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(dǎo)師,畢業(yè)于北京工業(yè)大學(xué),美國弗吉尼亞理工學(xué)院暨州立大學(xué)訪問學(xué)者,主要研究方向?yàn)槲锫?lián)網(wǎng)、優(yōu)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(diǎn)，成績依舊很好，全班第一，年級第三。我的成績進(jìn)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(zhì)量轉(zhuǎn)錄組數(shù)據(jù)，共生成50，126個裝配的unigenes，并對公共蛋白數(shù)據(jù)庫進(jìn)行注釋，然后進(jìn)行GO、COG、KEGG分類，檢測到21 089例假定的簡單序列重復(fù)(SSRs).這些轉(zhuǎn)錄組數(shù)據(jù)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(jī)制，促進(jìn)發(fā)現(xiàn)與次級代謝途徑及其調(diào)控有關(guān)的基因，以及未來青錢柳的基因表達(dá)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(wǎng)上，看見南方一家煤礦發(fā)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(duì)將水抽于，下去后，發(fā)現(xiàn)僵死的礦工們像雕塑。我一晚上沒有睡好覺?！?/p>

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(hù)名錄，這對保康來說，是一件大好事，極大地提升了?？档奈幕瘍?nèi)涵，打響了文化品牌。我第一時間打電話把這個好消息告訴給王述金，電話的那頭傳來的是老王悲喜交加的哭泣聲。

OD values of hRPE cells treated with Chit-DC nanoparticles of different concentrations for 24 and 48h were shown in Table 1. There was no significant difference in OD value between the control group (drug concentration of 0 mg/L) and the experimental group (

=0.381,

=0.281;

＞0.05). No changes in the cell morphology were observed under a light microscope (Figure 1), suggesting that Chit-DC nanoparticles had no toxicity on hRPE cells.

Synthesis technique of Cur/CHit-DC nanoparticles was performed mainly as described by Zheng

.

青海衛(wèi)拉特蒙古又稱“德都蒙古”，是青海世居少數(shù)民族之一，主要是以衛(wèi)拉特蒙古和碩特部為主要族源，并附以自13世紀(jì)以來陸續(xù)移居青海的蒙古各部的統(tǒng)稱。他們歷經(jīng)800余年的歷史變遷形成了今天獨(dú)具特色的風(fēng)俗習(xí)慣和文化特征。飲食文化就是其中之一，青海衛(wèi)拉特蒙古傳統(tǒng)飲食可分為：[tsaa：n id：](即白食)、[ulɑ：n id：](即紅食)和[br id：](灰食)三種，白食可理解為奶食；紅食可理解為肉食；灰食可理解為面食、菜蔬等輔助食品。

對于常規(guī)水電站，根據(jù)電站運(yùn)行水頭、所在地理位置差異等因素，機(jī)組技術(shù)供水方式主要有自流減壓供水、水泵供水、二次循環(huán)供水等。對于循環(huán)供水系統(tǒng)，由于其水源沒有取自機(jī)組流道，故其往往不需要設(shè)置汛期備用水源。而對于自流減壓供水或水泵供水，其水源多來自機(jī)組流道。有部分電站則采用沉淀池供水做為機(jī)組冷卻水的備用水源，當(dāng)汛期來臨，水庫水質(zhì)較差時，機(jī)組技術(shù)供水切換到沉淀池供水。

The 3

to 6

generations of cells at logarithmic growth stage were inoculated in 25 mL flask at a density of 5×10

/mL,and 24h later, 100 μL DMEM-F12 medium containing 10%FBS was for further culture for 24h. The final concentration of 5, 10, 20, 40 μg/mL Cur and Cur/Chit-DC nanoparticles were treated for 24h. The control group only added culture medium.Adherent cells were collected for flow cytometry analysis and cell cycle analysis. The experiment was repeated three times for each group.

雞腺病毒病主要發(fā)生在1～3周齡的雞群中。被感染的雞通常會在3周齡后出現(xiàn)死亡現(xiàn)象。從感染到死亡時間極短，能在短時間內(nèi)達(dá)到死亡高峰，具有較大的威脅。雞腺病毒病具有普遍性、易垂直感染、水平傳播以及人工授精傳播的特征，以下進(jìn)行詳細(xì)介紹。

The mRNA integrity test of extracted VEGF showed three bands of 5S, 18S, and 28S (Figure 2). Semi-quantitative RT-PCR gel diagram (Figure 3) image analysis to obtain the relative OD of VEGF in each group (Table 4, Figure 4) showed that there was no difference in VEGF mRNA expression between Chit-DC nanoparticles and control group after 24h treatment (

＞0.05). The mRNA expression of VEGF in Cur/Chit-DC and Cur group was lower than that in control group(

＜0.05).

DISCUSSION

Chitosan is the only cationic basic aminopolysaccharide discovered. It is a kind of natural polymer and suitable for preparation of nano-controlled release system as carrier material. In nature, chitosan is widely found in lower organisms such as fungi, shells of shrimps, crabs and insects. It is widely used in food, medicine, bioengineering and so on

.In this experiment, the Cur/Chit-DC nanoparticles synthesized used Chit-DC as the drug carrier, and Chit-DC used EDC as the coupling agent to graft deoxycholic acid onto chitosan to synthesize amphiphilic modified chitosan. The polymer can form nanoparticles in aqueous solution. CCK-8 method was used to compare the inhibition rate of proliferation of hRPE cells with different concentrations of Chit-DC nanoparticles group and the control group. The results showed that there was no significant difference between the groups with different concentrations of Chit-DC nanoparticles and the control group,and no change in cell morphology was observed under inverted microscope. These results indicated that Chit-DC nanoparticles as a drug carrier did not affect the growth of hRPE cells,suggested that Chit-DC nanoparticles had no toxicity on hRPE cells and can be used as a drug nanocapsule in this study. Liu

found that there was no significant difference between cytotoxicity and free Cur-coated chitosan/polycaprolactone nanoparticles in human cervical cancer cells, Hela cells, human choroidal melanoma cells, and OCM-1 cells.

In this paper, we synthesized Cur/Chit-DC nanoparticles, and analyzed their properties. It was found that the drug-loading capacity of the Cur/Chit-DC nanoparticles was 27.5%, which was close to the drug-loading capacity (24%) of the Cur albumin nanoparticles prepared by Zhang

by using defusing method. The release of the drugs from nanoparticles revealed two stages

: burst release and sustained release.Equilibrium was reached after 96h of release, and the cumulative release amount of Cur/Chit-DC nanoparticles was 31.6%. In the first 20h, the drug release in the sudden release stage reached 19.9%. The slow release of the Cur nanoparticles was confirmed by diffusion release experiments

. CCK-8 method was used to detect the proliferation effects of different doses of Cur/Chit-DC nanoparticles and Curs on hRPE cells at different time points. It was found that both Cur/Chit-DC nanoparticles and Cur had inhibitory effects on the cells, but there were differences in the inhibition rate of cell proliferation. In the initial period (1-4d), the inhibition rate of hRPE cells in the Cur/Chit-DC group was lower than that in the Cur group. However, with the prolongation of time,the difference in the inhibition rate of hRPE cells between two groups decreased gradually and the last the two groups showed the same inhibition rate of hRPE cells. Cur, as a traditional Chinese medicine with a long history, has a wide range of pharmacological effects, no obvious toxic, wide range of drug sources and low price. At present, the United States Food and Drug Administration (FAD) has regarded it as a new anticancer drug in the 21

century

. In addition, clinical trials have been conducted to gradually study the pharmacokinetics of Cur and the bioeffective dose for cancer treatment

. The research on its application in ophthalmology mainly focuses on its antiangiogenesis effect, which may play an important role in the treatment of pterygium and diabetic retinopathy

. Gong

studied the effect of Cur on the proliferation of human embryonic retinal pigment epithelial cells cultured

,and found that Cur could significantly inhibit the proliferation of human embryonic retinal pigment epithelial cells. The results obtained in this study were consistent with the above findings, indicating that the Cur/Chit-DC nanoparticles could inhibit the proliferation of hRPE cells. The sustained release mechanism of nanomaterials is generally considered as follows: 1) the drug connected to or adsorbed on the particle surface is separated from the nanoparticles; 2) the drugs in the nanodrug micelles are constantly diffused outward; 3) the nanomicelles themselves are constantly degraded, and the drugs encapsulated in the micelles are constantly released

.In the results of this experiment we believed that the Cur/Chit-DC nanoparticles did not enter cells or a small amount entered cells at the initial stage (1-4d). The reason may be that a small amount of drugs adsorbed on the surface of the nanoparticles fall off from the surface, and the drugs encapsulated in the nanoparticles slowly diffuse outward from the inside, or a small amount of drug nanoparticles entering the cells are degraded by intracellular enzymes to release drugs.Therefore, although both Cur/Chit-DC nanoparticles and Cur have inhibitory effects on hRPE cells. However, the inhibition rate of Cur/Chit-DC nanoparticles on hRPE cells was lower than that of Cur. With the prolongation of the action time,more and more Cur/Chit-DC nanoparticles entered the cells,and the nanoparticles were degraded continuously, the drug release and the concentration of effective drugs on the cells gradually increased. The difference of inhibition rate between the two groups on hRPE cells was gradually narrowed. At the later stage (5-6d), the Cur/Chit-DC nanoparticles were almost degraded, and the cumulative drug concentration reached the maximum, approaching and reaching the level of the action of Cur on hRPE cells. At present, there are many studies on the nano sustained release function of different drugs, but there are few reports on the use of Chit-DC as drug carrier to carry Cur. Li

studied the effect of novel Cur nanoparticles on the proliferation and apoptosis of Lewis lung cancer (LLC)cells and observed that Cur nanoparticles could significantly improve the inhibitory effect of free Cur on the proliferation of LLC cells

. Huang

applied 5-FU-PLA-NPS and 5-FU active agents to act on gastric cancer cells respectively in the study of 5-FU immunodrug loading preparation and anti-gastric cancer effect, and observed that the total amount of 5-FU-PLA-NPS released continuously increased with the extension of time, and its effect on gastric cancer cells was continuously enhanced. The results obtained in the above experiments are consistent with the findings of our study,which indicates that the nanomaterials synthesized in the experiments are also ideal long-acting sustained-release drugs.Apoptosis is an important reason for inhibiting cell proliferation

. In order to understand the changes of Cur and Cur/Chit-DC nanoparticles on hRPE cell cycle, we used flow cytometry to detect the apoptosis of hRPE cells, and the results showed that in the range of 5-40 μg/mL, the percentage of cells in G0-G1 phase increased and the percentage of cells in S phase decreased in both Cur group and Cur/Chit-DC nanoparticles group, indicating that the hRPE cells enter the cell proliferation stage decreased. Cur can block the hRPE cell cycle in S phase. It may be one of the important reasons why Cur inhibits cell proliferation

. Li

studied the LLC cells treated with Cur and Cur nanoparticles at the same concentration for 24h, and the apoptosis rates of LLC cells in Ct group, Cur group and Cur-NPS group were 4.94%, 7.43%,and 67.69%. It was also found that Cur could block LLC cell cycle in S phase. Therefore, the results obtained in this experiment are consistent with the above findings.

Hypoxia can induce VEGF expression in RPE cells both

and

, and VEGF can promote intraocular angiogenesis

. Vasquez

found that Cur not only inhibited the upregulation of VEGF in hypoxia-induced RPE cells, but also inhibited the angiogenesis of choroid vascular cells. At the same time, Mrudula

also found that Cur could inhibit the expression of VEGF in the retina of diabetic rats, thus inhibiting fundus neovascularization.We proposed a hypothesis: Can Cur inhibit the proliferation of fundus neovascularization by affecting the secretion level of VEGF in hRPE cells? Whether nano drugs have the same effect? We conducted experiments to clarify these questions.The effects of Cur/Chit-DC nanoparticles and Cur on VEGF mRNA expression level of hRPE cells were detected by semiquantitative RT-PCR. The results showed that both of them could decrease the expression level of VEGF mRNA in hRPE cells. The results are consistent with the above findings.However, whether Cur nanoparticles can reduce VEGF protein expression level of hRPE cells remains to be confirmed by further study. This study proved a research basis for further preparation of Cur/Chit-DC nanoparticles with targeted function by coupling specific VEGF antibody and

animal experiments. It provides a preliminary theoretical basis for finding a new, durable, safe and effective drug for fundus neovascular disease.

同阿里巴巴、京東及亞馬遜等國內(nèi)外電商巨頭合作，大力發(fā)展B2C跨境電子商務(wù)。在南寧綜合保稅區(qū)建設(shè)面向我國國內(nèi)市場的東盟傳統(tǒng)優(yōu)勢產(chǎn)品海外保稅倉以及面向東盟市場的中亞、西亞傳統(tǒng)優(yōu)勢產(chǎn)品海外保稅倉，將南寧建設(shè)為東盟產(chǎn)品進(jìn)入我國市場以及中西亞產(chǎn)品進(jìn)入東盟市場的重要信息平臺、物聯(lián)網(wǎng)中心與物流節(jié)點(diǎn)。

Supported by Hainan Provincial Natural Science Foundation of China (No.819MS133).

None;

None;

None;

None;

None.

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