Saussurea tridactyla Sch. Bip.-derived polysaccharides and flavones reduce oxidative damage in ultraviolet B-irradiated HaCaT cells via a p38MAPK-independent mechanism

Introduction

The solar ultraviolet (UV) spectrum can be divided into three segments: ultraviolet C, 200–290 nm; ultraviolet B (UVB), 290–320 nm, and ultraviolet A, 320–400 nm.¹ UVB is more genotoxic and approximately 1,000 times more capable of causing sunburn than ultraviolet A,^2,3 which causes a number of detrimental effects to the skin, such as inflammation, immunosuppression, premature skin aging (photoaging), and skin cancer.^4,5 UVB primarily causes direct DNA damage via the generation of reactive oxygen species (ROS)^4–9 such as hydrogen peroxide and the hydroxyl radical, which have harmful effects on the skin and may lead to oxidative DNA damage, lipid peroxidation, and protein modification.^10,11 Other studies have demonstrated that accumulation of ROS within UVB-treated keratinocytes may promote apoptosis.¹² An enzymatic antioxidant defense system, composed of catalase (CAT) and superoxide dismutase (SOD),¹³ plays an important role in the repair of ROS-induced DNA damage.

UVB-induced apoptosis is mediated by apoptotic signals activating two main pathways: intrinsic (mitochondrial) and extrinsic (death receptor-dependent).¹⁴ ROS alters the mitochondrial permeability transition.¹⁵ The ratio between antiapoptotic Bcl-2 and pro-apoptotic Bax has been suggested as a marker of determining the susceptibility to apoptosis, through the maintenance of mitochondrial integrity and inhibition of the activation of the caspase cascade.¹⁶ UVB can increase the population of apoptotic cells, decrease mitochondrial membrane potential (Δψ), and increase intracellular Ca²⁺.¹⁷ It has been shown JNK, ERK1/2, and p38 MAPK levels increase in keratinocytes after 30 minutes of UVB irradiation.¹⁸

“Snow lotus” is a well-known herbal medicine in People’s Republic of China, widely prescribed for the treatment of rheumatoid arthritis, stomachache, and dysmenorrhea.¹⁹ There are many types of snow lotuses available in People’s Republic of China, of which 12 species and one variety are often used as crude drugs. The chemical compositions of Saussurea involucrata Kar. et Kir., Saussurea Hand. Mazz., Saussurea medusa Maxim., and Saussurea tridactyla Sch. Bip. are being actively investigated.²⁰ S. tridactyla Sch. Bip., which grows in the Qinghai-Tibetan plateau,²¹ is the only Asteraceae species with normal growth and reproduction at altitudes of 5,000 m or higher. Additionally, this plant is a source of several compounds that play an important role in the detoxification and promotion of blood circulation,²² which include polysaccharides, flavonoids, alkaloids, phenols, tannins, and essential oils.²³ S. tridactyla Sch. Bip.-derived polysaccharides contain seven kinds of monosaccharide: xylose, arabinose, rhamnose, galactose, glucose, galactose uronic acid, and mannose, which show obvious scavenging effects on anion free radicals and hydroxyl-free radicals.^24–26 Studies have also shown that the S. tridactyla Sch. Bip. water extract exhibits antioxidant effects, which may increase SOD activity and glutathione (GSH) content, and reduce malondialdehyde (MDA) content in mouse tissue.^27–29

The present study is the first to investigate whether S. tridactyla Sch. Bip.-derived polysaccharides and flavones reduce cell apoptosis in human keratinocyte (HaCaT) cells via the p38MAPK pathway, with protective effects against UVB irradiation-induced damage.

Materials and methods

S. tridactyla Sch. Bip.-derived polysaccharides and flavones

According to a previously used protocol,^30,31 95% ethanol was added to 50 g of S. tridactyla Sch. Bip. (solid to liquid ratio of 1:12). The Soxhlet extraction method with a filter and volatile organic solvent was used. The filter residue was added to distilled water (solid to liquid ratio of 1:12) in a water bath at 85°C, with heating for 2 hours and stirring every 15 minutes. After removing the supernatant from the precipitate with 95% ethanol in a 1:3 ratio, static set overnight to recycling supernatant ethanol, the precipitate was washed with anhydrous ethanol and dried to obtain S. tridactyla Sch. Bip.-derived polysaccharides. Low and high concentration (10 and 40 mg/mL, respectively) solutions of S. tridactyla Sch. Bip.-derived polysaccharides were prepared by dissolving the extract in 10% fetal bovine serum Dulbecco’s Modified Eagle’s Medium (DMEM) culture medium, and filtering using a 0.22 μm pore size filter.

According to a previously used protocol,³² 70% ethanol was added to 100 g of S. tridactyla Sch. Bip. (liquid to solid ratio of 13:1 [mL/g]). The solution was twice sonicated at 320 W, 60°C with extraction for 50 minutes, and the extract was concentrated by freeze-drying to obtain S. tridactyla Sch. Bip.-derived rough flavones. Low and high concentration (2 and 6 mg/mL, respectively) solutions of S. tridactyla Sch. Bip.-derived flavones were prepared by dissolving the extract in 10% fetal bovine serum DMEM culture medium and filtering using a 0.22 μm pore size filter.

Cell culture

Cell culture was performed according to previous literature.^9,33 HaCaT cells were obtained from the Nanjing KeyGen Biotech Development Co. Ltd. Cells were maintained at 37°C in an incubator (Shanghai Science Instrument Co. Ltd.) with a humidified atmosphere of 5% CO₂. The cells were grown in DMEM containing 10% fetal calf serum (Biological Industries) with streptomycin (10 units/mL) and penicillin (10 units/mL). The study protocols were approved by the Affiliated Hospital of Qinghai University Institutional Ethical Committee, Xining, People’s Republic of China.

UVB irradiation

UVB irradiation was performed as previously described.³ Cells were washed with phosphate-buffered saline (PBS) and irradiated with UVB light (30 and 60 mJ/cm²). In parallel, nonirradiated cells were treated similarly and kept in the dark. For irradiation, TL20W/12 ultraviolet UVB light tube (Shanghai JIETU Trading Company, Shanghai, People’s Republic of China) was used. The UVB output was measured by an UVB irradiance monitor (UV-340A; Beijing Precise Instruments Co. Ltd., Beijing, People’s Republic of China).

Experimental groups and treatment

HaCaT cells were cultured in vitro and divided into a low radiation UVB (L-UVB) group (30 mJ/cm² radiation for 1 hour) and a high radiation UVB (H-UVB) group (60 mJ/cm² radiation for 1 hour).

S. tridactyla Sch. Bip.-derived polysaccharides L-UVB and H-UVB groups were subdivided into a control group, UVB radiation group (UVB), S. tridactyla Sch. Bip.-derived polysaccharides low-dose group (10 mg/mL), and S. tridactyla Sch. Bip.-derived polysaccharides high-dose group (40 mg/mL).

S. tridactyla Sch. Bip.-derived flavones L-UVB and H-UVB groups were subdivided into a control group, UVB group, S. tridactyla Sch. Bip.-derived flavones low-dose group (2 mg/mL), and S. tridactyla Sch-Bip.-derived flavones high-dose group (6 mg/mL).

Different concentrations of S. tridactyla Sch-Bip.-derived polysaccharides and flavones were added to the cells for 1 hour before exposure to UVB light at doses of 30 and 60 mJ/cm². After 1 hour of irradiation, cells were incubated at 37°C for 18 hours in 10% fetal bovine serum DMEM culture medium.

First, we investigated whether treatment with S. tridactyla Sch. Bip.-derived polysaccharides and flavones reduced UVB-induced oxidative damage in HaCaT cells. Next, we investigated the protective effect of S. tridactyla Sch-Bip.-derived polysaccharides and flavones on HaCaT cells exposed to UVB irradiation and studied the mechanisms involved.

Cell viability assay

The effect of treatment with S. tridactyla Sch. Bip.-derived polysaccharides and flavones on the viability of HaCaT cells was assessed as previously described.³⁴ Cells were seeded in a 96-well plate at a density of 1×10⁶ cells/well. MTT stock solution (Promega, Madison, WI, USA) was added to each well to yield a total reaction volume of 20 μL. After the cells were incubated for 3 hours, the absorbance at 490 nm was read on a full wavelength microplate reader (X-Mark; Bio-Rad Laboratories Inc., Hercules, CA, USA).

Trypan blue staining

Cells were seeded in a 96-well plate at a density of 1×10⁶ cells/well; each well was washed three times with 0.01 mol/L PBS, and 0.4% trypan blue was added to each well in each group to a total volume of 50 μL for 3 minutes. The cells were then visualized with fluorescence microscopy (IX71; Olympus Corporation, Tokyo, Japan), and the photomicrographs were saved and merged with Image Pro Plus 6.0 software (Olympus America Inc., Melville, NY, USA).

Determination of SOD, GSH, CAT, and MDA levels

After 18 hours of incubation in DMEM medium containing 10% fetal bovine serum, the SOD activity, GSH content, MDA content, and CAT activity of HaCaT cells were evaluated using SOD kit, GSH kit, MDA kit, and CAT kit (Jiancheng Biotechnology, Nanjing, People’s Republic of China), respectively, according to the manufacturer’s instructions.

Apoptosis assays and determination of intracellular Ca²⁺ levels and mitochondrial membrane potential (Δψ) by flow cytometry

The apoptosis assays were performed and intracellular Ca²⁺ levels and mitochondrial membrane potential (Δψ) of HaCaT cells were determined by flow cytometry, as described previously.^17,35,36 First, apoptosis was detected by Dead Cell Apoptosis Kit with Annexin V Alexa Fluor^® 488 & PI for Flow Cytometry (replacement for PHN1008, PHN1010; Thermo Fisher Scientific, Waltham, MA, USA), cells in 6-well plates were digested and washed with PBS, and 10⁶ cells/pipe were centrifuged and the supernatant discarded. A 100 μL volume of 1× annexin-binding buffer was added; cells were pulsed with 5 μL annexin V and 1 μL PI working fluid and incubated for 15 minutes. Finally, 400 μl of 1×annexin-binding×Buffer was added and detected using flow cytomety. Next, HaCaT cells were digested in a 6-well plate and washed with Hanks Balanced Salt Solution (HBSS), 10⁷ cells/pipe were added to 4 μL fluo-am4 (Thermo Fisher Scientific, Waltham, MA, USA), incubated at 37°C for 30 minutes, and washed twice with HBSS. Cells were suspended and detected by flow cytometry. Finally, cells were digested and washed with HBSS 6-well plate, 10⁷ cells/pipe were added to 0.5 mL of culture medium and 0.5 mL of JC-1 (Life Technologies) working fluid, incubated for 20 minutes, and centrifuged at 600× g at 4°C for 3 minutes by low temperature centrifuge (Sigma, St Louis, MO, USA). The supernatant was discarded and cells were washed twice with HBSS. Cells were then suspended and detected by flow cytometry.

Western blotting

Western blotting was performed as described previously.³⁷ Each group of HaCaT cells was extracted in radio immunoprecipitation assay (RIPA) lysis buffer (Beijing apply Gene Technology Co., Ltd., Beijing, People’s Republic of China), and centrifuged at 12,000× g at 4°C for 10 minutes by using a low temperature centrifuge (Sigma). The protein concentration of the supernatant was determined using a full wavelength microplate reader (X-Mark, Bio-Rad Laboratories Inc.).

The lysates (40 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 3% bovine serum albumin, incubated with the primary antibody overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. The primary antibodies and horseradish peroxidase-conjugated secondary antibodies are shown in Table 1. The bands were detected using a Chemiluminescent Kit (Beijing apply Gene Technology Co., Ltd.), followed by exposure to X-ray film and densitometry detection using Image J 5.0 software (National Institutes of Health, Bethesda, MD, USA). β-actin was used as a loading control.

Table 1 Antibodies and conditions used in Western blotting

Immunofluorescence staining

Each group of HaCaT cells was washed with 0.01 mol/L PBS followed by fixing by 4% paraformaldehyde (pH 7.4) in a 24-well plate. Cells were incubated with 0.3% Triton X-100 for 30 minutes at 37°C, then washed with 0.01 mol/L PBS three times for 5 minutes, and treated with 3% bovine serum albumin at 37°C for 1 hour at room temperature. Cells were incubated with the following primary antibodies: rabbit polyclonal antibody p-38 (1:80, Abcam, Cambridge, UK), rabbit polyclonal antibody p-53 (1:50, Abcam), rabbit polyclonal antibody Bcl-2 (1:100, Abcam), rabbit polyclonal antibody Bax (1:50, Abcam), and rabbit polyclonal antibody active-caspase-3 (1:100, Abcam) overnight at 4°C. The cells were then incubated with corresponding fluorescence dye-conjugated secondary antibodies (Zhong Shan Jinqiao Biological Technology Co., Ltd., Beijing, People’s Republic of China) for 1 hour at room temperature, protected from light. To provide a negative control, control serum was used instead of the primary antibody. The cells were visualized by fluorescence microscopy (BX5; Olympus Corporation); the photomicrographs were saved and merged using Image Pro Plus 6.0 software (Olympus America Inc.).

Statistical analysis

All data were expressed as mean ± standard deviation and analyzed by one-way analysis of variance with Tukey–Kramer post hoc tests. A P-value of less than 0.05 was considered statistically significant.

Results

Antioxidative protective effects of S. tridactyla Sch. Bip. polysaccharides and S. tridactyla Sch. Bip. flavones on HaCaT cells after UVB

Cell viability assay

The effects of S. tridactyla Sch. Bip.-derived polysaccharides and flavones on cell viability, as indicated by the results of the cell viability assays, are shown in Figures 1 and 2. In the L-UVB and H-UVB groups, the absorbance was significantly lower in the UVB groups than in the control group (P<0.05), and the absorbance was significantly higher in the polysaccharides 10 mg/mL, polysaccharides 40 mg/mL, flavones 2 mg/mL, and flavones 6 mg/mL groups than in the UVB group (P<0.05). In L-UVB and H-UVB groups, the absorbance significantly increased in polysaccharides 40 mg/mL compared with polysaccharides 10 mg/mL group (P<0.05) and the absorbance significantly increased in the flavones 6 mg/mL compared with the flavones 2 mg/mL group (P<0.05).

Figure 1 Cell viability assay of Saussurea tridactyla Sch. Bip.-derived polysaccharides. Notes: (A) Cell viability assay of Saussurea tridactyla Sch. Bip.-derived polysaccharides in L-UVB group. (B) Cell viability assay of Saussurea tridactyla Sch. Bip.-derived polysaccharides in H-UVB group. #P<0.05, UVB group vs control group. *P<0.05, 10 mg/mL group vs UVB group, 40 mg/mL group vs UVB group. **P<0.05, 40 mg/mL group vs 10 mg/mL group. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B; UVB group, UVB radiation group.

Figure 2 Cell viability assay with Saussurea tridactyla Sch. Bip.-derived flavones in (A) L-UVB group and (B) H-UVB group. Notes: #P<0.05, UVB group vs control group. *P<0.05, 2 mg/mL group vs UVB group, 6 mg/mL group vs UVB group. **P<0.05, 6 mg/mL group vs 2 mg/mL group. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B; UVB group, UVB radiation group.

Trypan blue staining

The results of trypan blue staining for S. tridactyla Sch. Bip.-derived polysaccharides and flavones are shown in Figures 3 and 4. In the L-UVB group, the number of blue cells was increased as compared to the control group. The cells showed mild swelling and floating, indicating that some of these cells had died in the UVB group; however, cell morphology could still be discerned. In the polysaccharides 10 mg/mL, polysaccharides 40 mg/mL, flavones 2 mg/mL, and flavones 6 mg/mL groups, the number of blue cells was low compared with the UVB group, and the degree of cell edema was alleviated. In the polysaccharides 40 mg/mL group, cell morphology resembled that of the control group, and the number of blue cells was lower than in the polysaccharides 10 mg/mL group. In the flavones 6 mg/mL group, cell morphology resembled the control group, and the number of blue cells was lower than in the flavones 2 mg/mL group. In the H-UVB group compared with control group, cells showed obvious swelling, cell death fragments and chip phenomenon were clearly observed, and cell morphology was blurred. In the polysaccharides 10 mg/mL, polysaccharides 40 mg/mL, flavones 2 mg/mL, and flavones 6 mg/mL groups, the morphology of cells was restored, the number of death cells had reduced, and cell death fragments and chip phenomenon was obviously improved. In the polysaccharides 40 mg/mL group, cell morphology resembled that in the control group, and the number of blue cells was significantly lower than in the polysaccharides 10 mg/mL group; and in the flavones 6 mg/mL group, cell morphology resembled the control group, and the number of blue cells was significantly lower than in the flavones 2 mg/mL group.

Figure 3 Trypan blue staining of Saussurea tridactyla Sch. Bip.-derived polysaccharides. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B.

Figure 4 Trypan blue staining of Saussurea tridactyla Sch. Bip.-derived flavones. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B.

Determination of SOD, GSH, CAT, and MDA levels

The effects of S. tridactyla Sch. Bip.-derived polysaccharides and flavones on SOD activity, GSH content, CAT activity, and MDA content are shown in Figures 5 and 6. In L-UVB and H-UVB groups, activity of SOD, content of GSH, and activity of CAT were significantly decreased, and content of MDA was significantly increased in the UVB compared with the control group (P<0.05); activity of SOD, content of GSH, activity of CAT were significantly increased, and content of MDA was significantly decreased in the polysaccharides 10 mg/mL, polysaccharides 40 mg/mL, flavones 2 mg/mL, and flavones 6 mg/mL groups compared with the UVB group (P<0.05), and SOD activity, GSH content, and activity of CAT were significantly increased, and content of MDA was significantly decreased in the polysaccharides 40 mg/mL compared with polysaccharides 10 mg/mL group (P<0.05). In the L-UVB group, activity of SOD, content of MDA, and activity of CAT were significantly decreased, while the content of GSH was significantly increased in the flavones 6 mg/mL compared with flavones 2 mg/mL group (P<0.05). In the H-UVB group, activity of SOD, content of GSH, content of MDA, and activity of CAT were significantly decreased in flavones 6 mg/mL compared with flavones 2 mg/mL group (P<0.05).

Figure 5 SOD activity (A), GSH content (B), MDA content (C), and CAT activity (D) of Saussurea tridactyla Sch. Bip.-derived polysaccharides. Notes: (A1, B1, C1, D1) show the results of SOD activity, GSH content, MDA content and CAT activity in Saussurea tridactyla Sch. Bip.-derived polysaccharides in L-UVB group. (A2, B2, C2, D2) show the results of SOD activity, GSH content, MDA content and CAT activity in Saussurea tridactyla Sch. Bip.-derived polysaccharides in H-UVB group. #P<0.05, UVB group vs control group. *P<0.05, 10 mg/mL group vs UVB group, 40 mg/mL group vs UVB group. **P<0.05, 40 mg/mL group vs 10 mg/mL group. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB group, UVB radiation group; UVB, ultraviolet B; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde; CAT, catalase.

Figure 6 SOD activity (A), GSH content (B), MDA content (C), and CAT activity (D) of Saussurea tridactyla Sch. Bip.-derived flavones. Notes: (A1, B1, C1, D1) show the results of SOD activity, GSH content, MDA content and CAT activity in Saussurea tridactyla Sch. Bip.-derived flavones in L-UVB group. (A2, B2, C2, D2) show the results of SOD activity, GSH content, MDA content and CAT activity in Saussurea tridactyla Sch. Bip.-derived flavones in H-UVB group. #P<0.05, UVB group vs control group. *P<0.05, 2 mg/mL group vs UVB group, 6 mg/mL group vs UVB group. **P<0.05, 6 mg/mL group vs 2 mg/mL group. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B; UVB group, UVB radiation group; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde; CAT, catalase.

Protective mechanisms of S. tridactyla Sch.-Bip.-derived polysaccharides and flavones in UVB-irradiated HaCaT cells

Apoptosis assays and determination of intracellular Ca²⁺ levels and mitochondrial membrane potential (Δψ) by flow cytometry

S. tridactyla Sch. Bip.-derived flavones: through these three experiments, we found that in the H-UVB group, HaCaT cells were obviously swollen, cell death fragments and chip phenomenon were obvious, and cell morphology was blurred in the UVB group as indicated by trypan blue staining; therefore, we chose the L-UVB group for the following experiments. In the L-UVB group, the absorbance significantly increased in the 6 mg/mL compared with 2 mg/mL group, as detected by MTT (P<0.05). Also, the activity of SOD, content of MDA, and activity of CAT were significantly decreased, while the content of GSH was significantly increased in the 6 mg/mL compared with 2 mg/mL group (P<0.05); therefore we chose the S. tridactyla Sch. Bip.-derived flavones 6 mg/mL group for the following experiments.

The levels of apoptosis, intracellular Ca²⁺ levels, and mitochondrial membrane potential (Δψ) for S. tridactyla Sch. Bip.-derived polysaccharides and flavones, as measured by flow cytometry, are shown in Figures 7 and 8.

Figure 7 Apoptosis assays (A), intracellular Ca2+ (B), and mitochondrial membrane potential (Δψ) (C) indicated by flow cytometry of Saussurea tridactyla Sch. Bip.-derived polysaccharides. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B.

Figure 8 Apoptosis assays (A), intracellular Ca2+ (B), and mitochondrial membrane potential (Δψ) (C) indicated by flow cytometry of Saussurea tridactyla Sch. Bip.-derived flavones. Abbreviation: UVB, ultraviolet B.

The annexin V/propidium iodide staining (Figures 7A and 8A) revealed that UVB irradiation of HaCaT cells leads to an increase in the population of apoptotic cells as compared to the control group, while in the L-UVB and H-UVB groups, the population of apoptotic cells decreased in the polysaccharides 10 mg/mL and polysaccharides 40 mg/mL groups compared with UVB group. And in the S. tridactyla Sch. Bip.-derived flavones group, the population of apoptotic cells decreased in the 6 mg/mL group compared with the UVB group.

The intracellular Ca²⁺ measurements (Figures 7B and 8B) revealed that intracellular Ca²⁺ fluorescence increased, indicating that the content of intracellular Ca²⁺ had increased in the UVB group compared with the control group. The level of intracellular Ca²⁺ fluorescence decreased, indicating that the content of intracellular Ca²⁺ had decreased in the polysaccharides 10 mg/mL and polysaccharides 40 mg/mL groups compared with the UVB group in the L-UVB and H-UVB groups. And in the S. tridactyla Sch. Bip.-derived flavones group, intracellular Ca²⁺ fluorescence decreased indicating that the content of intracellular Ca²⁺ was decreased in the 6 mg/mL compared with the UVB group.

The mitochondrial membrane potential (Δψ) (Figures 7C and 8C) revealed that green fluorescence was increased indicating that the mitochondrial membrane potential (Δψ) was decreased in the UVB compared with control group; while in the groups of L-UVB and H-UVB, green fluorescence was decreased, and the mitochondrial membrane potential (Δψ) was increased in the polysaccharides 10 mg/mL and polysaccharides 40 mg/mL groups compared with the UVB group. And in the S. tridactyla Sch. Bip.-derived flavones group, green fluorescence was decreased, and the mitochondrial membrane potential (Δψ) was increased in the 6 mg/mL compared with UVB group.

Western blotting

The results of Western blotting for p-38, p-53, Bcl-2, Bax, and active-caspase-3 in each group, after treatment with S. tridactyla Sch. Bip.-derived polysaccharides and flavones, are shown in Figures 9 and 10. In the L-UVB group, the expressions of p-38, p-53, and Bcl-2 were significantly decreased, and the expressions of Bax and active-caspase-3 were significantly increased in the UVB compared with control group (P<0.05); the expression of Bcl-2 was significantly increased, and the expressions of Bax and active-caspase-3 were significantly decreased in the 10 mg/mL compared with UVB group (P<0.05). However, the expressions of p-38 and p-53 were decreased in the 10 mg/mL compared with UVB group (P>0.05); the expressions of p-38, p-53, and Bcl-2 were significantly increased, and the expressions of Bax and active-caspase-3 were significantly decreased in the 40 mg/mL compared with UVB group; the expressions of p-53 was significantly increased, and the expressions of Bax were significantly decreased in the 40 mg/mL compared with 10 mg/mL group (P<0.05). In the H-UVB group, the expressions of p-38, p-53, and Bcl-2 were significantly decreased, and the expressions of Bax and active-caspase-3 were significantly increased in the UVB compared with control group (P<0.05); the expressions of p-53 and Bcl-2 were significantly increased, and the expressions of p-38, Bax, and active-caspase-3 were significantly decreased in the 10 and 40 mg/mL groups compared with UVB group (P<0.05); the expression of Bcl-2 was significantly increased, and the expressions of p-38, p-53, Bax, and active-caspase-3 were significantly decreased in the 40 mg/mL compared with 10 mg/mL group (P<0.05). And in the S. tridactyla Sch. Bip.-derived flavones group, the expressions of p-38, p-53, and Bcl-2 were significantly decreased, and the expressions of Bax and active-caspase-3 were significantly increased in the UVB compared with control group (P<0.05); the expressions of p-38, p-53, and Bcl-2 were significantly increased, and the expressions of Bax and active-caspase-3 were significantly decreased in the 6 mg/mL compared with UVB group significantly.

Figure 9 Expressions of p-38, p-53, Bcl-2, Bax, and active-caspase-3 indicated by Western blotting of Saussurea tridactyla Sch. Bip.-derived polysaccharides. Notes: #P<0.05, UVB group vs control group. *P<0.05, 10 mg/mL group vs UVB group, 40 mg/mL group vs UVB group. **P<0.05, 40 mg/mL group vs 10 mg/mL group. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B; UVB group, UVB radiation group.

Figure 10 Expressions of p-38, p-53, Bcl-2, Bax, and active-caspase-3 indicated by Western blotting of Saussurea tridactyla Sch. Bip.-derived flavones. Notes: #P<0.05, UVB group vs control group. *P<0.05, 6 mg/mL group vs UVB group. Abbreviations: UVB, ultraviolet B; UVB group, UVB radiation group.

Immunofluorescence staining

The results of immunofluorescence staining for p-38, p-53, Bcl-2, Bax, and active-caspase-3 in each group after treatment with S. tridactyla Sch. Bip.-derived polysaccharides and flavones are shown in Figures 11 and 12. In the L-UVB and H-UVB groups, the expressions of p-38, p-53, and Bcl-2 were lower, and the expression levels of Bax and active-caspase-3 were higher in the UVB than in the control group; the expressions of p-38, p-53, and Bcl-2 were higher, and the expressions of Bax and active-caspase-3 were lower in the 10 mg/mL and 40 mg/mL groups than in the UVB group.

Figure 11 The expressions of p-38, p-53, Bcl-2, Bax, and active-caspase-3 indicated by immunofluorescence staining of Saussurea tridactyla Sch. Bip.-derived polysaccharides. Abbreviations: L-UVB, low radiation UVB; H-UVB, high radiation UVB; UVB, ultraviolet B.

Figure 12 The expressions of p-38, p-53, Bcl-2, Bax, and active-caspase-3 indicated by immunofluorescence staining of Saussurea tridactyla Sch. Bip.-derived flavones. Abbreviation: UVB, ultraviolet B.

And in the S. tridactyla Sch. Bip.-derived flavones group, the expressions of p-38, p-53, and Bcl-2 were lower, and the expressions of Bax and active-caspase-3 were higher in the UVB than in the control group; the expressions of p-38, p-53, and Bcl-2 were increased, and the expressions of Bax and active-caspase-3 were decreased in the 6 mg/mL compared with the UVB group.

Discussion

Several previous studies have demonstrated suppression of UV irradiation- and/or peroxide-induced cell injury and melanogenesis in human skin cells.^38,39 UVB irradiation injures cellular components of skin tissues and cells via the generation of ROS, which stimulates the oxidative stress response of the skin, resulting in the depletion of cellular antioxidants, increased damage to cellular components (eg, lipid membrane, protein, and DNA), photoaging, inflammation, immunosuppression, and apoptosis.^40,41 An enzymatic antioxidant defense system composed of CAT and SOD¹³ and GSH protein repairs the skin by reducing photoaging due to the most common free radical, MDA, which is a frequently used marker of lipid peroxidation. Therefore, we assessed the degree of injury in HaCaT cells after UVB irradiation by using SOD activity, GSH content, CAT activity, and MDA content as indicators of such damage. In this study, we observed that S. tridactyla Sch. Bip.-derived polysaccharides and flavones increased cell viability by MTT, decreased the number of apoptotic cells, alleviated the degree of cell edema, restored the morphology of cells (as indicated by trypan blue staining), and increased SOD activity, GSH content, CAT activity, and decreased MDA content. These results suggest that S. tridactyla Sch. Bip.-derived polysaccharides and flavones exhibit protective effects against oxidative damage in UVB-irradiated HaCaT cells.

Other studies have demonstrated that accumulation of ROS in UVB-treated keratinocytes may promote apoptosis, resulting in mitochondrial permeability transition.^12,15 In our study, we confirmed that S. tridactyla Sch. Bip.-derived polysaccharides and flavones can alleviate apoptosis in HaCaT cells through a mitochondrial pathway that decreases intracellular Ca²⁺ fluorescence and increases the mitochondrial membrane potential (Δψ).

It has been shown that the JNK, ERK1/2, and p38 MAPK pathways are activated in response to UVB irradiation in keratinocytes.¹⁸ The present study focused on the p38MAPK pathway, which inactivates p-53, triggering the apoptotic cascade by the formation of the pro-apoptotic caspase-3 complex. In our study, we confirmed using Western blotting and immunofluorescence staining that polysaccharides and flavones from S. tridactyla Sch. Bip. reduce apoptosis in HaCaT cells by increasing the expression of Bcl-2, and decreasing the expressions of Bax and active-caspase-3. However, the increase in the expressions of p-38 and p-53, as observed by Western blotting and immunofluorescence staining, suggests that this antiapoptotic effect is not achieved through the p38MAPK pathway. To our knowledge, this is the first study demonstrating that while S. tridactyla Sch. Bip.-derived polysaccharides and flavones can reduce cell apoptosis and exert protective effects against UVB irradiation-induced cell damage, these effects do not involve the p38MAPK pathway. These data provide new directions for the application of S. tridactyla Sch. Bip.-derived polysaccharides and flavones in drug development and for future investigation of the mechanisms by which these compounds exert their protective effects.

Acknowledgment

The study is supported by Basic Research Project of Qinghai Province (application) (grant 2013-Z-730).

Disclosure

The authors report no conflicts of interest in this work.

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