Effects of curcumin nanoparticles on proliferation and VEGF expression of human retinal pigment epithelial cells

INTRODUCTION

Retinal hypoxia in fundus neovascular disease can lead to the release of various cytokines, and vascular endothelial growth factor (VEGF) is considered to be the most closely related factor to neovascular disease. Hypoxia can induce VEGF expression in retinal pigment epithelial(RPE) cells both

and

, and VEGF can promote intraocular angiogenesis

. It is important to find a drug that can effectively inhibit the proliferation of RPE cells and reduce the level of VEGF. Curcumin (Cur) has a variety of curative effects

. Studies on the application of Cur in ophthalmology have shown that Cur can inhibit the proliferation of human RPE (hRPE) cells and the expression of VEGF in the retina of diabetic rats

, so it can play its pharmacological role in the prevention and treatment of fundus neovascular lesions.However, Cur is difficult to dissolve in water, is rapidly metabolized in the body, has a short half-life in the body,and requires long-term and repeated use. This brings a lot of inconvenience and pain to patients, so it is of great clinical significance to develop a new dosage form of Cur. Nano Chinese medicine refers to the active components, active parts,active drugs and compound preparations of Chinese medicine with particle size less than 100 nm that are manufactured by nanotechnology

. Compared with traditional Chinese medicine, it has its advantages

: It increases the solubility of the drug; More likely to target the retina through the bloodretina barrier; With sustained release; Reduce side effects and so on. In this topic, Cur is mainly encapsulated into chitosan nanoparticles grafted by deoxycholic acid (Chit-DC) through nanotechnology to prepare drug-carrying nanoparticles. It is hoped that the nanoparticles have the function of slow release of drugs, improve the efficacy of drugs, maintain the drug concentration for a long time, and reduce the pain caused by repeated administration of drugs to patients. At the same time,the effects of Cur/Chit-DC and Cur on the proliferation and VEGF mRNA expression levels of hRPE cells cultured

were observed and compared, providing a preliminary theoretical basis for finding a new, sustained-release, safe and effective drug for the prevention and treatment of fundus neovascular diseases.

MATERIALS AND METHODS

Materials and Main Instruments and Equipment

Cur (purchased from SIGMA Company), fetal bovine serum (FBS; Biological Industries Company), Vimentin monoclonal antibody (Boshide Company), cell counting kit-8 (CCK-8; Dojindo Company), Trizol (TaKaRa Company),reverse transcription-polymerase chain reaction (RT-PCR) kit(TaKaRa Company) and hRPE cells (the 3

to 6

generations of cells were selected for the experiment provided by the Affiliated Eye Hospital of Sun Yat-sen University).

李國瑞(通信作者) 男,1980年7月出生,山西夏縣人,博士,副教授,碩士生導師,畢業于北京工業大學,美國弗吉尼亞理工學院暨州立大學訪問學者,主要研究方向為物聯網、優化算法.

In the treatment of hRPE cells, both Cur/Chit-DC nanoparticles and Cur had inhibitory effects on hRPE cells(

＜0.01), and the inhibition rate increased with the increase of drug concentration and the prolongation of action time.At the initial stage (1-4d), the inhibition rate of the Cur/Chit-DC nanoparticles group on hRPE cells was lower than that of the corresponding concentration of Cur group, and the difference was statistically significant (

＜0.05). With the further prolonging of the treatment time (5-6d), the inhibition rate of hRPE cells in Cur/Chit-DC group was similar to that in Cur group, and there was no significant difference in statistical analysis (

＞0.05).

Surgical microscope(Zeiss, Germany), optical inverted microscope (Leica MPS-30, Germany), fluorescence microscope Axioplan2 Imaging(Zeiss, Germany), flow cytometry (BD, USA), Wellscan MK3 Microplate meter (USA) Labsystem Dragon, Gene Amp2700 PCR instrument (US Syn Gene), Constant voltage constant current Electrophoresis instrument (US Bio-RAD), Gene GEN IUS gel Imager (US Syn Gene).

Experimental Method

CCK-8 method was used to detect the effects of Cur and Cur/Chit-DC nanoparticles at different concentrations and time points on the proliferation of hRPE cells (Table 2).

The drug reached equilibrium after 96h of release from the nanoparticles, and the cumulative drug release amount was 31.6%

.

張紹凡依舊跟我同班，她依然是眾星捧月的焦點，成績依舊很好，全班第一，年級第三。我的成績進步很大，雖然暫時不足以撼動張紹凡的“班一寶座”，但于我自己卻是全新的開始。

A: Total amount of Cur added (mg); B: Amount of unloaded Cur drug (mg); obtained after centrifugation, precipitation, and drying; C: Total input of chitosan derivatives (mg).

Cytological experiment

hRPE cells in the logarithmic growth phase were prepared in 2500/mL cell suspensions in a Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM-F12) with 10% FBS.Then, 100 μL cell suspensions were added to each well and inoculated into four culture plates for 24h, each plate with 96 holes. After the cells grew a monolayer, 100 μL of Chit-DC nanoparticles with final concentrations of 5, 10, 20,and 40 μg/mL were added to the cells. At the same time, the negative control (without drug addition) and blank control(with only the culture medium) were set up, with four multiple wells set up in each group. Then, the 96-well plates were cultured for 24 and 48h, and 100 μL 10% FBS, DMEM-F12 medium, and 10 μL CCK-8 were added to each well for 3h. The optical density (OD) value of each well at 450 nm was detected using a microplate tester. The experiment was repeated three times for each group. At the same time, the morphological changes in the cells were observed under a light microscope.

Effects of Cur/Chit-DC nanoparticles and Cur on the proliferation of hRPE cells

hRPE cells in logarithmic growth phase were prepared into 2500/mL cell suspensions using 10% FBS DMEM-F12. The 100 μL cell suspensions were added to each well and inoculated into 4 culture plates for 24h, and each plate had 96 holes. After the cells grew to monolayer, 100 μL of Cur and Cur/Chit-DC nanoparticles with final concentration of 5, 10, 20, 40 μg/mL were added into the cells respectively (the corresponding dose of Cur in Cur/Chit-DC nanoparticles was calculated based on drug loading rate of 27.5%). At the same time, a blank control with only culture medium was set, and each group had 4 repeating wells. Then the 96-well plates were cultured for 1,2, 3, 4, 5, and 6d, and then add 100 μL DMEM-F12 medium containing 10% FBS and 10 μL CCK-8 to each well for 3h.OD value of each well was detected by microplate tester(450 nm). The experiment was repeated three times for each group.

The inhibition rate of cell proliferation was calculated according to CCK-8 OD value, and the time and dose effect curves were plotted. Proliferation inhibition rate (%)=(control group OD valu-experimental group OD value)/control group OD value×100%.

Flow cytometry results(Table 3) showed that in the range of 5-40 μg/mL, the percentage of cells in G0-G1 phase increased in both Cur and Cur/Chit-DC nanoparticles, while the percentage of cells in S phase decreased. It indicated that the proliferation period of hRPE cells was reduced.

在本研究中，利用Illumina Hiseq 4000平臺獲得了青錢柳葉的高質量轉錄組數據，共生成50，126個裝配的unigenes，并對公共蛋白數據庫進行注釋，然后進行GO、COG、KEGG分類，檢測到21 089例假定的簡單序列重復(SSRs).這些轉錄組數據提供了一個有價值的公共基因組資源，對于理解青錢柳葉次生代謝機制，促進發現與次級代謝途徑及其調控有關的基因，以及未來青錢柳的基因表達譜和功能基因組研究都著十分重要的作用.

Effects of Cur, Chit-DC nanoparticles and Cur/Chit-DC nanoparticles on VEGF mRNA in hRPE cells

Gel image scanning analysis software was used to analyze the electrophoresis results and determine the OD value of the electrophoresis bands. The relative VEGF content is expressed as the ratio of VEGF to the PCR product content of β-actin from the same specimen (OD value of VEGF/OD value of β-actin).

Primer sequence VEGF RT sense: 5’GACA AGAAAATCCCTGTGGGC3’, RT anti-sense: 5’AACGCG AGTCTGTGTTTGC3’, theoretical length of amplified fragment was 102 bp; Internal reference β-actin RT sense:5’CTCAAGTTGGGGGACAAAAA3’, RT anti-sense:5’GATGAGATTGGCATGGCTTT3’, theoretical length of amplified fragment was 428 bp.

女警官一震，動情了。動了真情的女警官，像個美麗的母親。她摘下警帽，放在并攏的膝頭上，說：“昨天，我在網上，看見南方一家煤礦發生透水事故，一百六十九人遇難。一位老礦工，將年輕礦工扛在自己的肩膀上，拼命往高舉；年輕礦工在驚慌中，一只手抓住棚梁，另一只手伸下去，要拽起被他騎在身下的老礦工。水太大，巷道灌滿了。救援隊將水抽于，下去后，發現僵死的礦工們像雕塑。我一晚上沒有睡好覺。”

The total RNA of each treatment group was extracted by Trizol one-step method. Four specimens were taken from each group. According to the instructions of the RTPCR kit, the target gene was amplified by polymerase chain reaction using cDNA obtained after reverse transcription as the template. PCR reaction conditions: 94℃ pre-denaturation for 3min; denaturation at 94℃ for 30s; annealing at 55℃ for 30s;elongation at 72℃ for 1min; a total of 33 cycles were carried out, and the last cycle was extended at 72℃ for 8min.

The experiment was divided into four groups: 1) Control group: DMEM-F12 culture medium; 2) Cur group: the concentration of Cur is 10 μg/mL;3) Cur/Chit-DC nanoparticle group: the concentration of Cur in Cur/Chit-DC nanoparticles is 10 μg/mL; 4) Chit-DC nanoparticle group: the final concentration of Chit-DC was the same as that of group 3.

SPSS18.0 software package was used for statistical processing. One-way analysis of variance of multiple groups of quantitative data was used for statistical analysis.

＜0.05 was considered statistically significant.

After centrifugation, the lower layer of the liquid precipitated into the unloaded drugs, which were dissolved in 20% ethanol phosphate buffer (pH=6.2), and their content was determined

ultraviolet (UV) spectrophotometry (λ=433 nm). The formula for calculating the drug loading capacity and loading efficiency of the drug-loaded nanoparticles was as follows:

RESULTS

The drug loading capacity and loading efficiency of the synthesized Cur/Chit-DC nanoparticles were determined

UV spectrophotometry, as follows: drug load (Cur)=27.5%,load rate (Cur)=55%.

一個月零三天后，也就是6月10日，《沮水巫音》被列入國家級非遺保護名錄，這對保康來說，是一件大好事，極大地提升了保康的文化內涵，打響了文化品牌。……