hPP10-Cu，Zn-SOD融合蛋白的制备、穿膜效应及其抗氧化、抗炎症功效

doi:10.12122/j.issn.1673-4254.2024.06.06

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1059-1069.doi: 10.12122/j.issn.1673-4254.2024.06.06

• • 上一篇

hPP10-Cu，Zn-SOD融合蛋白的制备、穿膜效应及其抗氧化、抗炎症功效

张洁¹(), 姚君妍³, 杨英桂⁴, 王飞⁶, 郑清友⁴, 李欣⁵, 柳长柏²()

^1.运城护理职业学院，山西运城 04400
^2.三峡大学肿瘤微环境与免疫治疗湖北省重点实验室，湖北宜昌 443002；南方医科大学深圳医院3. 麻醉手术中心
^3.南方医科大学深圳医院，麻醉手术中心
^4.南方医科大学深圳医院，泌尿外科
^5.南方医科大学深圳医院，临床医学创新中心，广东深圳 518100
^6.海南省人民医院泌尿外科，海南海口 570100

收稿日期:2023-12-20 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 柳长柏 E-mail:zhangjie5229@163.com;cbliu@ctgu.edu.cn
作者简介:张洁，硕士，讲师，E-mail: zhangjie5229@163.com
基金资助:
国家自然科学基金(81902788);深圳市科技计划(JCYJ20210324130801004);海南省重点研发项目基金(SQ2021SHFZ0739)

Cell membrane-penetrating capacity of hPP10-Cu, Zn-SOD fusion protein and its antioxidant and anti-inflammatory activity

Jie ZHANG¹(), Junyan YAO³, Yinggui YANG⁴, Fei WANG⁶, Qingyou ZHENG⁴, Xin LI⁵, Changbai LIU²()

^1.Yuncheng Vocational Nursing College, Yuncheng 04400, China
^2.China Three Gorges University, Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Yichang 443002, China
^3.Anesthesia Surgery Center, Shenzhen Hospital of Southern Medical University Shenzhen 518100, China
^4.Department of Urology, Shenzhen Hospital of Southern Medical University Shenzhen 518100, China
^5.Clinical Innovation and Research Center, Shenzhen Hospital of Southern Medical University, Shenzhen 518100, China
^6.Department of Urology, Hainan Provincial People's Hospital, Haikou 570100, China

Received:2023-12-20 Online:2024-06-20 Published:2024-07-01
Contact: Changbai LIU E-mail:zhangjie5229@163.com;cbliu@ctgu.edu.cn
Supported by:
National Natural Science Foundation of China(81902788)

摘要/Abstract

摘要：

目的探究人源性细胞膜穿透肽hPP10携带人源性抗氧化蛋白Cu，Zn-SOD的穿膜效应并观察其抗氧化、抗炎功效。方法利用RT-PCR技术扩增人源性SOD基因片段、酶切连接法构建重组质粒pET15b-Cu，Zn-SOD，pET15b-hPP10-Cu，Zn-SOD；利用镍柱亲和层析法、分子筛方法纯化Cu，Zn-SOD、hPP10-Cu，Zn-SOD融合蛋白并利用Western blotting法鉴定其正确性；利用免疫荧光法、荧光共定位实验、Western blotting法鉴定hPP10-Cu，Zn-SOD在细胞中的穿膜效应；利用Western blotting法鉴定hPP10-Cu，Zn-SOD融合蛋白穿膜时间梯度和浓度梯度效应；利用SOD酶活性测定试剂盒检测hPP10-Cu，Zn-SOD穿膜后的SOD酶活性；利用MTT法检测hPP10对细胞活性的影响。利用H₂O₂建立HEK293氧化应激细胞模型，通过流式细胞分析术（FCM）分析hPP10-Cu，Zn-SOD穿膜后细胞凋亡情况；并RT-qPCR分析hPP10-Cu，Zn-SOD穿膜后凋亡相关因子的表达情况；通过ROS检测分析hPP10-Cu，Zn-SOD穿膜后抗氧化能力。TPA诱导建立小鼠耳部炎症模型（5只/组，共4组），然后RT-qPCR分析hPP10-Cu，Zn-SOD穿膜后，NFκB，IL-1β、IL-6、TNFα因子的转录情况；Western blotting检测NFκB p65、p-NFκB p65、IL-1β、TNFα基因的表达；免疫组化分析炎性因子p-NFκB p65、TNFα基因的表达。结果成功构建了重组质粒pET15b-Cu，Zn-SOD，pET15b-hPP10-Cu，Zn-SOD，并获得Cu，Zn-SOD蛋白和hPP10-Cu，Zn-SOD融合蛋白；免疫荧光试验结果表明5 µmol/L浓度的hPP10-Cu，Zn-SOD转导HEK293细胞具有明显的穿膜效应；荧光共定位实验显示融合蛋白hPP10-Cu，Zn-SOD入胞后定位在细胞膜内，部分蛋白定位在细胞核内；Western blotting实验结果表明hPP10-Cu，Zn-SOD转导Hela（ P<0.05），HEK293（ P<0.01）细胞具有浓度依赖性，细胞内hPP10-Cu，Zn-SOD存在可持续24 h；5 µmol/L的hPP10-Cu，Zn-SOD转导细胞具有较好的抗氧化活性（ P<0.01）；MTT结果显示高达10 µmol/L浓度的hPP10-Cu，Zn-SOD对于细胞活力的影响小。在氧化应激模型中，FCM结果显示hPP10-Cu，Zn-SOD融合蛋白孵育细胞降低了细胞的早期凋亡（ P<0.01）和晚期凋亡（ P<0.05）；RT-qPCR结果显示hPP10-Cu，Zn-SOD融合蛋白孵育细胞使Bcl2、Mcl1、Deptor等抗凋亡因子升高（ P<0.05），降低了Bad、P21、P27等促凋亡因子的表达（ P<0.05）；ROS结果显示hPP10-Cu，Zn-SOD融合蛋白孵育细胞显著降低了细胞内的活性氧含量（ P<0.01）。在炎症模型中，hPP10-Cu，Zn-SOD融合蛋白可显著降低IL-1β、IL-6、TNFα因子的转录（ P<0.05）；p-NFκB p65、IL-1β及TNFα的表达都呈现了抑制作用（ P<0.05）；hPP10-Cu，Zn-SOD融合蛋白处理组较Cu，Zn-SOD蛋白处理组，降低了炎性因子p-NFκB p65及TNFα基因的表达（ P<0.05）。结论融合蛋白hPP10-Cu，Zn-SOD具有明显的穿膜入胞、抗氧化、抗炎功效，且对细胞毒副作用很小。这些结果为hPP10作为新的递送载体奠定基础，也为hPP10-Cu，Zn-SOD应用于护肤品等提供了理论依据。

关键词: 人源性细胞膜穿透肽hPP10, 铜锌超氧化物歧化酶Cu，Zn-SOD, 抗氧化, 抗炎, 酶活力测定

Abstract:

Objective To investigate the cell membrane-penetrating capacity of human cell-penetrating peptide hPP10 carrying human antioxidant protein Cu-Zn superoxide dismutase (Cu, Zn-SOD) and assess the antioxidant and anti-inflammatory activity of these fusion proteins. Methods The fusion protein hPP10-Cu, Zn-SOD was obtained by genetic engineering and identified by Western blotting. The membrane-penetrating ability of the fusion protein was evaluated by immunofluorescence assay, fluorescence colocalization assay and Western blotting, its SOD enzyme activity was detected using a commercial kit, and its effect on cell viability was assessed with MTT assay. In a HEK293 cell model of H2O2-induced oxidative stress, the effect of hPP10-Cu, Zn-SOD on cell apoptosis was analyzed with flow cytometry and RT-qPCR, and its antioxidant effect was assessed using reactive oxygen species (ROS) assay; its anti-inflammatory effect was evaluated in mouse model of TPA-induced ear inflammation by detecting expression of the inflammatory factors using RT-qPCR, Western blotting and immunohistochemistry. Results The fusion protein hPP10-Cu, Zn-SOD was successfully obtained. Immunofluorescence assay confirmed obvious membrane penetration of this fusion protein in HEK293 cells, localized both in the cell membrane and the cell nuclei after cell entry. hPP10-Cu, Zn-SOD at the concentration of 5 µmol/L exhibited strong antioxidant activity with minimal impact on cell viability at the concentration up to 10 µmol/L. The fusion protein obviously inhibited apoptosis and decreased intracellular ROS level in the oxidative stress cell model and significantly reduced mRNA and protein expression of the inflammatory factors in the mouse model of ear inflammation. Conclusion The fusion protein hPP10-Cu, Zn-SOD capable of penetrating the cell membrane possesses strong antioxidant and anti-inflammatory activities with only minimal cytotoxicity, demonstrating the value of hPP10 as an efficient drug delivery vector and the potential of hPP10-Cu, Zn-SOD in the development of skincare products

Key words: human cell membrane penetrating peptide hPP10, Cu-Zn superoxide dismutase Cu, Zn-SOD, antioxidant, anti-inflammatory, enzyme activity assay

张洁, 姚君妍, 杨英桂, 王飞, 郑清友, 李欣, 柳长柏. hPP10-Cu，Zn-SOD融合蛋白的制备、穿膜效应及其抗氧化、抗炎症功效[J]. 南方医科大学学报, 2024, 44(6): 1059-1069.

Jie ZHANG, Junyan YAO, Yinggui YANG, Fei WANG, Qingyou ZHENG, Xin LI, Changbai LIU. Cell membrane-penetrating capacity of hPP10-Cu, Zn-SOD fusion protein and its antioxidant and anti-inflammatory activity[J]. Journal of Southern Medical University, 2024, 44(6): 1059-1069.

图/表 11

表1 SOD酶活检测体系

Tab.1 SOD enzyme activity detection system (mL)

Reagent	Control group	Assay group
Reagent I	1.0	1.0
Control supernatant	a*
Sample group supernatant		a*
Reagent II	1.0	1.0
Reagent III	1.0	1.0
Reagent IV	1.0	1.0
Mix thoroughly with a vortex mixer and place in a water bath at 37 ℃ for 40 minutes
Color developing agent	2.0	2.0

表2 RT-qPCR分析所用的引物序列

Tab.2 Primer sequences for quantitative real-time PCR analysis

Gene	Primer sequences
Bcl-2	Forward (5'-3')	GGTGGGGTCATGTGTGTGG
Bcl-2	Reverse (3'-5')	CGGTTCAGGTACTCAGTCATCC
Deptor	Forward (5'-3')	TTAGCAGACCGGGGCATTATT
Deptor	Reverse (3'-5')	GAAGGTGCCGTCATCCTTTCT
NF-κB	Forward (5'-3')	AGTATTCCTGGCGAGAAAG
NF-κB	Reverse (3'-5')	CTGTTCCTGGTCCTGTGTAG
IL-6	Forward (5'-3')	GACTTCCATCCAGTTGCCTTCT
IL-6	Reverse (3'-5')	AGACAGGTCTGTTGGGAGTGGTA
IL-1β	Forward (5'-3')	GAAATGCCACCTTTTGACAGTG
IL-1β	Reverse (3'-5')	TGGATGCTCTCATCAGGACAG
TNF-α	Forward (5'-3')	CAGGCGGTGCCTATGTCTC
TNF-α	Reverse (3'-5')	CGATCACCCCGAAGTTCAGTA

图1 pET15b- Cu, Zn-SOD、pET15b- hPP10-Cu, Zn-SOD重组质粒的构建和鉴定

Fig.1 Construction and identification of the recombinant plasmids pET15b-Cu, Zn-SOD and pET15b-hPP10-Cu, Zn-SOD. A: Total RNA. B: Human Cu, Zn-SOD gene fragment (462 bp). C: hPP10 gene fragment (about 60 bp). D: Identification of the recombinant plasmids (Lane 1: pET15b-hPP10-SOD; Lane 2: pET15b-SOD; Lane 3: pET15b-Cu, Zn-SOD digested by Xho I and BamH I (462 bp); Lane 4: pET15b-Cu, Zn-SOD digested by Nco I and Hind III (823 bp); Lane 5: pET15b-hpp10-Cu, Zn-SOD digested by Xho I and BamH I (462 bp); Lane 6: pET15b-Cu, Zn-SOD digested by Nco I and Hind III (892 bp).

图2 Cu, Zn-SOD、hPP10-Cu, Zn-SOD融合蛋白的诱导表达及纯化

Fig.2 Induced expression and purification of Cu, Zn-SOD and the fusion protein hPP10-Cu, Zn-SOD. Lane 1: Uninduced group; Lane 2: Induced group; Lane 3: Cu, Zn-SOD protein purified by imidazole; Lane 4: Cu, Zn-SOD protein purified by urea; Lane 5: Uninduced group; Lane 6: Induced group; Lane 7: hPP10-Cu, Zn-SOD protein purified by imidazole; Lane 8: hPP10-Cu, Zn-SOD protein purified by urea.

图3 His一抗和SOD一抗鉴定原核表达的纯化蛋白

Fig.3 Identification of purified proteins with His primary antibody ( A) and SOD primary antibody ( B).

图4 不同浓度的Cu, Zn-SOD蛋白及hPP10-Cu, Zn-SOD融合蛋白在HEK293细胞中的穿膜效应检测

Fig.4 Assessment of cell membrane-penetrating capacity of Cu, Zn-SOD protein and hPP10-Cu, Zn-SOD fusion protein at different concentrations in HEK293 cells (scale bar=50 μm).

图5 10 μmol/L的 Cu, Zn-SOD蛋白及hPP10-Cu, Zn-SOD融合蛋白穿膜后胞内定位检测

Fig.5 Intracellular localization of Cu, Zn-SOD protein and hPP10-Cu, Zn-SOD fusion protein at 10 μmol/L after cell membrane penetration (Arrow: hPP10-Cu, Zn-SOD).

图6 hPP10-Cu, Zn-SOD融合蛋白在Hela、HEK293细胞的穿膜效应检测

Fig.6 Cell membrane penetration by hPP10-Cu, Zn-SOD fusion protein in Hela and HEK293 cells. A: Concentration gradient effect of Cu, Zn-SOD protein transduction into Hela cells. B: Concentration gradient effect of hPP10-Cu, Zn-SOD fusion protein transduction into Hela cells (* P<0.05). C: Concentration gradient effect of Cu, Zn-SOD protein transduction into HEK293 cells. D: Concentration gradient effect of hPP10-Cu, Zn-SOD fusion protein transduction into HEK293 cells. ** P<0.01. E: Time gradient effect of hPP10-Cu, Zn-SOD fusion protein transduction into HEK293 cells.

图7 hPP10-Cu, Zn-SOD处理细胞后的酶活性及细胞活性分析

Fig.7 Analysis of enzyme activity and viability in cells treated with hPP10-Cu, Zn-SOD. A: Enzyme activities of hPP10-Cu, Zn-SOD treated cells detected by SOD kit. * P<0.05; ** P<0.01. B: The effect of hPP10-Cu, Zn-SOD treatment on cell viability detected by MTT assay.

图8 H2O2处理细胞后,hPP10-Cu, Zn-SOD对细胞凋亡的影响及抗氧化活性

Fig.8 Effect of hPP10-Cu, Zn-SOD on apoptosis and antioxidant activity in cells treated with H2O2. A: hPP10-Cu,Zn-SOD inhibits cell apoptosis induced by H2O2. B: hPP10-Cu, Zn-SOD inhibits early apoptosis induced by H2O2 (* P<0.05 vs H2O2+Cu, Zn-SOD). C: hPP10-Cu, Zn-SOD inhibited the early and late apoptosis induced by H2O2 (** P<0.01 vs H2O2+Cu, Zn-SOD). D: hPP10-Cu, Zn-SOD fusion protein promotes expression of anti-apoptotic factors and inhibits the transcription of pro-apoptotic factors (* P<0.05 vs H2O2+Cu, Zn-SOD). E: SOD fusion protein significantly reduces oxygen free radicals in the cells (** P<0.01 vs H2O2+Cu, Zn-SOD).

图9 hPP10-Cu, Zn-SOD降低TPA引起的炎症反应

Fig.9 hPP10-Cu, Zn-SOD reduces the inflammatory response induced by TPA. A: Modeling of ear inflammation by TPA in mice. B: Inhibitory effect of hPP10-Cu, Zn-SOD on TPA-induced ear inflammation detected by RT-PCR (* P<0.05 vs TPA+Cu, Zn-SOD). C: Inhibitory effect of hPP10-Cu, Zn-SOD on TPA-induced ear inflammation detected by Western blotting. D: Inhibitory effects of hPP10-Cu, Zn-SOD on TPA-induced ear inflammation analyzed by immunohistochemistry.

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hPP10-Cu，Zn-SOD融合蛋白的制备、穿膜效应及其抗氧化、抗炎症功效

Cell membrane-penetrating capacity of hPP10-Cu, Zn-SOD fusion protein and its antioxidant and anti-inflammatory activity

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