The protective effect of Sargassum on UVB-induced photodamage was evaluated by L929 cells and zebrafish models

Recently, the research team of Dr.
Chen Bei of Fujian Fisheries Research Institute and the research team of the School of Life Sciences of Xiamen University jointly published the "Photoprotective effects of Sargassum thunbergii on ultraviolet B-induced mouse L929" in BMC complementary and alternative medicine Fibroblasts and Zebrafish" research paper
.

This project was supported by the zebrafish experiment provided by Cyclosbio, which evaluated the protective effect of Sargassum on ultraviolet B-induced photodimulation through L929 cells and zebrafish models, aiming to explore the photoprotective effect
of Sargasso ethanol extract.

At present, with the gradual improvement of people's living standards, people's demand for delaying skin aging is becoming more and more intense, and the awareness of protecting the skin is gradually improving
.

The skin is the first barrier
that separates the human body from the external environment.

Sustained exposure to ultraviolet B (UVB) can trigger skin lesions
such as acute or chronic skin disorders, injuries, skin cancer, and skin aging by altering signaling pathways associated with inflammation, oxidative stress, and DNA damage.

How to prevent skin exposure to UV or relieve skin UV damage, thereby delaying skin photoaging is increasingly concerning
.

Seaweed is rich in bioactive compounds with antioxidant and anti-infective properties
.

By preparing Sargassum is enriched with phenol extract (STPE), its activity
against UVB damage is evaluated with L929 fibroblasts and zebrafish.

Extract and purify STPE
with 40% ethanol and macroporous resin XDA-7.

Analyze reactive oxygen species (ROS) and antioxidant markers such as superoxide dismutase (SOD), catalase (CAT) activity, and malondialdehyde (MDA) content
.

Determination of the effect of STPE on UVB-induced inflammation by inflammatory cytokine gene and protein expression; Determination of signaling molecule expression in nuclear factor-κB (NF-κB) pathways by Western blotting; Analyze and observe DNA aggregulation by Hoechst 33342 staining; In vivo evaluation
was performed by zebrafish model tail fin area and ROS measurements.

Results: The total polyphenol content of STPE was 72%, stPE reduced the ROS content in L929 cells, increased SOD and CAT activity, and significantly reduced MDA content, thereby effectively alleviating UVB radiation-induced oxidative damage
.

STPE inhibits mRNA and protein expression
of TNF-α, IL-6, and IL-1α.

Compared to UVB controls, STPE reversed DNA aggregulation
at concentrations of 20 and 40 μg/mL.

In addition, STPE inhibits the activation of the NF-κB signaling pathway in L929 cells after UVB irradiation, reducing DNA coagulation
.

1.
67 μg/mL STPE significantly increased the zebratail fn area, and 0.
8-1.
6 μg/mLSTPE significantly eliminated excess ROS
after UVB radiation.

Conclusion: STPE inhibits UVB-induced oxidative stress, inflammatory cytokine expression and DNA clotting by down-regulating the NF-κB signaling pathway, indicating that it can prevent UVB-induced photodamage and has the potential
for clinical development in the treatment of skin diseases.

● Review Among the types of ultraviolet (UV) that are not absorbed by the ozone layer, ultraviolet B (UVB) (wavelength 280-315 nm) is the main environmental risk factor
for the pathogenesis of skin injury.

The wavelength of UVB is shorter, 30-40 times stronger than the energy of UVA (320–400 nm
).

Many skin conditions, such as cancer, photoaging, sunburn, and pigmentation, are strongly associated
with UVB exposure.

Due to its short wavelength, UVB is mainly absorbed by the epidermis; However, it can also penetrate this skin layer to reach the dermis and affect the physiological function of fibroblasts
.

Research on UV-irradiated skin or dermal fibroblasts focuses on DNA mutations, inflammation, and photoaging
.

Marine organisms can reduce the damage caused by UV radiation through a variety of physiological and biochemical defense mechanisms such as physical or chemical barrier action, antioxidant effect, pigment inhibition effect, and repair function of nucleic acids and proteins
.

Recently, due to the growing demand for anti-aging, anti-allergic and whitening natural products in the skin health industry, marine bioactive substances have gradually been applied to the development of anti-photoaging cosmetics, such as marine polyphenols, marine polypeptides, marine polysaccharides, marine carophyllum, marine terpenoids, etc.
, these bioactive substances can usually work together through a variety of light protection mechanisms to alleviate the damage
of UV radiation to the skin.

The brown algae polyphenols, lycopodol and di-lysophilm extracted from conandocoum (E.
cava) can reduce the intracellular ROS content after UVB radiation, and the cell viability increases dose-dependent and DNA damage is significantly reduced after incubation of brown algae polyphenols.
The brown algae polyphenol DPHC extracted from ishige okamurae has strong protective properties
against UVB radiation by reducing intracellular ROS levels and reducing damaged DNA tail length, causing fibroblast morphological changes.

Over the past few decades, seaweed has been a great source of natural products that can be used in search of new compounds
with pharmacological and nutritional properties.

Black algae is an important economic brown algae distributed in the waters of China and Japan, containing many bioactive compounds such as indole derivatives, indole ketone alkaloids, quinone derivatives, polyphenols and polysaccharides
.

S.
thunbergii lives in the intertidal zone and undergoes a variety of changes due to tidal changes, including changes in
high solar irradiance, dryness, salinity and temperature.

Therefore, we speculate that the phenolic compounds in Aspergillus niger may have UV protection activity
.

However, to our knowledge, the photoprotective activity of Thunbergia streptococcal has never been reported before
.

Mouse L929 fibroblast line is reported to be an effective in vitro screening test system
for drug/chemical phototoxicity assessment.

Danio rerio is increasingly seen as a good model for evaluating the efficacy of UVB protective compounds on fin damage and ROS generation
.

In this study, we established a model of L929 cells exposed to UVB and zebrafish photodamage to study S.
Thunbergii is rich in UVB photoprotective activity
of phenolic extracts (STPE).

● Experiment Part 1.
1 Effect of STPE on Zebrafish Growth Collect ab wild zebrafish embryos that develop 2 days after fertilization (dpf), and each experimental group is randomly divided into 30 embryos
.

Embryos are treated with STPE concentrations of 5, 10, 25, 100, 250, 500, 1000 and 2000 μg/mL for 24 hours and exposed to an UV therapy instrument
.

After UVB exposure, all embryos are counted at 5 dpf for survival
.

1.
2 The repair effect of STPE on UVB irradiation zebra tail fin injury was collected in embryos developed at 2 dpf and treated with different concentrations of STPE (0.
56, 1.
67 and 5 μg/mL) for 24 hours
.

10 zebrafish randomly selected from each group were observed in their caudal fins under a dissecting microscope, analyzing the images to calculate the area
of their caudal fins.

The caudal fin area is used to assess the effect of STPE on the repair of skin damage caused by UV exposure in
zebrafish.

1.
3 Effects of STPE on ROS clearance from UVB irradiation zebrafish collected in embryos developing at 2 dpfTreat embryos with different concentrations of STPE (0.
8, 1.
2 and 1.
6 μg/mL) or EGCG (30 μg/mL) for 24 h
.

Measuring the fluorescence intensity of embryos (FI), the effect of STPE on zebrafish oxygen radical scavenging is calculated as follows:● Experimental results 1.
STPE reversed UVB-induced decrease in cell viability with 40% absolute ethanol extracted at 70 °C.
Thunbergii was purified twice with the porous adsorption resin XDA-7 to obtain STPE
.

The total polyphenol content of STPE was determined using the Folin-Ciocalteu method of colorimetry to 72%.


To determine STPE cytotoxicity, L929 cells are treated with different concentrations of STPE (0-320 μg/mL) for 24 hours
.

STPE concentrations of 10-40 μg/mL had no effect on cell viability compared to the untreated group (p> 0.
05; Figure 1a), although cytotoxicity was observed at 80 μg/mL (p<0.
05).


Based on this, 10–40 μg/mL STPE is used for subsequent assays
.

Cells are treated with different doses of UVB (0-320 mJ/cm2) and cultured for 24 hours to determine the appropriate dose
of irradiation to induce photodamage.

UVB reduces cell viability in a concentration-dependent manner (Figure 1b); 20 MJ/cm2UVB reduced cell viability by approximately 50% and was selected as the UVB dose in subsequent experiments
.

Cell viability is reduced to 60% after exposure to UVB alone compared to experimental controls (Figure 1c).


However, pretreatment of cells with STPE (10–40 μg/mL) reversed this effect in a concentration-dependent manner and differed significantly from
the UVB model group at 40 μg/mL.

Figure 1: STPE reverses the decrease in
cell viability caused by UVB.

(a) Effect of STPE and (b) UVB on L929 cell viability at different concentrations or doses, and (c) protective effect
of STPE on L929 cell damage by UVB (20 mJ/cm2).

The mean with "*" differs significantly (p< 0.
05)2.
STPE inhibits oxidative stress and lipid peroxidation to assess the clearance effect of UVB-induced STPE on intracellular ROS, measuring the clearance effect of STPE on intracellular ROS produced by UVB exposure (Figure 2a
).

Ros levels in cells exposed to UVB increased by a factor of 2.
35 compared to control cells
.

However, 24 h pretreatment of cells with increased concentration STPE results in a decrease
in intracellular ROS concentration dependence.

Figure 2: STPE inhibits UVB-induced oxidative stress and lipid peroxidation
.

Effects
of STPE on L929 cells (a) ROS and (b) MDA content and (c) SOD and (d) CAT activity.

With significant differences in means with "*" (p<0.
05) SOD and CAT are important antioxidant enzymes that protect tissues from oxidative damage
.

MDA generation is used to assess intracellular lipid peroxidation due to oxidative
stress.

The protection mechanism
of STPE was evaluated by measuring the levels of SOD, CAT and MDA.

Reduced SOD levels after UVB exposure compared to the control group; However, this is eliminated by STPE pretreatment of 20 and 40 μg/mL (Figure 2c
).

CAT activity is elevated after STPE treatment at 40 μg/mL (Figure 2d
).

The increase in MDA content induced by UVB (p<0.
05) (Figure 2 b) was reversed by 40 μg/mLSTPE pretreatment (p< 0.
05).


3The repair effect of STPE on UVB irradiation zebra tail fin damage In recent years, zebrafish have been widely used as a model of oxidative stress in vivo to study the protective effects
of ultraviolet radiation.

A schematic diagram of the protocol is shown in
Figure 3a.

This study first explored the effect of STPE on the growth of zebrafish in wild-type AB strains, thus providing data on the concentration of STPE in the large safety of ZUI for subsequent experiments
.

After UV exposure, STPE causes high mortality
at concentrations of 10 μg/mL and above.

No significant abnormalities were observed at 5 μg/mL STPE; Therefore, this is the large experimental concentration
of zui used in the zebrafish ultraviolet damage model.

Figure 3: Evaluation of stPE's photoprotective effect
on UVB irradiation of zebrafish.

Use zebrafish embryos
2 days after fertilization (DPF).

a Schematic diagram
of the protocol performed in this study.

b and c are UVB-induced deformed fin phenotypes that can be attenuated
by STPE.

d is the inhibitory effect
of STPE on UVB-induced ROS in zebrafish.

Differences with p-values less than 0.
05 are considered statistically significant and denoted
by "*".

STPE has the potential to repair zebrafish skin damage exposed to UVB radiation, as shown in Figures 3b and c, where the zebrafish's caudal fin exhibits a rough and wrinkled phenotype
after exposure to ultraviolet radiation.

The repair effect of tea polyphenols on zebrafish skin damage was 29%, indicating that positive control treatment significantly repaired zebrafish skin damage
.

Treatment of zebrafish with 0.
56, 1.
67 and 5 μg/mL of STPE resulted in caudal fin area, which had an effect on skin damage repair of 3%, 24%, and 6%,
respectively, compared with the control model.

Overall, STPE has a significant restorative effect
on zebrafish skin injuries.

4 THE EFFECT OF STPE ON ROS SCAVETING OF UVB RADIATION ZEBRAFISH THE EFFECT OF STPE ON UVB-MEDIATED ROS INDUCTION IN ZEBRAFISH IS shown in
Figure 3d.

The fluorescence value of 6271±264 (p<0.
001) in the UVB group was higher than that of the normal control group (1169±124), indicating that the model was successfully established
.

The fluorescence value of the 30 μg/mL EGCG-positive control group (1122±39) decreased by 82% compared to the UVB group (p<0.
001).


The STPE group had fluorescence values of 1818 ± 78, 1834 ± 116 and 950 ±30 at concentrations of 0.
8, 1.
2, and 1.
6 μg/mL, respectively, and the intracellular ROS accumulation was reduced by 71, 71, and 85% compared with the UVB group (p<0.
001).


These findings suggest that STPE has a significant clearance effect
on ROS in zebrafish models.

● Discuss this study from S.
Thunbergii middle scoreExtracts rich in phenols are isolated and L929 cells are used in vitro and zebrafish models are used in vivo to assess their protective effect
against UVB-induced photodamage.

Experimental data show that the protective effect of UVB is mediated by the regulation of the NF-κB signaling pathway by stPE's antioxidant, anti-inflammatory, and anti-apoptotic abilities (figure below
).

This research contributes to an understanding
of the protective effects of STPE skin.

We will continue our efforts to obtain a highly purified and stable STPE molecule for further use as a potential protective agent to reduce skin damage
caused by UVB.