The effect of Dendrobium officinale extract in inhibiting the senescence of H2O2‐induced fibroblasts based on the senescence‐associated secretory phenotype

Dendrobium officinale is widely used for a long time in China, with effect of antioxidation, antitumor, enhancing immunity and so on. In recent years, Dendrobium officinale has been gradually used in cosmetics due to its powerful beauty effects.


| Preparation of Dendrobium officinale extract (DOE)
Fresh Dendrobium officinale was extracted with deionized water at 95°C - 105°C.The filtrate was obtained by separation of solid and liquid.0.5% (w/w) active carbon powder (≥200 mesh) was added to the filtrate for over 30 min and was removed by separation of solid and liquid.The filtrate was lyophilized in a lyophilizer (Songyuan Freeze Dryer, China).Then DOE was obtained.

| Establishment of the senescent model of H 2 O 2 -induced fibroblasts
The control group and H 2 O 2 -induced group were set up in the experiment.Fibroblasts in logarithmic phase were selected and seeded into 6-well plates, and were treated according to groups when the plates were spread about 40% - 60%.According to the rate of plate laying is 80% - 90%, fibroblasts cultured for five passages were seeded into 6-well plates and incubated for 24 h under 5%CO 2 at 37°C.Then fibroblasts were washed twice with PBS. 1 mL RNAiso Plus was added to each well, the fibroblasts were lysed by blowing.RNA was extracted, reverse transcribed to cDNA, and then tested by CFX96 Touch Real-time PCR Detection System (BioRad, America).The primer sequence of p16 is showed in Table 1. 2 -△△CT was used to calculate and relative gene expression was presented as fold change in relation to the control group (Control).

| Cell viability assays
The cell viability was determined by MTT assay.According to the rate of plate laying is 80% - 90%, the fibroblasts induced by

| Statistical analysis
All data were represented by mean ± SD (n = 3) and were analyzed by one-way analysis of variance (ANOVA) using "Graph Pad Prism 9 Software (USA)".A p-value of <0.01 and <0.05 was considered as a statistically significant difference.][10][11] One of the marks of cell senescence is that the activity of SA-β-gal is Enhanced.3][14][15] In a variety of normal tissues, the expression of p16 gene is inhibited, and it can only be activated when the tissue is damaged or the cell has stress reaction.[18][19][20][21][22][23] After fibroblasts were induced by H 2 O 2 , the senescent cells are stained blue (Figure 1).The positive rate of SA-β-gal staining (Figure 2) and p16 mRNA expression (Figure 3) were significantly higher than control group.It indicated that 400 μM H 2 O 2 could induce the senescence of fibroblasts successfully.

| Cell viability
The MTT assay was used to evaluate cytotoxicity of DOE on H 2 O 2induced fibroblasts.Both the two concentrations of DOE had no significant effect on the viability of H 2 O 2 -induced fibroblasts (Table 2).It indicated that the evaluated concentrations of DOE had no toxicity to H 2 O 2 -induced fibroblasts.

| SASP factors assays
SASP is a typical characteristic of cell senescence, including cytokines, chemokines, growth factors, proteases, etc. IL-6 is one of the main cytokines in SASP signaling and plays a direct mediating role in inflammation.MCP-1, a member of chemokine CC subfamily, has a mediated effect on inflammatory response and can also induce the generation of inflammatory mediators. 24MMP-1 is a proteolytic enzyme that promotes cell invasion and metastasis. 25e content of IL-6, MCP-1 and MMP-1 were tested by ELISA kit.
Based on the cytotoxicity results, two concentrations of DOE were selected for SASP factors assays.As shown in, Figure 4A

| DISCUSS ION
Fibroblasts are an important part of the dermis, which can synthesize and secrete collagen, elastin and other extracellular matrix (ECM) to maintain skin elasticity and prevent the senescence of skin.
Traditionally, it is believed that the senescence of skin fibroblasts leads to the decrease of the number of fibroblasts, the change of their morphology, the weakening or decline of the function of secreting and synthesizing ECM.Eventually, skin aging appears.It is speculated that the senescence of fibroblasts may be the main

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et al. officinale on skin.In this study, H 2 O 2 was used to establish the senescent model of human skin fibroblasts, and the antiaging effect of Dendrobium officinale was evaluated by SASP factors.

from H 2 O
2 processing step.Then the obtained fibroblasts were cultured for five passages and were collected for SA-β-gal staining and testing of p16 mRNA expression.Fibroblasts cultured for five passages were stained with SA-βgal staining kit.According to the rate of plate laying is 80% - 90%, they were seeded into 6-well plates and incubated for 24 h under 5%CO 2 at 37°C.Then fibroblasts were washed once with PBS. 1 mL SA-β-gal staining fixative solution was added to each well.After 15 min the fibroblasts were washed by PBS for three times.1 mL SAβ-gal staining working solution was added to each well.Then the fibroblasts were incubated overnight at 37°C, and the 6-well plates were sealed with sealing film to prevent evaporation.They were observed, photographed and counted under an ordinary light microscope (Olympus, Japan).The positive fibroblasts of SA-β-gal staining is blue.The number of positive fibroblasts and total fibroblasts in five different areas in each film was counted to calculate the proportion of the positive fibroblasts of SA-β-gal.

5 |
H 2 O 2 according to point 2.3 were seeded into 96-well plates and incubated under 5%CO 2 at 37°C.Then DOE was diluted two concentrations (2.5%, 5%, m/V) by normal medium and treated by 200 μL per well.The solvent control group (SC) was treated by 200 μL normal medium.The positive control group (PC) was treated by 200 μL 10% V/V DMSO in normal medium.The wells without cells but only 200 μL normal medium were used as the blank control group (BC).Three wells were used for each concentration of the aforementioned drugs.After 24 h, the medium was removed and 200 μL MTT solution (0.5 mg/mL) was added to each well and incubated at 37°C in dark Place for 4 h.The MTT solution was then removed, and 150 μL DMSO was added to each well.Cell viability was calculated using the following formula: cell viability (%) = ([OD] test -[OD] BC )/ TA B L E 1 The primer sequence of p16.SASP factors assays According to the rate of plate laying is 80% - 90%, fibroblasts induced by H 2 O 2 according to point 2.3 were seeded into 6-well plates and incubated for 24 h.Then fibroblasts were treated by DOE and incubated for 24 h.The supernatants were collected and used to test the content of IL-6, MCP-1 and MMP-1 by ELISA kits.The PC of IL-6 content assay was 0.1 mg/g DXMS.The PC of MCP-1 content assay and MMP-1 content assay was 100 ng/mL TGF-β1.

3 | RE SULTS 3 . 1 |
Establishment of the senescent model of H 2 O 2 -induced fibroblasts H 2 O 2 is a commonly used inducer of cell senescence.It can affect cellular DNA directly, then lead to specific DNA damage response (DDR), thereby activate p53/p21 or p16/pRb pathways, induce cell -4C the content of IL-6, MCP-1 and MMP-1 decreased significantly after the treatment with DXMS or TGF-β1.Also The content of IL-6, MCP-1 and MMP-1 decreased significantly with the treatment with different concentrations of DOE.It shows that DOE can inhibit the secretion of SASP factors in senescenct fibroblasts.

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et al. factor of skin aging.Therefore, slowing down or preventing the senescence of skin fibroblasts is one of the effective means to prevent skin aging.Fibroblasts are also a common cell type for studying the mechanism of cellular senescence in vitro.Recent SASP studies suggest that senescent fibroblasts can also enhance senescent phenotype and induce senescence of adjacent fibroblasts by secreting SASP factors.Therefore, further study to obtain SASP inhibitors of senescent fibroblasts can provide a new idea for the development of antiaging agents for skin.In this study, fibroblasts were induced by H 2 O 2 , and the rate of the positive cells of SA-β-gal staining and p16 mRNA expression were used as indicators to evaluate the establishment of senescent model of fibroblasts.Three representative SASP factors, IL-6, MCP-1 and MMP-1, were selected to evaluate the effects of DOE on the secretion of SASP from H 2 O 2 -induced fibroblasts.The results showed that DOE could inhibit secretion of SASP from senescent fibroblasts.The effect of 0.25% DOE was better than 0.5% DOE.However, with only two concentrations, the relationship between concentration and efficacy cannot be determined.It was speculated that the two concentrations were not the best for DOE to inhibit the secretion of SASP by senescent fibroblasts, and further studies on the dose-effect relationship were needed.However, this did not affect our discovery that DOE can inhibit the secretion of SASP from senescent fibroblasts, which clarified the new antiaging mechanism of DOE, and provided more sufficient basis and more modern scientific support for its application in skin beauty.CON CLUS IONS Traditional Chinese medicine (TCM) is a great treasure of China.Dendrobium officinale ranks first among the "nine immortal herbs" and has high value of beauty research.In this study, it was found that Dendrobium officinale could effectively inhibit the secretion of SASP.It shows that Dendrobium officinale has good antiaging ability and is a natural skin antiaging agent for skin.In the 21st century, green and natural are still important guidelines for global sustainable development.We deeply study TCM, especially valuable Dendrobium officinale, and use it in cosmetics.It will certainly be pursued and loved by people all over the world, and will also contribute to the sustainable development of the world.F I G U R E 2 SA-β-gal staining of H 2 O 2 -induced fibroblasts.The results represent mean ± SD from three parallel experiments.p < 0.01 (**) means significantly different from the control value.F I G U R E 3 p16 mRNA expression of H 2 O 2 -induced fibroblasts.The results represent mean ± SD from three parallel experiments.p < 0.01 (**) means significantly different from the control value.TA B L E 2 Cell viability.
The results represent mean ± SD from three parallel experiments.SC represents solvent control.