Qiu Shuting,Pan Zhi,Jiang Xiao

Shenzhen Synergy Testing Co.,Ltd.,China;

Hu Fan,Zhang Hao

Guangzhou Xingji Yuedong Co.,Ltd.,China

Wu Fengjuan

Guangzhou Juzi Youpin Co.,Ltd.,China

Abstract The low level red light irradiation dose that produced positive response on different cell indexes was selected through experiments.In fibroblast detection model,the low level red light irradiation dose that had positive response to the four indicators of mitochondrial membrane potential,ATP release,cell migration and collagen synthesis was screened.The experimental results showed that low level red light with a irradiation dose of 2 J/cm2 had a positive response to the above four indexes in fibroblast.The test model on fibroblast was later transferred to dermal papilla cells for further experimental verification.The results showed that low level red light with a irradiation dose of 2 J/cm2 also had a positive response to the four indicators of dermal papilla cells,namely,mitochondrial membrane potential,ATP release,cell proliferation rate and collagen synthesis.It is proved that the low level red light with a irradiation dose of 2 J/cm2 could improve the mitochondrial function,cell migration and collagen synthesis of fibroblast and dermal papilla cell,thus providing energy for cell activities,improving cell repair ability and cell anti-aging ability.

Key words low level red light;dose screening;mitochondrial function;collagen synthesis;cell migration

Low level light therapy (LLLT) refers to the optical therapy that utilizes low doses (generally less than 100 J/cm2)[1]of visible or near-infrared light to produce photobiological regulation and alter biological activity in biological tissues,resulting in positive effects of body[2].In recent years,studies have shown that LLLT has positive effects on promoting the production of Adenosine triphosphate(ATP) in cells,stimulating the synthesis of collagen in cells,promoting blood circulation,and reducing cell/skin inflammation[3,4].Joo[5]et al.found that low level light (30 J/cm2) at 660 nm showed positive effects on human dermal papilla cells after irradiation,especially in the indexes of cell proliferation and cell migration.Choi[6]et al.found that after irradiation with 660 nm low level red light,the cellular activity of fibroblasts increased and their healing ability enhanced.Based on the above characteristics of LLLT,LLLT is widely used in reducing pain and inflammation,enhancing tissue repair,promoting wound healing,promoting regeneration of different tissues and nerves[3,7],preventing tissue damage[8],anti-aging and preventing hair loss[9,10].In 2007,the US FDA approved LLLT devices with a wavelength of 655 nm for the treatment of male hair loss,and later approved LLLT devices with wavelengths of 678 and 650 nm,respectively[11].The LLLT instrument produced by Dynamonics (Salt Lake City,UT) uses light with wavelengths of 660 and 880 nm to achieve anti-aging effects[3].

Cellular senescence will bring about the decline of cell metabolism and biological activity,which is reflected in the decline of cell mitochondrial function,the decline of cell repair ability and the reduction of collagen synthesis,thus leading to the visible facial aging and hair loss[12,13].Therefore,cellular anti-aging is necessary for physical anti-aging and hair loss prevention.Mitochondrial function (mitochondrial membrane potential,ATP release and etc) can reflect the energy supply of cells and affect cell vitality and activities (such as cell proliferation,cell metabolism)[14].The increase of cell migration rate and cell proliferation rate,to some extent,indicates the improvement of cell vitality and repair ability.[15-17]An increase in the synthesis of collagen in cells can increase the collagen content inside the cells,thereby achieving skin plumpness,anti-aging,and hair fixation and detachment prevention effects[18-21].

At present,many studies have shown that low level red light has a promoting effect on mitochondrial function and collagen synthesis in cells.However,few studies have mentioned the correlation between different irradiation doses and different cells or cell indicators,as well as methods to improve cell level related effects by screening irradiation doses.Based on the status quo,this study screened and validated low level red light doses that could improve cell function,by using mitochondrial membrane potential,ATP release amount,cell migration rate/cell proliferation rate and collagen synthesis as indicators to measure the low level red light cell model (referring to the combination of irradiation dose and test methods at the cell level).

1 Experiment

1.1 Materials,reagents and equipment

Primary skin fibroblasts,primary dermal papilla cells and low sugar culture medium,were obtained from Guangdong Boxi Biotechnology Co.,Ltd.PBS buffer solution,was procured from Beijing Solaybao Technology Co.,Ltd.Mitochondrial Membrane potential test kit,enhanced ATP test kit,BCA protein test kit and Hochest33342,were procured from Shanghai Biyuntian Biotechnology Co.,Ltd.Collagen I Test Kit (CUSABIO),was procured from Wuhan Huamei Bioengineering Co.,Ltd.Anti Collagen I antibody,was procured from Protein Group Inc.Goat pAb to Rb IgG(Alexa Fluor?488),was procured from Abcam Plc.Goat Serum,was procured from Beijing Zhongshan Jinqiao Biotechnology Co.,Ltd.MSCM medium,was procured from Sciencell Research Laboratories,inc.Newborn bovine serum,was procured from Lanzhou Rongye Biotechnology Co.,Ltd.

CO2incubator (Thermo Fisher Scientific);Ultra Clean Workbench (Suzhou Antai Air Technology Co.,Ltd.);Inverted microscope (Olympus Corporation);Fluorescence microscope (Olympus Corporation);Microplate reader(BioTech,Berten Instruments,USA);Fluorescence microplate reader (Synergy HTX,Berten Instruments,USA);Low Temperature High Speed Centrifuge (Hunan Xiangyi Centrifuge Instrument Co.,Ltd);Analytical balance (Sartorius Company);Spectral illuminometer(OHSP350S,Hangzhou Hongpuguangse Technology Co.,Ltd);LED panel (wavelength 630±20 nm;panel size:300×195×10 mm;Lamp beads: 288PCS),produced and provided by Guangdong Boxi Biotechnology Co.,Ltd.

1.2 Method

1.2.1 Cell culture and irradiation

Primary fibroblasts and primary dermal papilla cells were incubated in a culture incubator (37 ℃,5% CO2,95% RH) for 24 h,respectively,ensuring that the cell planking rate of dermal papilla cell was 30% to 40%.Then replaced the culture medium with low sugar DMEM medium containing 10% newborn bovine serum and continued to incubate in the incubator (37 ℃,5% CO2,95% RH) for 24 h.

The cell culture orifice plate was taken out from the incubator.Then the cells were irradiated with different doses on the Aseptic technique platform.The size of the LED panel was larger than that of the orifice plate (125×85 mm),ensuring that the light from the light plate was able to evenly cover the whole orifice plate.Before irradiation,a spectral illuminometer was used to measure the irradiance dose on the orifice plate.After irradiation,the orifice panel was buckled and placed back in the incubator (37 ℃,5% CO2,95% RH) for incubation.In the indicator testing of fibroblast,four irradiation doses were set,namely 0,1,2,and 8 J/cm2.Among them,0 J/cm2was the blank control group (BC group).In the indicator test of dermal papilla cells,two irradiation doses were set,namely 0 and 2 J/cm2.Among them,0 J/cm2is the blank control group (BC group).The specific grouping and testing conditions are shown in Table 1.

Table 1.The setting of test conditions for different cells

1.2.2 Mitochondrial Membrane potential and ATP release

The cells incubated for 6 h after irradiation were taken out and the mitochondrial membrane potential and ATP release amount were measured respectively.The mitochondrial membrane potential was detected according to the operating instructions of the mitochondrial membrane potential detection kit (JC-1).The detection of ATP release amount was carried out according to the operation manual of the enhanced ATP detection kit.

1.2.3 Cell migration rate of fibroblasts

After 24 h cell incubation,a cell scratch test was operated on cells.Then cells were washed 3 times with PBS and normal cell culture medium was added in it.Taken photos under a 4x microscope.After completion,the cells were irradiated under the LED light panel at different irradiation doses according to steps 1.2.2.After irradiation,cells were incubated in a culture incubator (37 ℃,5% CO2) for 24 h and then was taken photos under a 4x microscope.

1.2.4 Cell proliferation ability of dermal papilla cells

After irradiated with different doses of red light,cells were incubated for 24 h.Later,discarded the supernatant and added MTT working solution (0.5 mg/mL),cells incubated in dark at 37 ℃ for 4 h.After incubation,the supernatant was discarded and 150 μL of DMSO was added in each well.The absorbance was read at 490 nm.Calculate the relative cell proliferation ability according to the following formula.The relative cell proliferation activity of cells=(sample absorbance -zero set absorbance)/(solvent reference absorbance -zero set absorbance) × 100%.

1.2.5 Collagen synthesis in fibroblasts

Fibroblasts: After irradiating cells with LED panel at different irradiation doses,cells were incubated in the incubator for 24 hours.After being fixed with 4% paraformaldehyde,the collagen I synthesis of cells was detected by immunofluorescence staining.

Dermal papilla cells: After being irradiated with LED panel at doses of 0 and 2 J/cm2,dermal papilla cells were incubated for 24 h.The cell supernatant in orifice plate was collected and frozen at -80 ℃.The release of collagen I in the cell supernatant was measured according to the operating instructions of the collagen I ELISA kit.

1.3 Stability test

In the testing of 1.2,each test was underwent three parallel batches to ensure the stability and reliability of the research results.

1.4 Statistical processing

The obtained data was analyzed by the software Image J Pro.The graphs were drew by GraphPad Prism,and the results were represented as Mean±SD.Comparison between groups was conducted using t-test statistical analysis.All statistical analyses were double tailed withp<0.05 indicating significant differences,denoted by *;P<0.01 is considered to have extremely significant differences,represented by * *;P<0.001 is considered to have a significant difference,denoted by * * *.

2 Result and discussion

2.1 Dose screening of low level red light in fibroblasts

2.1.1 Mitochondrial function

The detection results of mitochondrial membrane potential at different irradiation doses are shown in Figure 1.Compared with the BC group,fibroblasts irradiated with a irradiation dose of 2 J/cm2showed an obvious increase trend of mitochondrial membrane potential (<0.05).Although the mitochondrial membrane potential of the 8 J/cm2group was higher than that of the BC group,the data deficient were lack of statistical significance.Mitochondrial membrane potential in 1 J/cm2group was slightly lower than that in BC group.It suggested that after 2 J/cm2irradiation,the cell mitochondrial membrane potential of fibroblasts was significantly increased,which could bring about the improvement of cell mitochondrial function.

Figure 1. Results of mitochondrial membrane potential of fibroblasts incubated for 6 h after different doses of irradiation

The ATP release detection results of fibroblasts are shown in Figure 2.After irradiation and 6 hours incubation,the groups showed a positive response in terms of ATP release.The groups of 2 J/cm2(p<0.05) and 8 J/cm2(p<0.01) showed significant improvements,indicating a significant promoting effect on ATP synthesis and release in fibroblasts.From the results of Figure 1 and 2,it preliminarily determined that the red light irradiation of 2 J/cm2increased the mitochondrial membrane potential and ATP release of cells,thereby significantly improving the mitochondrial activity of fibroblasts.

Figure 2. Results of ATP release from fibroblasts incubated for 6 hours after different doses of irradiation

In order to further determine the positive effect of 2 J/cm2red light irradiation on cell mitochondrial activity,three batches of stability experiments were set up (Figure 3 and 4).As shown in Figure 3,the cell mitochondrial membrane potential showed a significant increase at 2 J/cm2(p<0.05).The group of 2 J/cm2had a higher ATP release (p<0.01) than BC group in Figure 4.The results further illustrated that the mitochondrial membrane potential and ATP release of fibroblasts have been significantly improved by red light irradiation of 2 J/cm2,and the results were reliable and stable.

Figure 3. Three batches of stability test results of mitochondrial membrane potential of fibroblasts

Figure 4. Three batches of stability test results of ATP release of fibroblasts

According to the above data,the red light irradiation of 2 J/cm2could significantly promote the increase of mitochondrial membrane potential and ATP release of fibroblasts,thus improving the mitochondrial activity of cells,and providing more energy for various activities of cells,such as cell proliferation,cell migration,tissue repair and cell metabolism[22-25].

2.1.2 Cell migration

Cell scratch test simulates the process of cell migration in vivo to a certain extent,which can judge the migration and repair ability of cells.The results of cell migration ability of fibroblasts under different irradiation doses are shown in Figure 5 and 6.

Figure 5. Cell scratch results of fibroblasts incubated for 0 h and 24 h after different doses of irradiation

After irradiation and 24 h incubation,cell migration occurred to varying degrees in BC group and irradiation group (Figure 5).The scratch area of irradiation groups were smaller than that of the BC group,indicating that the cell migration of the irradiation groups were higher.Compared with the BC group,the average cell migration rate in the irradiation groups were significantly increased(Figure 6).The average cell migration rates in the groups of 1,2,and 8 J/cm2were 1.42 times (P<0.005),1.56 times (P<0.005) and 1.26 times (P<0.01) higher than that in the BC group,respectively.It reflected that with 2 J/cm2red light irradiation,the cell migration rate of fibroblasts was the fastest,indicating stronger cell repair ability.

Figure 6. Average relative cell migration rate of fibroblasts incubated for 24 hours after different doses of irradiation

In the 3 batches experiments of cell scratch test,the average cell migration rate of the 2 J/cm2group was 1.46 times (P<0.01),1.31 times (P<0.01),and 1.30 times (P<0.01) higher than those of the BC groups,respectively (Figure 7).Therefore,compared to the BC group,the cell migration rate after 2 J/cm2red light irradiation was significantly increased and had statistical significance.

Based on the above dose screening results and the three batches stability validation results,it could be concluded that the irradiation dose of 2 J/cm2could significantly and stably increase the migration rate of fibroblasts,thereby promoting cell proliferation,promoting cell viability and improving cell repair ability[26].

2.1.3 Cell collagen synthesis

By conducting collagen I immunofluorescence testing in fibroblasts after irradiation and 24 h incubated,the collagen synthesis levels of different irradiation groups can be obtained.Compared with the BC group,the irradiation groups had a more obvious fluorescence coloration,indicating that the irradiation groups had a higher collagen synthesis amount (Figure 8).As shown in Figure 9,compared with the BC group,the collagen synthesis of 1 J/cm2(P<0.01) and 2 J/cm2(P<0.01) groups increased significantly,which preliminarily confirmed that both 1 and 2 J/cm2were able to significantly increase the collagen synthesis of fibroblasts.Due to 2 J/cm2is the optimal dose among the three indicators (cell mitochondrial membrane potential,ATP release and cell migration) and showed significantly positive effect in collagen synthesis,therefore 2 J/cm2was selected for subsequent testing in this experiment to maintain consistency in experimental conditions of the low level red light fibroblast model.

Figure 8. Diagram of collagen I immunofluorescence staining results incubated for 24 hours after irradiation

Figure 9. Quantitative histogram of collagen I immunofluorescence staining under different red light irradiation doses

As shown in Figure 10,it suggested that collagen I synthesis level in 2 J/cm2groups were significantly higher than those in the BC groups.The collagen I synthesis level in 2 J/cm2groups were 3.62 times (P<0.005),2.67 times (P<0.005) and 3.76 times (P<0.005) higher than those in the BC group,respectively.

After dose screening experiments and three batches stability experiments of collagen synthesis,it was proved that 2 J/cm2red light irradiation could stably and significantly upregulate the expression of collagen I,achieving the goal of increasing the synthesis of collagen in cells.

Based on the above data,it can be concluded that the irradiation dose of 2 J/cm2can steadily and significantly improve the functional activity of mitochondria(mitochondrial Membrane potential and ATP release),enhance the cell migration rate and improve collagen synthesis in fibroblasts,so as to increase cell energy,cell activity,cell repair ability and collagen content.

2.2 Low level red light model validating in dermal papilla cells

Hair papilla cells are special interstitial components located at the base of hair follicles,playing a dominant role in hair follicle development and periodic growth regulation.The improvement of cellular activity and function of dermal papilla cells can to some extent enhance hair follicle activity,achieving the goal of hair growth and preventing hair loss.In the above experiments,it had been proven that 2 J/cm2red light irradiation can promote the cellular function of fibroblasts.Verify the efficacy of cell indicators by applying a 2 J/cm2irradiation dose on dermal papilla cells.A 2 J/cm2irradiation group and a blank control (BC) group were set up to verify whether the 2 J/cm2irradiation dose had a positive response to the cellular function of dermal papilla cells.

2.2.1 Mitochondrial function

The mitochondrial function test results in dermal papilla cells are shown in Figure 11.When the irradiation dose is 2 J/cm2,the mitochondrial membrane potential and intracellular ATP release of dermal papilla cells are significantly increased,which are 1.14 times (p<0.05) and 5.36 times (p<0.005)of BC group,respectively,indicating that 2 J/cm2red light irradiation had promoted the mitochondrial activity of dermal papilla cells and provided cell energy for dermal papilla cells.

Figure 11. Effect of 2 J/cm2 red light irradiation on mitochondrial function of dermal papilla cells. (a) the mitochondrial membrane potential;(b) the release of ATP.

2.2.2 Cell proliferation ability

The experimental results of cell proliferation ability of dermal papilla cells are shown in Figure 12.When the irradiation dose was 2 J/cm2,the proliferation rate of dermal papilla cells was significantly increased and 1.19 times that of the BC group (p<0.01),indicating that the irradiation dose of 2 J/cm2had a promoting effect on the cell activity of dermal papilla cells and promoted cell proliferation.

Figure 12. Results of cell proliferation of dermal papilla cells

2.2.3 Collagen synthesis ability

The results of collagen synthesis achievement test in dermal papilla cells are shown in Figure 13.When irradiation dose was 2 J/cm2,the collagen synthesis of dermal papilla cells significantly increased and 1.60 times of the BC group (p<0.01),indicating that the irradiation dose of 2 J/cm2had a promoting effect on collagen synthesis of dermal papilla cells and promoted collagen synthesis of cells.

Figure 13. Histogram of collagen synthesis of dermal papilla cells

3 Conclusion

In fibroblast model,low level red light of 2 J/cm2significantly increased mitochondrial membrane potential,ATP release,cell migration rate and collagen synthesis.It indicated that the low level red light of 2 J/cm2was helpful to provide energy for cell activity,improve cell repair ability and collagen synthesis for fibroblasts.The 2 J/cm2low level red light model in fibroblasts was transferred to human dermal papilla cells for efficacy test.The results showed that 2 J/cm2low level red light significantly improved the mitochondrial membrane potential,ATP release,cell migration rate and collagen synthesis of dermal papilla cells.This indicated that the 2 J/cm2low level red light test model established on fibroblasts was also applicable to dermal papilla cells.The 2 J/cm2low level red light could promote mitochondrial function,cell proliferation and collagen synthesis of dermal papilla cells.

In summary,through irradiation doses screening experimental in cellular level,it was demonstrated that low level red light of 2 J/cm2could stably and significantly enhance the mitochondrial activity,cellular activity,cell repair ability,and collagen synthesis of human fibroblasts and dermal papilla cells,thereby increasing cell activity.The test results were able to provide certain guiding significance for the anti-aging and anti-detachment of human skin.In addition,the experimental method of 2 J/cm2low level red light on four cell indicators established a simple cell model of low level red light to verify the synergistic effect between low level red light and active substances at the cellular level.