The protective effect of Dendrobium officinale polysaccharides on photoaging fibroblasts by scavenging reactive oxygen species and promoting the expression of TGF-β1

RuiTang,Qia-Qia Li,Di Wang,Jing Chen,Jin-Hua Huang*,Qing-HaiZeng*

1Xiangya School of Medicine,Central South University,Changsha,Hunan,China.2Dermatological Department,The Third Xiangya Hospital of Central South University,Changsha,Hunan,China.

Background

Skin photoaging refers to the damage caused by long-term repeated exposure of the skin to ultraviolet(UV)rays.Long-term exposure of the skin to UV causes dermal collagen degeneration and progressive degradation and fragmentation of elastic fibers until they disappear;the skin becomes loose,dry,w rinkly,pigmented,and sensitive [1,2].The mechanism underlying skin photoaging is complicated.It has been found that various photosensitive substances or chromophores acting on the skin through UV exposure can induce the generation of reactive oxygen species(ROS),such as OH·and lipid peroxides,destroying the skin’s own antioxidant system and leading to an imbalance of oxidative stress[3,4].Oxidative stress caused by UV irradiation can promote the expression of matrix metalloproteinases(MMPs),reduce collagen synthesis and secretion,and cause skin damage[5-9].MMP-1 mainly degrades type I collagen-based skin extracellular matrix.During the process of photoaging,the expression of MMP-1 is increased,and this can be used as an indicator of skin photoaging[10].Among the many cytokines that induce collagen secretion,transforming grow th factor β (TGF-β)is currently the most recognized cytokine that induces the synthesis and secretion of extracellular matrix[11].In the TGF-bfamily,TGF-b1 has the highest expression,the strongest activity,and the most extensive range in somatic cell lines.Previous studies found that TGF-b1 could significantly increase the expression of type I procollagen mRNA in artificial skin and TGF-β1 can also reduce the expression of MMP-1 mRNA and inhibit collagen degradation[12-13].In the photoaging process,decrease in TGF-b1 can be used as an indicator of skin photo-aging.

In order to reduce UV damage to the skin,people should not only cover the skin to prevent UV radiation exposure,but also use antioxidants,anti-inflammatory agents,and other protective measures to prevent UV damage to the skin,and even repair damaged tissue.In recent years,studies found that many traditional Chinese medicines had a positive effect on the prevention and treatment of skin photoaging,with high efficiency and no side effects[14-16].Tiepishihu(Dendrobium officinaleKimura et Migo,TPSH)is an orchid and herbaceous plant that has a unique medicinal value.TPSH was first recorded inShennongbencaojingin the Qin and Han Dynasty of China(221 B.C.-25 A.D.)as being sweet,with the property of eliminating wind,cold,and dampness.Modern pharmacology studies have shown that,in addition to TPSH acting to regulate endocrine function, enhance resistance, and promote tissue regeneration,it also can scavenge oxygen free radicals and thus has a very prominent role in anti-aging therapy[17].ROS play a very important role in the photoaging of the skin;thus,we investigated whether TPSH could play a role in antagonizing photoaging by scavenging ROS.There have been no previous studies evaluating this question.This paper explored the protective effect ofDendrobiumofficinalepolysaccharides (DOP) on photoaging human skin fibroblasts and the specific underlyingmechanism.

Methods

Materials and reagents

DOP of 90%purity were purchased from Xi’an Wei Te Biotechnology Co.,Ltd.DMEM medium was purchased from Gibco.Fetal bovine serum was purchased from the United States Hyclone company.The β-galactosidase(SA-β-Gal)kit and reactive oxygen detection kit were purchased from Biyun Tian Biotechnology Research Institute.The MMP-1 detection kit and pre-human type I collagen C-terminal peptide (CICP)test kit were purchased from the Wuhan Bode Science and Technology Development Co.,Ltd.Biotech.The TGF-β1 test kitwas purchased from Wuhan Elaite Biotechnology Co.,Ltd.The UVB irradiation instrument(SS-07)was purchased from ShanghaiSigma Technology Co.,Ltd.

Fibroblastextraction and culture

The skin tissue of prepuce was obtained from a healthy young man after circumcision.The specimen was selected in advance,and the patient provided w ritten informed consent.The study procedurewas confirmed by the hospital ethics comm ittee.The skin tissue was placed in a complex soaked in iodine for 15 min.The dirt was cleaned off with PBS solution,and the subcutaneous tissue was cut.The tissue was then cut into pieces.The tissue pieceswere allowed to attach to a 35 mm Petridish by being left undisturbed for 10 min.Two to three drops of DMEM were periodically added to prevent the tissue block from drying.The dish was placed in a carbon dioxide incubator overnight.The next day,2 m L DMEM was slow ly added to the culture dish,and the liquid medium was changed once after 2-3 days.When many cells grew around the tissue block,the fibroblasts were digested with 0.25%trypsin,collected,and washed.The isolated fibroblasts were cultured in high-glucose DMEM containing 10%fetal bovine serum and 1%double antibody.When the cultured fibroblastsgrewinto a dense monolayer,they were subcultured at a ratio of 1:3.In this experiment,3-10 generationsof fibroblasts were used.

Experimental grouping and adm inistration

The same generation of fibroblasts were random ly divided into a normal control group,photoaging model group(60 m J/cm2UVB irradiation),and UVB irradiation+ DOP groups.Fibroblasts were incubated with serum-free medium for 24 h and then irradiated with 60 m J/cm2UVB to establish the fibroblastphotoaging model[18].The control group was not irradiated,and DOP were not added to the culture solution.For the photoaging model group,cells were irradiated with UVB,and DOP were not added to the culture solution.For the UVB irradiation+DOP groups,the cells were incubated with serum-free medium containing DOP at different concentrations for 24 h before UVB irradiation.AfterUVB irradiation,the cells continued to be cultured with the culture solution containing different concentrations of DOP for 24 h.DOP powder was dissolved in PBS solution at a concentration of 1 mg/m L,stored in a stock solution at 4 °C and diluted to the appropriate concentrationwith serum-free DMEM.

Cell culture and cell viability assay

The toxic effects of different concentrationsof DOPwere studied.Fibroblasts were incubated with different concentrations of DOP in serum-free medium for 24 h.MTT(20μL,5 mg/m L)solution was added and incubated at 37°C for 4 h.The absorbance value at 490 nm was read on amicroplate reader.

SA-β-Gal assay for detection of senescent cells

The cells were stained according to the instructions of the SA-β-Gal Staining Kit.The cells were observed at 200×magnification under a lightmicroscope.Fifteen fields of view were selected for each specimen,and at least 200 cells were random ly counted.The positive cell ratios were calculated as follows:positive cell ratios=blue dyed cells/totalnumber of cells×100%).

ROS detection by flow cytometry

The cells were treated and collected according to the kit’s operating instructions.The fluorescence intensity was measured by flow cytometry.The stronger the fluorescence intensity is,the higher the ROS level is.

ELISA for expression analysis of MMP-1,CICP,and TGF-β1

The spent culturemedium was aspirated and centrifuged at 3000 rpm for 10 min.Then,according to the instructions of theMMP-1,CICP,and TGF-β ELISA Kits,the OD valuewasmeasured at450 nm with a microplate reader,and the concentrations were calculated according to the standard curve.The concentrations of MMP-1,CICP,and TGF-β1 in fluid weremeasured.CICP can be used asan indirect measure to assess the contentof type I collagen.

Statistical analysis

Statistical analyses of experimental data were performed using SPSS 19.0.The experiment was repeated three times,and data were expressed as means±standard deviations;the differences between groups were analyzed using analysis of variance or t-test.WhenP<0.05,the differencewas considered statistically significant.

Results

Drug toxicity of DOPon fibroblasts

The MTT assay was used to determine cell activity of fibroblasts after being treated with DOP.The results showed no toxicity in the groups treated with 20,40,and 80 μg/m L of DOP.The cell activity in the 160 μg/m L concentration group was significantly lower than that in the control group(P=0.003)(Figure 1).The results showed that within a certain concentration,DOP is not toxic to fibroblasts,but high concentrations have toxic effects on fibroblasts.

DOP inhibits UVB irradiation-induced fibroblast injury

As shown in figure 2,the senescent cells presented blue color after detected with SA-β-Gal assay.The percentage of senescent cells in the control group was 18.67%,while that in the photoaging model group was 66.48%,indicating a significant difference between the two groups(P<0.001).The cell senescence rates of the DOP groups(20,40,80 μg/m L)were lower than that of the photoaging model group.The senescent cell rate decreased gradually with increasing DOP concentration;the rate in the DOP 20 μg/m L group was 55.82%.There was no statistically significant difference in the senescence rate between DOP 20 μg/m L group and photoaging model group(P=0.087).The rates in the DOP 40 and 80 μg/m L groups were 50.45%and 42.63%,respectively.Compared with the photoaging model group,the difference was statistically significant(P=0.025,P=0.004 respectively).

Figure1 Cytotoxicity of DOPon fibroblasts

Figure 2 DOPdepressed fibroblastsaging induced by UVB

Figure 3 The effects of DOPon the expression ofMMP-1,CICPand TGF-β1.

The effects of DOP on the expression of MMP-1,TGF-β1 and CICP

The results showed thatcompared with the control group,the expression of MMP-1 in fibroblasts significantly increased,and the expression of TGF-β1 and CICP decreased significantly after UVB irradiation(P<0.001,P=0.026 andP=0.017)(Figure 3).The expression of MMP-1 in the DOP groups(20,40,80 μg/m L)was significantly lower than that in the photoaging model group(P=0.038,P=0.007 andP=0.001 respectively);the expression of CICP in the DOPgroups(40,80μg/m L)was significantly higher than that in the photoaging model group(P=0.008 andP=0.002 respectively);compared with the photoaging model group,the expression of TGF-β1 (DOP 20,40,80 μg/m L group)increased gradually,and the difference was statistically significant(P=0.015,P=0.006 andP<0.001 respectively).

DOP inhibits the production of ROS in fibroblasts after UVB irradiation

Compared with the control group,the fluorescence intensity of ROS in the photoaging model group was increased(P<0.001),indicating the higher level of ROS.While after treated with increasing concentrationsof DOP,the fluorescence intensity of ROS was decreased,indicating the lower level of ROS.Flow cytometry data also showed that the ROS content in the photoaging model group was 2.3 times higher than thatof the control group;ROS levels in the DOP 20,40,and 80μg/m L groups were lower than those in the photoaging model group(P=0.029,P=0.017 andP=0.002).This suggests that a certain concentration of DOP can inhibit UVB-induced ROS production(Figure 4).

Figure4DOPdepressed the levels of UVB-induced ROS

Discussion

TPSH has a long history of medicinaluse.It is one of the most effective medicinal products among the more than 70 kinds of dendrobium in China.The polysaccharides and dendrobins are several times richer in TPSH than in other varieties of dendrobium.Dendrobium has a unique medicinal value,with hypoglycemic,anti-oxidation,anti-tumor,and anti-aging properties,while it has also been shown to improve immunity and have other beneficial effects[1,19-23].DOP is one of the main active components of TPSH,and has antioxidant and anti-aging effects,as it scavenges oxygen free radicals[24-29].ROS damage is currently recognized as one of themajor mechanisms leading to photoaging[30-36].We speculated that DOP could protect the photoaging of fibroblastsby inhibiting the production of ROS.

At 60 m J/cm2UVB irradiation,the cell activity was about 75%of that in the normal controlgroup.Therefore,60 m J/cm2was chosen as the irradiation dose for inducing the photoaging model.The toxicity of different concentrations of DOP(20,40,80,160 μg/m L)was explored.The results showed that 20,40,and 80μg/m L DOP had no cytotoxic effect on fibroblasts,and these concentrations were chosen for the study.The results of SA-β-Gal staining showed that 20,40,and 80 μg/m L DOP could reduce cell senescence induced by UVB irradiation,and the number of senescent cells in 40 and 80μg/m L DOP groups decreased significantly.Thus,DOPcan protect fibroblasts from photoaging.

UV can induce cells to produce ROS;ROS can directly damage cells.Increased ROS promotes the expression of MMPs and educes collagen secretion and synthesis by activating mitogen-activated protein kinase cell-mediated pathways including ERK,JNK,and p38 kinases[7,37].The four main types of MMPs include MMP-1,72,000 gelatinase(MMP-2),stromelysin-I(MMP-3),and 92,000 gelatinase (MMP-4).They cooperate to degrade extracellular matrix of dermal dermis with or without collagen components.The extracellular matrix of skin mainly contains type I collagen,which is mainly degraded by MMP-1.During the process of photoaging,the expression of MMP-1 is elevated,a large amount of collagen is degraded and disintegrated,and fibroblast proliferation and collagen synthesis are also inhibited[10,38].Studies have shown that inhibiting production of UV-induced ROS,and reducing the expression of MMP-1,MMP-2,and MMP-9,can protect the skin[14,16].The results showed that UVB irradiation reduced the expression of CICP in fibroblasts and increased the expression of MMP-1.However,the expression of MMP-1 in the DOP groups was inhibited and the expression of CICP was increased.Compared with the photoaging model group,the levels of ROS in the DOP groups were decreased,and ROS levels gradually decreased with increasing concentrations of DOP.DOP can reduce ROS levels in photoaging cells,inhibit the expression of MMP-1,and increase the expression of CICP,thereby protecting the photoaging cells.

TGF-β is a fibroblast chemokine that promotes fibroblast proliferation and produces type I and type III collagen[39-41].TGF-β1 is a major component of the TGF-β family.TGF-β combines with cell surface receptors to form heterologous trimers,which activate R-Smad protein and transmit signals to the cytoplasm.UV light can inhibit the synthesis of dermal collagen by affecting the transduction of TGF-β/Smad signaling pathway[42,43].TGF-β1 can induce the formation of extracellular matrix.Gambichleretal.found that the level of TGF-β1 protein in fibroblasts significantly decreased after exposure to UVA for 24 h[45,46];Sunet al.found that increasing the expression and secretion of type I procollagen by the TGF-β/Smad signaling pathway could prevent UV-induced acute skin damage and photoaging[47,48].In addition,Yong-Jinget al.found that TGF-β1 could reduce the expression of MMP-1 and MMP-3 mRNA and decrease type I procollagen degradation in skin fibroblasts during photoaging[13,44].We found that DOP could also promote the expression of TGF-β1.We speculate that DOP can reduce ROS and inhibit MMP-1 expression,thereby reducing type I collagen degradation.In addition,it can promote type I collagen production by up-regulating the expression of TGF-β1.In addition,the up-regulated expression of TGF-β1 by DOP may also reduce type I collagen degradation by inhibiting the expression ofMMP-1.

Conclusion

DOP may protect photoaged fibroblasts by scavenging UVB-induced ROS,inhibiting the secretion of MMP-1,increasing the expression of TGF-β1,and regulating the balance of collagen in the skin.

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