NORIKO Minmisw,MIAO Ming-Sn,KANG Le,LIU Hui-Jun,CUI Lin-Lin,HIROKI Knemisu,TERUMI Nito,HIROAKI Shinotsuk

a.Akyrise Ltd.,Tokyo 163-8001,Japan

b.Henan University of Chinese Medicine,Zhengzhou,Henan 450046,China

Keywords

Jin Bai Mei Yan Prescription (JBMYP)

HaCaT cell

Oxidative damage

Ultraviolet B (UVB)

Chinese medicine

Anti-aging

ABSTRACT

Objective Ultraviolet B (UVB) mainly acts on the skin epidermis,causing oxidative damage and apoptosis of keratinocytes.Jin Bai Mei Yan Prescription (JBMYP) comprises a variety of antioxidant traditional Chinese medicines (TCM).In this study,we aimed to evaluate the effects of JBMYP on the oxidative damage induced by UVB in human immortalized epidermal keratinocytes(HaCaT) cells.

Methods HaCaT cells were divided into six groups:control group,model (UVB) group,positive (UVB+vitamin E) group,UVB+JBMYP low dose group (160 μg/mL),UVB+JBMYP moderate dose group (800 μg/mL),and UVB+JBMYP high dose group(1 600 μg/mL).HaCaT cells were irradiated with UVB and treated with JBMYP for 24 h.Methyl thiazolyl tetrazolium (MTT) assay and real-time unlabeled cell function analyzer were used to assess the cell survival and proliferation rates,respectively.At the same time,the levels of intracellular reactive oxygen species(ROS),lipid peroxide malondialdehyde (MDA),glutathione (GSH),and hydroxyproline (HYP),as well as the activities of antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT) were evaluated using enzyme linked immunosorbent assay (ELISA).

Results Compared with the model group,the survival rate of HaCaT cells in each dosage group of JBMYP was significantly improved (P < 0.05).Further,JBMYP could promote the proliferation of HaCaT cells,leading to a reduction in the contents of MDA and ROS,and increase in the contents of SOD,CAT,GSH and HYP in HaCaT cells.

Conclusions JBMYP has enhanced protective effect on oxidative damage induced by UVB in HaCaT cells.

1 Introduction

Ultraviolet rays present in the sunlight,include longwave ultraviolet (UVA) and medium-wave ultraviolet(UVB) rays,of which the wavelength of mediumwave ultraviolet rays is 275-320 nm.In addition,UVB is the most important cause of radiation damage in the human immortalized epidermal keratinocytes(HaCaT).Long-term exposure of human skin to these harmful UVB rays accelerates the skin aging process.After receiving UVB irradiation,skin keratinocytes can induce the accumulation of reactive oxygen species (ROS) in their specific target cells,which further induces oxidative damage of the cells and even skin cancer[1].Therefore,it is of importance to conduct research on skin aging and establish effective anti-aging strategies and medicines for promoting and developing the health industry in order to improve the quality of life.Skin care products developed using traditional Chinese medicine (TCM) as raw materials possess characteristics such as high safety and less adverse reactions.

TCM has a long history of thousands of years in beauty-building and anti-aging industry.As early as in thePrescriptions for Fifty-two Diseases(Wu Shi Er Bing Fang,《五十二病方》) unearthed in Mawangdui,Hunan Province,a beauty prescription for removing warts and spots with TCM has been included in the list of prescriptions.More than 300 prescriptions for nourishing skin and promote anti-aging were included in theTaiping Royal Prescriptions(Tai Ping Sheng Hui Fang,《太平圣惠方》) andGeneral Medical Collection of Royal Benevolence(Sheng Ji Zong Lu,《圣濟(jì)總錄》) in the Song Dynasty.In the world-renowned medicine monographCompendium of Materia Medica(Ben Cao Gang Mu,《本草綱目》),there are more than 270 Chinese medicines described for nourishing skin and promoting antiaging.TCM cosmetology has been used till now and has further developed rapidly in recent years.They are very popular among the users for their characteristics,as they are safe,green and have less side effects.

Jin Bai Mei Yan Prescription (JBMYP) includes 15 Chinese medicines from commonly used clinical cosmetic medicines,namely Coicis Semen (Yi Yi Ren,薏苡仁),Armeniacae Semen Amarum (Ku Xing Ren,苦杏仁),Artemisiae Argyi Folium (Ai Ye,艾葉),Lonicerae Japonicae Flos (Jin Yin Hua,金銀花),Sophorae Flavescentis Radix (Ku Shen,苦參),Citri Reticulatae Pericarpium (Chen Pi,陳皮),Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草),Cinnamomi Cortex (Rou Gui,肉桂),Zingiberis Rhizoma Recens (Sheng Jiang,生姜),Allii Macrostemonis Bulbus (Xie Bai,薤白),Menthae Haplocalycis Herba (Bo He,薄荷),Lilii Bulbus (Bai He,百合),Ginkgo Semen (Bai Guo,白果),Puerariae Lobatae Radix (Ge Gen,葛根) and Eucommiae Folium (Du Zhong Ye,杜仲葉).Among these,Coicis Semen (Yi Yi Ren,薏苡仁),Armeniacae Semen Amarum (Ku Xing Ren,苦杏仁),Lonicerae Japonicae Flos (Jin Yin Hua,金銀花),Citri Reticulatae Pericarpium (Chen Pi,陳皮),Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草),Menthae Haplocalycis Herba (Bo He,薄荷),Lilii Bulbus (Bai He,百合) and Ginkgo Semen (Bai Guo,白果) have commonly been used in ancient prescriptions for beauty[2].Moreover,Coicis Semen (Yi Yi Ren,薏苡仁) in the prescription is sweet and cold.Its external application is good for strengthening the spleen,removing spots and nourishing the face,and thus it is usually used for removing facial spots and smoothening rough skin.Armeniacae Semen Amarum (Ku Xing Ren,苦杏仁) is rich in oils,which can moisturize the fur and skin.The two medicines in combination can stimulate the spleen,nourish the face and remove dampness.They are all principal medicines.Artemisiae Argyi Folium(Ai Ye,艾葉) is warm and dry,which is known for removing dampness and itchiness.Lonicerae Japonicae Flos (Jin Yin Hua,金銀花) and Sophorae Flavescentis Radix (Ku Shen,苦參) are bitter and cold.They together play a role in reducing heat,moistening the skin,relieve itching and not hurting the Yin,and they help the principal medicine in nourishing the face.They are the principal medicines,however the rest are the assistant medicines.It has also been reported that external application of Artemisiae Argyi Folium(Ai Ye,艾葉) can promote skin blood circulation,strengthen the metabolism,and promote the smooth discharge of metabolic wastes and toxins,leading to cosmetic and skin-moisturizing effects[3].Cinnamomi Cortex (Rou Gui,肉桂) also has cosmetic effects,such as skin whitening,anti-photoaging,scavenging free radicals and preventing acne,as it acts as a sunscreen[4].Flavescentis Radix (Ku Shen,苦參),Zingiberis Rhizoma Recens (Sheng Jiang,生姜),Allii Macrostemonis Bulbus (Xie Bai,薤白),Eucommiae Folium (Du Zhong Ye,杜仲葉) and Puerariae Lobatae Radix (Ge Gen,葛根) have been reported to have anti-inflammatory,antioxidant and free radical scavenging effects through oral administration[5].In order to explore and fully utilize the advantages of Chinese medicine cosmetology and explore its mechanism of action,in this study,we aimed to assess the protective effects of JBMYP on UVB-induced oxidative damage in HaCaT cells,in order to provide an experimental basis and reference for the application of anti-aging beauty products.

2 Materials and Methods [6-13]

2.1 Medicines and materials

Compositions of JBMYP are Coicis Semen (Yi Yi Ren,薏苡仁) 12 g,Armeniacae Semen Amarum (Ku Xing Ren,苦杏仁) 12 g,Artemisiae Argyi Folium (Ai Ye,艾葉) 9 g,Lonicerae Japonicae Flos (Jin Yin Hua,金銀花) 9 g,Sophorae Flavescentis Radix (Ku Shen,苦參)9 g,Citri Reticulatae Pericarpium (Chen Pi,陳皮) 6 g,Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草) 6 g,Cinnamomi Cortex (Rou Gui,肉桂) 6 g,Zingiberis Rhizoma Recens (Sheng Jiang,生姜) 5 g,Allii Macrostemonis Bulbus (Xie Bai,薤白) 6 g,Menthae Haplocalycis Herba (Bo He,薄荷) 5 g,Lilii Bulbus(Bai He,百合) 5 g,Ginkgo Semen (Bai Guo,白果) 5 g,Puerariae Lobatae Radix (Ge Gen,葛根) 5 g,and Eucommiae Folium (Du Zhong Ye,杜仲葉) 6 g.All these medicines were purchased from ZHANG Zhong-Jing Drugstore,Henan Province.The medicinal materials were soaked with 15 times of water at room temperature (18-25 °C) for 30 min until the water was completely absorbed by the interiors of the medicinal materials.The tank was heated for extraction,but the time was started only after the water reached the boiling state.A slight boiling was maintained at normal pressure,with temperature not less than 90 °C.The first extraction time was 40 min,with temperature at 60 °C.The second extraction time was 30 min with eight times of water.The residues of medicines were removed by passing the suspension through a folded 300-mesh gauze with eight folds.The filtrate was taken twice,which were combined together.Static precipitation was performed for not less than 12 h.The 300-mesh gauze was folded into eight folds for another filtration step.The supernatant was extracted and placed in a rotary evaporator.The pressure was maintained at 0.08-0.1 MPa and the temperature was kept not more than 65 degrees.The filtrate was concentrated to have 1 g/mL of raw medicine,and refrigerated at 4 °C for 24 h.Thereafter,the medicine was centrifuged at 10 000 rpm for 10 min.The supernatant was taken,and passed through a 0.22 μm filter with microporous membrane for preservation.

Vitamin E (positive medicine) is the principal raw material present in natural vitamin E sources,such as safflower seed oil,perilla oil,gelatin and glycerin.Function:beauty-building (remove chloasma) and delaying the aging process.Supplier:Yangshengtang pharmaceutical Co.,Ltd.(Batch No.20180407).Usage and dosage:1 tablet once per day.Specification:250 mg×200 tablets.

2.2 Reagent

The minimum essential medium (MEM) was purchased from Wuhan Procell Life Science and Technology Co.,Ltd.(Batch No.WH01111806SP).The fetal bovine serum (FBS) was purchased from Hyclone Company (Batch No.RB35934).The 0.25%trypsin was purchased from Gibco Company (Batch No.1906755).The Penicillin-Streptomycin Mixture was purchased from Beijing Solarbio Biotechnology Co.,Ltd.(Batch No.P1400).The dimethyl sulfoxide(DMSO) was purchased from Beijing Solarbio Biotechnology Co.,Ltd.(Batch No.1213C0342).The 1 x PBS buffer was purchased from Beijing Solarbio Biotechnology Co.,Ltd.(Batch No.P1020-500).The methyl thiazolyl tetrazolium (MTT) was purchased from Beijing Solarbio Biotechnology Co.,Ltd.(Batch No.413Y053).The Triton-100 was purchased from Beijing Solarbio Biotechnology Co.,Ltd.(Batch No.301G053).Superoxide dismutase (SOD) kit was purchased from Nanjing Jiancheng Institute of Bioengineering (Batch No.20180821).The reactive oxygen species (ROS) kit was purchased from Suzhou Calvin Biotechnology Co.,Ltd.(Batch No.201808013A).The hydroxyproline (HYP) kit was purchased from Suzhou Calvin Biotechnology Co.,Ltd.(Batch No.201808013A).The cell malondialdehyde (MDA) test box was purchased from Nanjing Jiancheng Institute of Bioengineering(Batch No.20180821).The catalase (CAT) kit was purchased from Suzhou Calvin Biotechnology Co.,Ltd.(Batch No.201808013A).The trace reduced glutathione hormone (GSH) kit was purchased from Nanjing Jiancheng Institute of Bioengineering (Batch No.20180821).

2.3 Cell line

HaCaT cells was purchased from Wuhan Procell Life Technology Co.,Ltd.The quantity obtained was 2×106cells/T25 (Catalog No.CL-0090).

2.4 Apparatus

The LDZX-50FBS Vertical Pressure Steam Sterilizer was purchased from Shanghai Shen'an Medical Apparatus and Instruments Factory.The BPG-9 140A precision air dry oven was purchased from Shanghai Yiheng Scientific Instruments Co.,Ltd.The RE-3 000 Rotary Thin Film Evaporator was purchased from Shanghai Yarong Biochemical Instrument Factory.The IC 1 000 Countstar Automatic Cell Counting Instrument was purchased from Shanghai Ruiyu Biological Technology Co.,Ltd.The 3 111 Carbon Dioxide incubator and Heraeus Multifuge X1R centrifuge were purchased from the German Thermo Company.The Cytation 3/CYT3 MFVDG Cell Imaging Microplate Detector was purchased from BioTek Instrument Co.,Ltd.The real time unlabeled cell function analyzer and E-Plate 16 PET were purchased from ACE Company.

2.5 Cell culture

Cells were first allowed to grow to reach the logarithmic phase,digested by trypsin for 2 min,centrifuged,and then counted by using the counting plate.Cells were inoculated into 96-well plates at a concentration of 1×104cells/well.Cells were inoculated into 6-well plates at a concentration of 1×106cells/well for each group.Cells were cultured at 37 °C in an incubator with constant supply of 5% CO2.

2.6 Index test

2.6.1 MTT assay for cell survivalCells in the 96-well plate were randomly divided into the following groups:control (culture medium) group; model(culture medium+UVB) group; positive control groups:culture medium+UVB+vitamin E (80 μg/mL)group,culture medium+UVB+JBMYP low dose(160 μg/mL) group,culture medium+UVB+JBMYP moderate dose (800 μg/mL) group,culture medium+UVB+JBMYP high dose (1 600 μg/mL) group.

The cells were incubated in the 96-well plate for 24 h.Then,the supernatant was discarded and the cells were washed twice with PBS by adding 200 μL PBS to each well.The control group was covered with aluminum paper and the plate was irradiated with UVB disinfection lamp tube.The radiation distance was set at 70 cm.The radiation dose was 30 J/m2and the plate was exposed for 20 min.After irradiating each group,PBS was discarded and 200 μL mixture(culture medium and JBMYP) was added into each dose group of JBMYP.The UVB and control groups were incubated with the same amount of culture medium.After incubation for 24 h,20 μL MTT(5 mg/mL) solution was added into each well,incubated for 4 h,and then the culture medium was discarded.Thereafter,150 μL of DMSO was added into each well.The plate was incubated at 37 °C for 10 min on an oscillator,and the optical density was measured at 492 nm wavelength as per the manufacturer’s instruction.

2.6.2 Detection of cell proliferation rate by real-time unlabeled cell analyzerThe E-Plate 16 was used to set up the control,UVB+vitamin E,UVB+JBMYP low dose,UVB+JBMYP moderate dose and UVB+JBMYP high dose groups.A total of 50 μL of FBS medium was added to each well,and real-time labeled cell analyzer was used to perform the scan and baseline detection.Then,the baseline E-Plate 16 was taken out and 100 μL FBS-containing cell suspension was added into each well.Cells were taken at a concentration of 1×105cells/mL and were incubated at room temperature for 30 min.Then the cell function was assessed by real-time unlabeled cell function analyzer.After 24 h,except for the control group,the UVB+vitamin E,UVB+JBMYP low dose,UVB+JBMYP moderate dose and UVB+JBMYP high dose groups were added with 5 μL of medicinal liquid,while equal volume of culture medium was added to the control group.After 72 h,the reading values were obtained.

2.6.3 Detection of ROS,MDA,CAT,SOD,GSH and HYP levels in HaCaT cellsIn the 6-well plate,2 mL cell suspension was added into each well.The concentration of cells used was 5×105cells/mL.Cells were incubated at 37 °C with 5% CO2for 24 h.Then the supernatant was absorbed and discarded.The cells were washed twice with PBS by adding 2 mL PBS to each well.The control group was covered with aluminum paper.Rest of the groups were irradiated with UVB disinfection lamp tube.The radiation distance was set at 70 cm.The radiation dose of 30 J/m2was provided for 20 min.After irradiation,PBS was discarded.Each dose group of UVB+JBMYP was added with 2 mL of medicinal liquid.The UVB and control groups were added with the same amount of culture medium.After incubation for 24 h,the supernatant was absorbed and discarded,and the cells were washed twice with PBS.Thereafter,the cells were added with 1 mL trypsin per well for digestion for 2 min,and subjected to centrifugation at 168 g for 10 min.Then,the supernatant was absorbed and discarded.The levels of ROS,MDA,CAT,SOD,GSH and HYP in HaCaT cells were detected according to the instructions on the enzyme linked immunosorbent assay (ELISA) kit.

2.7 Data and statistical analysis

Statistical analyses were performed using The Statistical Software Package for the Social Sciences,version 21.0 for Windows.The experimental results were expressed in the form of mean±SD.One-way ANOVA andttest were used for inter-group comparison.P< 0.05 is considered statistically significant.

3 Results

3.1 Effects of JBMYP on survival rate of HaCaT cells injured by UVB

According to the results of MTT assay,compared with the control group,the survival rate of UVB group was found to decrease significantly (P< 0.01).Compared with the UVB group,UVB+vitamin E group could significantly improve the survival rate of HaCaT cells (P< 0.01).UVB+JBMYP low dose group also showed improved survival rate of HaCaT cells(P< 0.05).Although there was no significant difference between UVB+JBMYP moderate dose and JBMYP high dose groups when compared with the control group,it also showed a trend for improving the survival rate of HaCaT cells.The results are shown in Figure 1 and Table 1.

3.2 Effects of JBMYP on the proliferation of HaCaT cells

Analyzed by using the real-time unlabeled cell analyzer,the effects of JBMYP on the proliferation of HaCaT cells was shown in Figure 2 and Table 2.At 24 h,there was no significant difference in the number of cells in each group.However,after 24 h of administration of medicine,that is,at 48 h,compared with the control group,UVB+vitamin E,UVB+JBMYP low dose and UVB+JBMYP moderate dose groups could significantly promote the proliferation of HaCaT cells (P< 0.01).At 72 h,compared with the control group,UVB+vitamin E and UVB+JBMYP low dose groups still significantly promoted the proliferation of HaCaT cells (P< 0.05).The results are shown in Figure 2.

3.3 Effects of JBMYP on MDA and ROS levels of HaCaT cells after UVB irradiation

As shown in Figure 3 and Table 3,compared with the control group,the MDA and ROS levels in the UVB group increased significantly (P< 0.01).Compared with the UVB group,UVB+vitamin E,UVB+JBMYP low dose,UVB+JBMYP moderate dose and UVB+JBMYP high dose groups had a tendency to reduce MDA level in HaCaT cells.Compared with the UVB group,UVB+vitamin E,UVB+JBMYP low dose,and JBMYP moderate dose groups could significantly reduce ROS level in HaCaT cells (P< 0.01).The high dose group of JBMYP also showed a tendency to decrease the ROS levels in HaCaT cells.

3.4 Effects of JBMYP on SOD,CAT and GSH levels of HaCaT cells after UVB irradiation

Compared with the control group,the levels of SOD,CAT and GSH in UVB group decreased significantly(P< 0.01,Table 4).Compared with the UVB group,UVB+vitamin E group significantly shown an increase in SOD and CAT levels in HaCaT cells(P< 0.01).The levels of SOD and GSH in HaCaT cells were significantly increased in UVB+JBMYP low dose group (P< 0.05),and the level of CAT was also significantly increased (P< 0.01).The other groups showed the trend of increasing the levels of SOD,CAT and GSH in HaCaT cells.The results are shown in Figure 4.

Table 1 Effects of JBMYP on the survival rate of HaCaT cells irradiated by UVB (±s,n=6)

Table 1 Effects of JBMYP on the survival rate of HaCaT cells irradiated by UVB (±s,n=6)

△△P < 0.01,compared with the control group; *P < 0.05,**P < 0.01,compared with the UVB group.

Group OD490 nm Survival rate (%)Control 0.121±0.017 100 UVB 0.078±0.007△△ 64.46△△UVB+Vitamin E 0.112±0.011** 92.56**UVB+JBMYP low dose 0.091±0.010* 75.21*UVB+JBMYP moderate dose 0.087±0.007 71.90 UVB+JBMYP high dose 0.080±0.030 66.12

Table 2 Effects of JBMYP on the proliferation of HaCaT cells (±s,n=3)

Table 2 Effects of JBMYP on the proliferation of HaCaT cells (±s,n=3)

*P < 0.05,**P < 0.01,compared with the control group.

Group 24 h (cell index) 48 h (cell index) 72 h (cell index)Control 2.86±0.03 2.76±0.1 1±0.13 UVB+Vitamin E 3.17±0.08 4.13±0.17** 2.03±0.06*UVB+JBMYP low dose 3.2±0.19 4.13±0.29** 1.99±0.11*UVB+JBMYP moderate dose 2.98±0.1 3.81±0.26** 1.55±0.26 UVB+JBMYP high dose 2.62±0.44 2.67±0.6 0.43±0.84

3.5 Effects of JBMYP on HYP level in HaCaT cells after UVB irradiation

Compared with the control group,the UVB group significantly showed a decrease in the level of HYP in HaCaT cells (P< 0.01,Table 5).Compared with UVB group,UVB+vitamin E and UVB+JBMYP low dose groups showed a significant increase in the level of HYP in HaCaT cells (P< 0.05).Both UVB+JBMYP moderate dose and UVB+JBMYP high dose groups showed a trend to increase the level of HYP in HaCaT cells.The results are shown in Figure 5.

4 Discussion

In recent years,with an increase in environmental pollution,ultraviolet radiation has become one of the major factors affecting human health.The keratinocytes covering the skin surface are the main points of action for UVB.Ultraviolet rays can induce photoaging and skin aging[14].

In the cell metabolism process,a certain amount of ROS are produced.Under physiological conditions,ROS are constantly produced and scavenged so that the body maintains a beneficial,harmless,lowlevel and stable level of these free-radical species,such that the body's reactive oxygen radical-antioxidant system is in a relatively balanced state.When external physical and chemical factors,such as ultraviolet radiations and chemical medicines act on the human body,they can induce the production of large number of free radicals,which clearly exceed the ability of the body’s antioxidant system to scavenge free radicals,making the above-mentioned balance system disordered.The accumulation of ROS can result in the formation of lipid peroxides,such as malondialdehyde (MDA),which can further damage the membrane structure of cells.The results of this study showed that JBMYP could decrease the levels of ROS and MDA in HaCaT cells and thereby protect the integrity of cell membrane structure and function[15-17].

Reductive glutathione (GSH) is one of the mostimportant non-enzymatic antioxidants produced in the body.It has many important physiological functions,such as scavenging free radicals,detoxifying,promoting iron absorption,and maintaining the integrity of erythrocyte membrane,biosynthesis of deoxyribonucleic acid,normal cell growth and development,as well as cellular immunity.It can also balance the oxidation and anti-oxidation system of the body along with SOD,CAT,HYP,and thus protect the body from free radical injury.In this study,JBMYP was shown to increase the activity of GSH,SOD,CAT and HYP,which might be involved in accelerating the scavenging process of free oxygen radicals and reduce the production of these free oxygen radicals,and thereby reducing the degree of lipid peroxidation damage induced by UVB radiation in HaCaT cells[18-20].

Table 3 Effects of JBMYP on MDA and ROS levels after UVB irradiation in HaCaT cells (±s,n=6)

Table 3 Effects of JBMYP on MDA and ROS levels after UVB irradiation in HaCaT cells (±s,n=6)

△△P < 0.01,compared with the control group; **P < 0.01,compared with the UVB group.

Group MDA (nmol/mg prot) ROS (IU/mL)Control 2.9±0.95 566.88±89.64 UVB 5.17±1.01△△ 1 228.13±191.11△△UVB+Vitamin E 3.83±1.6 956.88±84.39**UVB+JBMYP low dose 4.03±1.17 943.13±92.34**UVB+JBMYP moderate dose 4.16±1.01 998.13±75.31**UVB+JBMYP high dose 4.31±1.23 1 098.13±183.15

Table 4 Effects of JBMYP on SOD,CAT and GSH levels in HaCaT cells after UVB irradiation (±s,n=6)

Table 4 Effects of JBMYP on SOD,CAT and GSH levels in HaCaT cells after UVB irradiation (±s,n=6)

△△P < 0.01 compared with the control group; *P < 0.05,**P < 0.01,compared with the UVB group.

Group SOD (U/mg prot) CAT (U/mL) GSH (μmol/g prot)Control 191.96±36.98 79.42±10.87 61.79±16.43 UVB 94.7±21.3△△ 41.46±6.16△△ 29.5±9.68△△UVB+Vitamin E 178.41±32.44** 55.51±7.19** 45.56±13.52 UVB+JBMYP low dose 133.94±31.26* 56.21±5.76** 54.79±18.57*UVB+JBMYP moderate dose 122.02±22.73 51.36±7.9* 40.74±12.57 UVB+JBMYP high dose 102.72±24.47 44.08±6.3 35.19±16.48

Table 5 Effects of JBMYP on HYP level in HaCaT cells after UVB irradiation (±s,n=6)

Table 5 Effects of JBMYP on HYP level in HaCaT cells after UVB irradiation (±s,n=6)

△△P < 0.01,compared with the control group; *P < 0.05,compared with the UVB group.

Group HYP (μmol/L)Control 697.47±54.00 UVB 613.13±61.52△△UVB+Vitamin E 691.13±79.73*UVB+JBMYP low dose 690.47±67.00*UVB+JBMYP moderate dose 686.13±65.89 UVB+JBMYP high dose 667.47±53.22

In this study,HaCaT cells were treated with three doses of JBMYP (160 μg/mL,800 μg/mL and 1 600 μg/mL) seperately to observe the differences in cell states in three groups.The results showed that several indicators showed that the effect of low dose(160 μg/mL) of JBMYP is better than those of the moderate and high doses groups (800 and 1 600 μg/mL).It also suggests that JBMYP at very high concentrations does not have a better protective effect on HaCaT cells,which might be related to some cold medicines present in the JBMYP.Excessive amount of cold medicine concentration can induce some side effects,thereby inhibiting the proliferation of HaCaT cells.

The prolonged exposure to ultraviolet rays in daily life can cause cell damage and death in the body.The oxidative stress induced by ultraviolet rays causes excessive generation of intracellular ROS in skin cells,leading to cell damage and eventually apoptosis.In this study,human immortalized keratinocyte HaCaT cells irradiated by UVB were used for analysis.The results showed that the survival rate of HaCaT cells damaged by UVB radiation is significantly decreased,and the levels of MDA and ROS are significantly increased with a significant reduction in the levels of SOD,CAT,GSH and HYP upon administration of the JBMYP,showing a clear oxidation state.The degree of damage of HaCaT cells incubated with JBMYP was significantly reduced,indicating that JBMYP had photoprotective effect on keratinocytes damaged by UVB radiation.In summary,this study is a preliminary experimental model of JBMYP demonstrating the protective effects of JBMYP on HaCaT cells.Greater insights into the mechanism of action of JBMYP and the effect generated when directly applied on the skin will be the focus of the future research on this topic.

Acknowledgements

We thank for the funding support from the National Administration of Traditional Chinese Medicine(No.2017-149-11),National Base for International Cooperation (No.2016-65) and Henan Province Industry-University-Research Collaboration (No.182107000029) to conduct the Special Project on Standardization of Traditional Chinese Medicine.

Competing Interests

The authors declare no conflict of interest.