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

doi:10.12122/j.issn.1673-4254.2024.06.06

Abstract

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

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.

Figures/Tables 11

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

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

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).

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.

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

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).

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).

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.

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.

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).

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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