Effect of nano-selenium loaded with lycium barbarum polysaccharide on the proliferation of lens epithelial cells after UVB damage in vitro

INTRODUCTION

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MATERIALS AND METHODS

According to the results, after damaged with UV, SRA01/04 cells were incubated with 2.0 mg/mL LBP-SeNPs and photographed 24h later. The 2.0 mg/mL LBP-SeNPs showed a higher cell density at 0.5 µmol/L. As the concentration increased, cytoplasmic dehydration, cell crumbling and apoptotic vesicles appeared more, SRA01/04 cells density decreased (Figure 8).

The SV40 T-antigen-transformed HLECs,SRA01/04, was obtained from the Cancer Institute of the Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin and incubated at 37℃in a 5% COincubator.

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SRA01/04 suspensions were placed in 96-well cell culture plates at a concentration of approximately 5×10cells/mL and grouped according to the UV irradiation time (0, 10, 20, 30, 40, 50,60min), each group was replicated in 6 wells with a volume of 100 µL. After 24h of cell culture, the culture medium was sucked out and 20 μL phosphate buffered saline (PBS) was added to prevent cell apoptosis due to excessive dryness during irradiation. Cells were irradiated in an ultraviolet light box at a wavelength of 302 nm, and one group was uncovered every 10min until the end of 60min. After that, the cells continued to be cultured for 24h. Methylthiazol tetrazolium (MTT) was used to determine the survival rate of the cells.

Oxidative stress induced lens epithelial cell apoptosis plays an important role in cataract formation. It is very important to reduce the apoptosis of lens epithelial cells to control the occurrence and development of cataract. There were some studies on the use of selenium or SeNPs for the treatment of age-related cataracts. Zhuand Lubelieved selenium supplementation could slow the development of naphthalene cataract possibly by attenuating the oxidative stress in the lens. Postfound low serum selenium levels may constitute a potential risk factor of age-related cataract.Based on those, we prepared LBP-SeNPs with suitable particle size and good stability and investigated whether LBP-SeNPs could protect SRA01/04 cells from UV irradiation. Damage model was HLECs (SRA01/04) in logarithmic growth phase with UV irradiation.

According to the above experimental steps, SRA01/04 cells were damaged by ultraviolet radiation for 10min. Different concentrations of LBP-SeNPs were administered immediately after that steps.Cell activity was detected by MTT after 24h incubation. The LBP-SeNPs group with the highest cell proliferation rate was photographed to observe the cell status.

The particle sizes of the prepared LBP-SeNPs were examined using a nanoparticle size analyzer, and the average particle sizes corresponding to the LBP concentrations of 0.5, 1.0, 1.5, and 2.0 mg/mL are shown in Figure 2A, and the particle sizes of both loaded and unloaded LBP-SeNPs were in the range of 150-180 nm, and only the average particle size of 1.5 mg/mL LBP-SeNPs was less than 160 nm. As shown in Figure 2B,the particle size of LBP-SeNPs basically remained between 150-200 nm with time, indicating their stability.

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RESULTS

Data were analyzed using the SPSS 18.0 (IBM, Armonk, NY, USA). Data were presented as the mean±SD. For comparison of the different groups, statistical comparisons were performed by one-way analysis of variance and Student’stest. Differences were considered significant at<0.05.

SRA01/04 cells were observed under inverted microscope as shown in Figure 3. Most of the cells could be walled and the cell density reached more than 80% within 24h to 48h after SRA01/04 cells were passaged.After adhering to the wall, the cells gradually extended from the unadhered round shape to the final cell shape of shuttle shape or polymorphism, with clear nucleus and transparent cytoplasm and good growth status.

Lycium barbarum is a well-known herbal medicine with a variety of biological effects, including antioxidant and cytoprotective effects. Lycium barbarum polysaccharide(LBP) is the main component of lycium barbarum responsible for these biological activities. It is well documented that LBP can prevent the generation and accumulation of reactive oxygen species (ROS) and has protective effects against damage in a variety of cells and tissues through mitochondria-related pathways. Studies had shown that UVB irradiation can lead to cataract formation. Oxidative stress caused by ROS which produced by UVB irradiation to lens epithelial cells (LECs) is thought to be the main factor in the development of cataracts. Previous studies in our group had shown that LBP had a protective effect against oxidative damage in human lens epithelial cells (HLECs) and retinal ganglion cells. Nano-selenium (SeNPs), as a unique nanoscale elemental selenium, have high efficacy on diseased tissues due to their excellent pharmacokinetics and selectivity,low toxicity to normal tissues as well as high penetration to cells. In this experiment, we investigated the effect of nanoselenium loaded with LBP (LBP-SeNPs) on the proliferation of HLECs after UVB irradiation (Figure 1).

According to the literature, a UV lamp with a wavelength of 302 nm was placed on top of the incubator and irradiated SRA01/04 cells for 0,10, 20, 30, 40, 50, and 60min. After that, cells were incubated for 24h. MTT test was implemented for cell viability assay.As shown in Figure 4, cell survival was significantly lower in all experimental groups compared with the control group(=121.239,<0.01). The cell survival rate showed a gradient downward trend between 10min and 50min groups. Cell survival rate decreased as irradiation time increased. There was no statistical significance between 50min group and 60min group (=0.430). According to this result, we chose the cell survival rate around 80% (10min group) as the UV irradiation time for the next step of the experiment.

According to the previous steps,four gradient concentrations (0.5, 1.0, 2.0, 4.0 µmol/L) of four concentrations LBP-SeNPs (0.5, 1.0, 1.5, 2.0 mg/mL) were selected to interact with SRA01/04 damaged by UVB light.Cell proliferation rates were detected by MTT (Table 2; Figure 7).The cell proliferation rate of all the above experimental groups were greater than that of the control group (<0.05), indicating its cytoprotective effect.

SRA01/04 cells were treated with coumarin-loaded LBP-SeNPs for 24h and then stained with DAPⅠ. As shown in Figure 5, nucleus with blue fluorescence in combination with dye can be seen under fluorescence microscope. The LBP‐SeNPs loaded with coumarin fluorescent agent showed green fluorescent spots in the cytoplasm. This indicated that LBP-SeNPs had entered the cells in large quantities during this period.Six gradient concentrations (0.5, 1.0,2.0, 4.0, 8.0, and 16.0 µmol/L) of four concentrations LBPSeNPs (0.5, 1.0, 1.5, 2.0 mg/mL) were selected to interact with SRA01/04. Cell survival rates were detected by MTT(Table 1).The cell survival rates of the above experimental groups were lower than the control group (<0.05) and decreased with increasing drug concentration (<0.05; Figure 6). Based on this, we selected 0.5, 1.0, 2.0, and 4.0 µmol/L as the relatively safe drug concentrations for the next experimental step.

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The1 mL sodium selenite was added to the solution containing different concentrations of LBP (0.5, 1.0, 1.5, 2.0 mg/mL)at room temperature and pressure. After that, 1 mL reduced vitamin C solution and 2 mL water were added. The solution was stirred for 8h and dialysis for 12h, LBP-SeNPs solution was collected. The particle size and stability of LBP-SeNPs were determined by nanoparticle size analyzer. The LBPSeNPs used in this experiment were prepared and tested by the Institute of Chemical Materials of Jinan University.

DISCUSSION

Studies had confirmed that LBP, as a bioactive compound,had good antioxidant propertiesand similar effects on eye diseases. Published studies had associated LBP intake with a number of therapeutic effects, including antiaging,metabolic effects, neuroprotective effects in neurodegeneration and neurotoxicity. Duestablished an age-related macular degeneration model using blue light-induced oxidative stress damage in human retinal pigment epithelial cells, and found LBP could reduce cellular oxidative stress damage by enhancing mitochondrial membrane potential through LBP pretreatment. The latest LBP chemical component analysis demonstrated all glycopeptides in LBP act to eliminate lipid peroxidation. Thus, LBP might decrease cell injury induced by oxygen free radicals and protect cell development and differentiation as an antioxidant.

SRA01/04 cells were cultured for 24h and then administered in groups: 1) Experimental group: SeNPs loaded with different concentrations of LBP(0.5, 1.0, 1.5, 2.0 mg/mL), the stock solution was diluted in a gradient so that the concentrations were 16, 8, 4, 2, 1, and 0.5 µmol/L, respectively, and three replicate wells were set up for each concentration of each group of drugs, with 100 µL of drug added to each well; 2) Control group: An equal amount of complete culture solution was added to each well; 3) Blank group: Instead of planting cytosol, the plates were planted with complete culture solution, with 2 replicate wells, and the remaining steps were the same as the control group. After drug administration, the cells were continued to be incubated for 24h to observe cell morphology and MTT to detect cell activity.

First, we established a safe concentration range of LBPSeNPs., proliferation rate of SRA01/04 cells decreased with higher concentrations of LBP-SeNPs. Although the concentration of selenium nanoparticles which showed drug toxicity was not consistent at different concentrations of LBP‐SeNPs, each group showed different degrees of drug toxic effects at 8.0, 16.0 µmol/L. We selected the concentration regions of 0.5, 1.0, 2.0, 4.0 µmol/L as the relatively safe drug concentration range for the next experimental step.

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Damaged HLECs (SRA01/04) were treated with LBP-SeNPs.It was verified that LBP-SeNPs could entered through the HLECs membrane into the cytoplasm. Cell proliferation rate was higher between the same concentration of LBP-SeNPs loaded with more LBP. Low concentration of LBP-SeNPs had a better protective effect compared with high concentration of LBP-SeNPs. The proliferation rate of SRA01/04 cells treated with 0.5 µmol/L of 2.0 mg/mL LBP-SeNPs was the highest.We found that more cytoplasmic dehydration, cell crumpling,apoptotic vesicles appeared and cell density decreased when LBP-SeNPs concentration increased. This results is different with Qi. who suggested that antioxidant and protection of human crystalline epithelial cells by LBP could increase with increasing concentration up to 400 mg/mL and decrease with increasing concentration beyond 400 mg/mL.Maybe LBP combined with SeNPs changed LBP’s original characteristics. But the ability of LBP-SeNPs to protect against oxidative damage is indisputable.

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Further studies are needed to determine whether reducing the concentration of selenium nanoparticles and increasing the drug loading of LBP in LBP-SeNPs will improve cell survival.Protective effect of LBP‐SeNPs on animal models will also be further carried out.

Supported by the National Natural Science Foundation of China (No.81970806); Medical Scientific Research Foundation of Guangdong Province of China (No.A2019098).

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