Protocol for Studying Honokiol Regulation of Transforming Growth Factor Beta 1/Smad Signaling in Ultraviolet A–induced Skin Photoaging Models

Summary

This protocol investigates whether honokiol ameliorates UVA-induced photo-aging in human dermal fibroblasts and mouse skin by activating the TGF-β1/Smad pathway and reducing MMP expression.

Abstract

Honokiol (HNK), a key component derived from Magnolia officinalis, has the potential to treat skin diseases. At present, the impact and mechanism of action of HNK on Ultraviolet A (UVA)-induced photodamage in the skin are unclear. A photo-aging damage model of HDF cells and hairless mouse skin was constructed by UVA induction. The impact of HNK on the viability of normal HDF cells and UVA-induced HDF cells was examined through the Cell viability assay. SOD, GSH, CAT, and MDA levels in HDF cells were measured using different kits, and ROS levels were measured using DCFH-DA and Dihydroethidium. SA-β-galactosidase and TUNEL staining detected senescence and apoptosis in HDF cells, and flow cytometry assessed cell cycle progression. Histopathological damage to the skin was evaluated through pathological staining. In addition, immunofluorescence and Western blot were used to assess Ki67, collagen, matrix metalloproteinase (MMP), and TGF-β1/Smad pathway-associated protein levels. HNK (2.5-10 µM) treatment had no adverse impact on normal HDF cell viability but attenuated UVA-induced HDF cell damage, reducing oxidative stress and apoptosis. UVA induced senescence of HDF cells, induced S-phase arrest, downregulated Ki67, decreased collagen content, and increased MMP expression, while HNK effectively attenuated these UVA-induced abnormalities. In addition, UVA caused deeper wrinkles, decreased water content and collagen content, and increased epithelial thickness in mouse skin, whereas subcutaneous injection of HNK attenuated pathological damage to skin tissue and increased water and collagen content. Furthermore, HNK activated the TGF-β1/Smad pathway, and the TGF-β1 inhibitor SB431542 impaired the protective effect of HNK against photo-aging damage in HDF cells. In conclusion, HNK has the potential to be a drug for ameliorating photo-aging damage by reducing MMP expression through activation of the TGF-β1/Smad pathway, thereby inhibiting UVA-induced photo-aging in HDF cells and mouse skin.

Introduction

The process of skin aging is intricate and divided into two main forms: natural aging and photo-aging¹. Studies indicate that as much as 80% to 90% of skin aging is caused by exogenous factors in the environment (e.g., ultraviolet rays, air pollution), of which long-term ultraviolet radiation-induced photo-aging has the most serious effects^2,3. Ultraviolet radiation is divided into UVC, UVB, and UVA. Since UVC cannot penetrate the ozone layer, current research on photo-aging focuses on UVA (320-400 nm) and UVB (280-320 nm)^4,5

Protocol

All cell culture experiments were performed in accordance with institutional biosafety guidelines and conducted under Biosafety Level 2 (BSL-2) laboratory conditions. Animal experiments were performed after approval from the Animal Ethics Committee of the 988th Hospital of the Joint Logistic Support Force of the Chinese People’s Liberation Army (approval number: 2023-089).

All experimental procedures were conducted in strict accordance with institutional and national guidelines for the care and use of laboratory animals. At the end of the experiment, mice were euthanized via an overdose of sodium pentobarbital (100 mg/kg, intraperiton....

Results

HNK alleviates oxidative stress damage in HDF cells caused by UVA
The impacts of various concentrations of HNK treatment for 24 h or 48 h on the viability of HDF cells were examined by Cell viability assay to screen for the appropriate concentration and treatment duration. The results showed that 2.5, 5, and 10 µM of HNK did not significantly affect the viability of normal HDF cells after 24 h or 48 h of treatment, demonstrating that HNK is non-toxic to normal cells at these doses. Notably, HNK at co.......

Discussion

Ultraviolet light is one environmental factor that harms cells and organisms. Previous studies have reported that UVA, with its high penetrating power, is the main type of ultraviolet light that reaches the Earth's surface³⁸. Fibroblasts are important functional cells in the dermis, and their aging can directly or indirectly cause skin aging³⁹. UVA-induced photo-aging damage in the HDF cell model and the hairless mouse model is widely used in the study of skin photo-aging

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ALT activity assay kit	Beyotime Biotechnology, Shanghai, China	P2711M	alanine transaminase activity detection kit
Amplex Red CRE assay kit	Beyotime Biotechnology, Shanghai, China	S0291S	creatinine detection kit
AntiFade mounting medium	Beyotime Biotechnology, Shanghai, China	P0126	fluorescence signal preservation mounting medium
AST activity assay kit	Beyotime Biotechnology, Shanghai, China	P2715S	aspartate transaminase activity detection kit
BCA protein assay kit	Beyotime Biotechnology, Shanghai, China	P0012	protein concentration determination kit
BSA	Sigma-Aldrich, St. Louis, MO, USA	V900933	blocking reagent for non-specific binding
BUN Assay kit	Beyotime Biotechnology, Shanghai, China	S0574S	blood urea nitrogen detection kit
CAT Assay kit	Beyotime Biotechnology, Shanghai, China	S0051	catalase activity detection kit
CCK-8 reagent	Sigma-Aldrich, St. Louis, MO, USA	96992	cell viability assay solution which is a water-soluble tetrazolium salt
CDK2 antibody	Invitrogen, Carlsbad, CA, USA	PA5-79024	primary antibody for CDK2 detection
Chemiluminescent agent ECL	MedChemExpress, Monmouth Junction, NJ, USA	HY-K1005	enhanced chemiluminescence detection reagent
Collagen I antibody	Invitrogen, Carlsbad, CA, USA	PA1-26204	primary antibody for collagen I detection
Cyclin A2 antibody	Invitrogen, Carlsbad, CA, USA	PA5-34682	primary antibody for Cyclin A2 detection
DAPI staining solution	Sigma-Aldrich, St. Louis, MO, USA	D9542	nuclear staining dye
DCFH-DA fluorescent probe	MedChemExpress, Monmouth Junction, NJ, USA	HY-D0940	reactive oxygen species fluorescent probe
Differentiation solution	Beyotime Biotechnology, Shanghai, China	C0161s	reagent for hematoxylin differentiation step
Dihydroethidium probe	Beyotime Biotechnology, Shanghai, China	S0063	reactive oxygen species probe
Fibroblast culture medium	Sunncell Biotechnology Co., Ltd., Wuhan, Hubei, China	SNPM-H342	complete culture medium for fibroblast growth
FITC-labeled goat anti-rabbit IgG	Invitrogen, Carlsbad, CA, USA	F-2765	fluorescein isothiocyanate (FITC)-labeled secondary antibody
Flow cytometry	BD biosciences, San Jose, CA, USA	BD FACSCaliburTM	flow cytometer
FlowJo software	BD biosciences, San Jose, CA, USA	v10.8	flow cytometry analysis software
Fluorescence microscope	Leica, Heidelberg, Germany	DM IL LED	microscope for fluorescence imaging
GAPDH antibody	Invitrogen, Carlsbad, CA, USA	PA1-987	internal control antibody for protein normalization
gel imaging system	Invitrogen, Carlsbad, CA, USA	iBright CL1500	imaging system for protein detection
goat anti-rabbit IgG	Invitrogen, Carlsbad, CA, USA	31460	secondary antibody for immunodetection
Graphpad Prism 9.0 software	GraphPad Software, San Diego, CA, USA	Prism 9.0	graphing software
GSH Assay kit	Beyotime Biotechnology, Shanghai, China	S0053	glutathione content detection kit
HE staining kit	Beyotime Biotechnology, Shanghai, China	C0105S	histological staining kit for tissue morphology analysis
HNK	MedChemExpress, Monmouth Junction, NJ, USA	HY-N0003	honokiol, bioactive compound
Human dermal fibroblasts	Sunncell Biotechnology Co., Ltd., Wuhan, Hubei, China	SNP-H342	primary human skin fibroblast cells
IBM SPSS Statistics 26.0 software	IBM Corporation, Armonk, NY, USA	IBM SPSS Statistics 26.0	statistical analysis software
ImageJ software	Wayne Rasband, National Institute of Mental Health, USA	version 1.54h	image analysis software
Ki67 antibody	Invitrogen, Carlsbad, CA, USA	MA5-14520	primary antibody for cell proliferation marker detection
Light microscope	Leica, Heidelberg, Germany	DM3000	optical microscope for brightfield imaging
Masson trichrome staining kit	Solarbio, Beijing, China	G1340	collagen and connective tissue staining kit
MDA Assay kit	Beyotime Biotechnology, Shanghai, China	S0131S	malondialdehyde level detection kit (lipid peroxidation marker)
Microplate Reader	Thermo Fisher Scientific, Waltham, MA, USA	1410101	absorbance measurement instrument for cell-based assays
MMP-1 antibody	Invitrogen, Carlsbad, CA, USA	PA5-27210	primary antibody for matrix metalloproteinase-1 detection
MMP-1 ELISA kit	Enzyme-linked Biotechnology, Shanghai, China	ml038199	quantitative detection of MMP-1 in biological samples
MMP-3 antibody	Abcam, Cambridge, MA, USA	ab52915	primary antibody for matrix metalloproteinase-3 detection
MMP-3 ELISA kit	Enzyme-linked Biotechnology, Shanghai, China	ml105322	quantitative detection of MMP-3 in biological samples
Neutral gum	Beyotime Biotechnology, Shanghai, China	C0173	mounting medium
P16 antibody	Invitrogen, Carlsbad, CA, USA	PA5-20379	primary antibody for p16 protein detection
P21 antibody	Invitrogen, Carlsbad, CA, USA	MA5-14949	primary antibody for p21 protein detection
P53 antibody	Abcam, Cambridge, MA, USA	ab131442	primary antibody for p53 protein detection
paraformaldehyde	Solarbio, Beijing, China	P1110	cell and tissue fixation reagent
PI	Beyotime Biotechnology, Shanghai, China	ST512	propidium iodide for DNA staining in cell cycle analysis
Pro-collagen I C peptide assay kit	TAKARA, Tokyo, Japan	MK101	collagen synthesis detection kit
p-Smad2 antibody	Invitrogen, Carlsbad, CA, USA	44-244G	primary antibody for phosphorylated Smad2 detection
p-Smad3 antibody	Invitrogen, Carlsbad, CA, USA	44-246G	primary antibody for phosphorylated Smad3 detection
PVDF membranes	Invitrogen, Carlsbad, CA, USA	88520	protein transfer membrane for western blotting
RIPA lysis buffer	Beyotime Biotechnology, Shanghai, China	P0013B	protein extraction buffer for cell and tissue lysates
RNaseA	Beyotime Biotechnology, Shanghai, China	ST579	ribonuclease for RNA removal during DNA staining
SA-β-galactosidase staining kit	Beyotime Biotechnology, Shanghai, China	C0602	cellular senescence detection kit
SB431542	MedChemExpress, Monmouth Junction, NJ, USA	HY-10431	TGF-β1 signaling pathway inhibitor
SDS-PAGE gels	Invitrogen, Carlsbad, CA, USA	WG1403BX10	polyacrylamide gels for protein electrophoresis
SKH1 hairless mice	Vitalriver, Beijing, China		animal model for skin photo-aging studies
Smad2 antibody	Invitrogen, Carlsbad, CA, USA	51-1300	primary antibody for Smad2 detection
Smad3 antibody	Invitrogen, Carlsbad, CA, USA	51-1500	primary antibody for Smad3 detection
SOD Assay kit	Beyotime Biotechnology, Shanghai, China	S0101S	superoxide dismutase activity detection kit
TGF-β1 antibody	Abcam, Cambridge, MA, USA	ab215715	primary antibody for TGF-β1 detection
Triton X-100	Sigma-Aldrich, St. Louis, MO, USA	X-100	non-ionic detergent for cell membrane permeabilization
TUNEL assay solution	Beyotime Biotechnology, Shanghai, China	C1086	apoptosis detection reagent for DNA fragmentation
Xylene	Sigma-Aldrich, St. Louis, MO, USA	247642	organic solvent for tissue deparaffinization