Cytokine mediated tissue fibrosis[J]. Biochim Biophys Acta, 2013, 1832(7): 1049- 1060. DOI

17	Li Y, Zhao J, Yin Y, et al. The role of IL-6 in fibrotic diseases: Molecular and cellular mechanisms[J]. Int J Biol Sci, 2022, 18(14): 5405- 5414. DOI

18	Khan K, Xu S, Nihtyanova S, et al. Clinical and pathological significance of interleukin 6 overexpression in systemic sclerosis[J]. Ann Rheum Dis, 2012, 71(7): 1235- 1242. DOI

19	Khanna D, Denton CP, Jahreis A, et al. Safety and efficacy of subcutaneous tocilizumab in adults with systemic sclerosis (faSScinate): A phase 2, randomised, controlled trial[J]. Lancet, 2016, 387(10038): 2630- 2640. DOI

20	Bahcecioglu IH, Koca SS, Poyrazoglu OK, et al. Hepatoprotective effect of infliximab, an anti-TNF-alpha agent, on carbon tetrachloride-induced hepatic fibrosis[J]. Inflammation, 2008, 31(4): 215- 221. DOI

21	Messadi DV, Doung HS, Zhang Q, et al. Activation of NFkappaB signal pathways in keloid fibroblasts[J]. Arch Dermatol Res, 2004, 296(3): 125- 133.

22	Zhu G, Cai J, Zhang J, et al. Abnormal nuclear factor (NF)-kappaB signal pathway and aspirin inhibits tumor necrosis factor alpha-induced NF-kappaB activation in keloid fibroblasts[J]. Dermatol Surg, 2007, 33(6): 697- 708.

23	Li X, Sun Z, Peng G, et al. Single-cell RNA sequencing reveals a pro-invasive cancer-associated fibroblast subgroup associated with poor clinical outcomes in patients with gastric cancer[J]. Theranostics, 2022, 12(2): 620- 638. DOI

24	Solé-Boldo L, Raddatz G, Schütz S, et al. Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming[J]. Commun Biol, 2020, 3(1): 188. DOI

25	Griffin MF, desJardins-Park HE, Mascharak S, et al. Understanding the impact of fibroblast heterogeneity on skin fibrosis[J]. Dis Model Mech, 2020, 13(6): dmm044164. DOI

26	Sato Y, Yanagita M. Resident fibroblasts in the kidney: A major driver of fibrosis and inflammation[J]. Inflamm Regen, 2017, 37, 17. DOI

Options

文章导航

模态框（Modal）标题

摘要

本文引用格式

Abstract

1 资料与方法

1.1 组织标本来源和免疫组织化学检测

1.2 人皮肤原代成纤维细胞分离

1.3 细胞培养

1.4 细胞转染和分组

1.5 实时荧光定量PCR

表1 IL-6、TNF-α、PRSS23引物序列

1.6 蛋白免疫印迹法检测PRSS23和激活型Caspase-3表达

1.7 流式细胞检测

1.8 酶联免疫吸附试验

1.9 统计学分析

2 结果

2.1 SSc患者皮肤成纤维细胞中PRSS23的表达

图1 SSc患者皮肤组织成纤维细胞中PRSS23的表达

表2 PRSS23在SSc患者皮肤组织成纤维细胞中的表达

2.2 PRSS23抑制皮肤成纤维细胞凋亡

图2 转染PRSS23质粒的皮肤成纤维细胞过表达PRSS23

表3 PRSS23在转染PRSS23质粒的皮肤成纤维细胞中的表达

图3 过表达PRSS23抑制皮肤成纤维细胞凋亡

表4 PRSS23抑制皮肤成纤维细胞凋亡率和相关凋亡蛋白的测定

2.3 PRSS23促进皮肤成纤维细胞分泌炎症因子

图4 过表达PRSS23促进皮肤成纤维细胞分泌促炎因子IL-6和TNF-α

表5 PRSS23对皮肤成纤维细胞炎症因子分泌的影响

3 讨论

参考文献