Anti-inflammatory and antiaging properties of chlorogenic acid on UV-induced fibroblast cell

Background Skin aging is the most common dermatological problem caused by intrinsic and extrinsic factor, such as exposure to (ultraviolet) UV rays. Chlorogenic acid (CA) is a phenolic compound which is known for its antioxidant properties against oxidative stress. Objective This study investigates the antiaging and anti-inflammatory properties of CA on UV-induced skin fibroblast cells. Methods Anti-inflammatory properties of CA were assessed by measuring inflammatory-related proteins IL-1β and TNF-α, while antiaging properties of CA were assessed by measuring reactive oxygen species (ROS), apoptosis, live and necrotic cells, and COL-3 gene expression level. Results Treating UV-induced skin fibroblast cells with CA decreased the level of ROS, IL-1β, TNF-α, apoptotic cells, and necrotic cells and increased live cells and COL-3 gene expression. Conclusion CA has the potential as the protective compound against inflammation and aging by decreasing the level ROS, pro-inflammatory cytokines IL-1β and TNF-α, apoptotic cells, and necrotic cells and by increasing live cells and COL-3 gene expression.


INTRODUCTION
Skin aging is the most common dermatological problem, especially for women. While aging could happen as a result of intrinsic mechanisms, the external causes also play a big role in causing skin aging (Baumann, 2007;Farage et al., 2008). The external causes in aging are mostly from environmental factors i.e., sun (ultraviolet), infrared and heat, smoking, air pollution, and low antioxidant diet (Vierkötter & Krutmann, 2012;Addor, 2018). Ultraviolet (UV) exposure is one of the factors that play an important role in affecting all body systems, especially for the skin as the outer barrier against the external

Total protein measurements
The total protein measurement was conducted to assess the IL-1β and TNF-a level by mg protein. The BSA stock was obtained by dissolving 2 mg of BSA in 1,000 ml ddH 2 O (A9576, Lot. SLB2412; Sigma, St. Louis, MO, USA). The BSA solution was obtained from diluting the BSA stock. The standard solution with 20 ml in number and 200 ml of Quick Start Dye Reagent 1 Â (5000205; Biorad, Hercules, CA, USA) were added to each well. The plate was incubated for 5 min at room temperature. The result was read at 595 nm (Widowati et al., 2019a;Lister et al., 2020).

Measurement of apoptotic, live, necrotic cells
The measurement of apoptotic, live, and necrotic cells was assessed by flow cytometry analysis. The cells were placed in a 2 Â 6-well plate (n = 500.000) and were incubated for 2 h. The growth medium was discarded in 4 days and harvested and centrifuged for 5 min at 1,600 rpm. The pellet was added with 500 µl FACS buffer and centrifuged. The pellet was added with 100 µl FACS buffer and stained by annexin and propidium iodide (PI). The stained cells were incubated for 1 h and analyzed using flow cytometry (Lister et al., 2020;Widowati et al., 2019b).

Statistical analysis
All the data are presented as the mean ± standard deviation. The data were analyzed using Shapiro-Wilk test followed by Mann-Whitney-Wilcoxon test and independent t-test. P-values < 0.05 were considered significant.

IL-1β level
The IL-1β level of the conditioned medium of the treated fibroblast cell with CA was measured using the ELISA method and compared to the negative control and UV-induced fibroblast cell (positive control). Treatment with DMSO 1% (vehicle control) was not significantly different with the negative control. The result shows a significant decreasing level of IL-1β compared to the positive control (Fig. 1A). Total protein assay was present to measure the IL-1β level by mg protein. The CA treatment showed a significant decrease in the IL-1β level compared to the positive control (Fig. 1B).

TNF-a level
Treatment with DMSO 1% (vehicle control) on fibroblast cells toward TNF-α level was not significantly different compared to the negative control. Treatment with 6.25 and 25 mg/ml CA showed a significant decreasing difference compared to positive control (Fig. 1C). Total protein assay was present to measure the TNF-a level by mg protein. The CA treatment showed a significant decrease in the TNF-a level compared to the positive control (Fig. 1D). Total protein assay was present to measure the TNF-a level by mg protein. The result showed a significant decrease in the TNF-a level compared to both controls (Fig. 1D).

ROS level
The ROS level of UV-induced fibroblast cells was measured using flow cytometry with DCFDA single staining. The dot blots show the population of the analyzed cells, and the peak shows the positive ROS cells. The normal control, vehicle control, positive control, treatment with CA 6.25 µg/ml, and treatment with CA 25 µg/ml show 0.02%, 0.05%, 16,01%, 4.95%, and 6.86% positive ROS levels, respectively (Fig. 2). Both cells treated with 6.25 and 25 mg/ml showed a significant decreasing difference compared to positive control (Fig. 3).

Apoptosis level
The apoptosis level was analyzed using flow cytometry. The surface markers PI and annexin were used as stains. Figure 4 shows the representative of dot blots effect CA 6.25 and 25 mg/ml towards apoptosis cells. Flow cytometry analysis reveals the percentage of live cells, necrotic cells, early apoptosis, and late apoptosis of cells. Figure 5 shows the comparison between each treatment on live of cells (a), necrosis of cells (b), death of cells (early apoptosis) (c), and apoptosis (late apoptosis) (d).

COL-3 gene expression
The COL-3 gene expression was examined using qRT-PCR. Table 1 presents the primer and annealing temperature used for performing COL-3 gene expression, while Table 2 shows the concentration and RNA purity. The COL-3 gene expression shown in Fig. 6   revealed the CA treatment 6.25 and 25 mg/ml showed a significant increase in the COL-3 gene expression compared to the positive control.

DISCUSSION
UV-induced aging mechanism triggered free radicals in the form of increased intracellular ROS level, leading to oxidative stress, which then triggered inflammation leading to cell death (apoptosis). BJ fibroblast cells are the model of aging cells induced by UV (Girsang et al., 2019c). Fibroblast cells play a role in tissue granulation and scar formation during the inflammation process. The result of the anti-inflammatory test shows treating UV-induced BJ cells with CA decreased the IL-1β and TNF-a levels related to the aging model (Fig. 1). The inflammation triggered by UV rays resulted in the increase of the  pro-inflammatory cytokine group IL-1β and TNF-a, which are used as inflammatory markers for anti-inflammatory assessment (Chung et al., 2009). The presence of ROS as one of the inflammation trigger also being one of the direct UV-exposures effect in cells (Tanigawa et al., 2014). The accumulation of ROS leads to cell death known as apoptosis, which results in the decreasing level of healthy cells. The result of the assessment with fluorescence intensity as an indicator by DCFDA staining showed that CA decreased the level of ROS compared with the positive control ( Figs. 2 and 3). The previous study about CA also stating the reducing ROS level induced by lead (Pb) and hydrogen peroxide (H 2 O 2 ) effect by this substances (Girsang et al., 2019c;Hoelzl et al., 2010), CA has antiaging property by inhibiting enzyme related skin aging i.e., MMP-1 and human skin fibroblast elastase (HSFE) according in silico analysis (Girsang et al., 2019b).
UV exposure of BJ-cells also affects the level of apoptosis. The oxidative stress caused by UV rays promotes apoptosis in healthy cells (Kannan & Jain, 2000). Flow cytometry analysis revealed the lowest number of live cells in UV-induced BJ cells. Treatment with CA on UV-exposed cells resulted in increased number of live cells and decreased Figure 7 Proposed mechanism of CA protective effect towards UV-induced skin fibroblast cells. UV exposures causing an increasing level of ROS leading to an increase of pro-inflammatory cytokines IL-1β and TNF-a leading to inflammation. The presence of ROS triggered the production of caspase 9 (Casp-9) along with the production of Casp-8 by the presence of TNF-a leading to apoptosis. These two condition of inflammation and apoptosis leading to aging in cells. On another side, UV-exposures decreasing the COL-3 gene expression causing the loss of fibril pattern leading to aging as well. The presence of CA inhibits the production of ROS level thus suppressing the pro-inflammatory cytokines IL-1β and TNF-a. The presence of CA also increasing the level of COL-3 gene expression.
Full-size  DOI: 10.7717/peerj.11419/ fig-7 cell death, necrosis, and apoptosis (Figs. 4 and 5). The protective effect of CA toward apoptosis is also reported in rat liver with oxidative stress triggered by methotrexate (Ali et al., 2017). While CA could potentially decrease the levels of ROS, IL-1β, and TNF-a, the measurement of COL-3 gene expression can also be conducted to determine the repairing effect of CA toward aging. COL-3 gene is related to the pattern of tendon fibril thickness and is more abundant in younger individuals than aged individuals of the animal model (Ribitsch et al., 2019). UV exposure could also decrease the COL-3 gene expression level (Chen et al., 2019). The result of this research showed the effect of UV exposure and the treatment of CA toward COL-3 gene expression level. Treatment with CA increased the COL-3 gene expression level compared to the aging model (Fig. 6). According to these results, we proposed the mechanism of anti-inflammatory and antiaging properties of CA against UV-induced BJ cells (Fig. 7).

CONCLUSION
This study shows that CA has the potential as the protective compound against inflammation and aging by reducing ROS, pro-inflammatory cytokines IL-1β and TNF-a, apoptosis, and necrotic cells and by increasing live cells and COL-3 gene expression.