Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro

INTRODUCTION

Diabetic retinopathy is the primary cause of adult blindness in the world. Obesity, which is due to weight gain caused by increased adipose tissue, is usually related to lifestyle-related cardiovascular and metabolic diseases, for instance, diabetes, hypertension, and hyperlipidemia which lead to a higher risk of developing vascular disease or atherosclerosis.Adipose tissue is a vital endocrine organ that secretes many bioactive substances, such as leptin, chemerin and apelin

,collectively known as adipocytokines

. Evidence suggests that adipocytokines play a pathophysiological role in complications related to obesity and diabetes

. Leptin is an adipocytokine that acts directly on the hypothalamus, regulating energy intake and consumption

. Leptin receptors are expressed not only in the hypothalamus but also in various peripheral tissues

. Leptin concentrations in the peripheral blood of obese people is proportional to the degree of obesity,suggesting that leptin may play a pathophysiological role in obesity-related complications

. In addition, leptin is closely related to energy metabolism and insulin resistance

. Serum leptin concentrations were found to be significantly higher in hyperplastic diabetic retinopathy patients than non-proliferative retinopathy patients

. In proliferative diabetic retinopathy patients, intravitreous leptin concentrations were higher

,suggesting that leptin may play a role in proliferative diabetic retinopathy. The most crucial pathological change of proliferative diabetic retinopathy is the formation of retinal neovascularization

. However, it remains unclear whether leptin is involved in the process. Thus, this study aimed to clarify the role and mechanism of action of leptin on retinal neovascularization

.

MATERIALS AND METHODS

The Matrigel tube formation experiment was used to detect the formation of tubular structures in control, 100 ng/mL leptin- and AG-490- (100 ng/mL leptin+10 μmol/L AG-490) treated cells. We found that inhibition of the JAK/STAT pathway by AG-490 inhibited the number of tube formations in RF/6A cells induced by 100 ng/mL leptin, significantly (

=80.50,

=0.001; Figure 7).

RF/6A cells in the logarithmic growth phase were plated at a density of 5×10

/mL in 96-well plates (100 μL cell suspension per well). Cells were treated as described above for 24h, then cultured at 37

C for a further 24h. Next, cells were cultured for 4h at 37

C with 10 μL CCK8 in each well. Plates were read at OD 450 using an Enzyme standard instrument (ThermoFisher Scientific, MULTISKAN MK3).

In the final stages of varieties of ocular diseases, including diabetic retinopathy and retinal vein occlusion, retinal neovascularization often leads to catastrophic vision loss. In this study, it was demonstrated that leptin can stimulate RF/6A cells' proliferation, migration, and tube formation

.As an adipocytokine, leptin was found to be expressed nearly exclusively in adipose tissue in mammals

. It acts directly on the hypothalamus to regulates energy intake and expenditure.It has been shown that leptin expressed in retina, choroid,sclera, vitreous and tears

. Serum levels of leptin were closely related to obesity and obesity-associated microvascular complications

. Our findings are similar to previous studies

. Cell migration, proliferation, and tube formation are vital factors in the formation of new blood vessels

.Our data suggest that leptin could promote new blood vessels formation. Furthermore, our findings confirm that leptin could activate the JAK2/STAT3 signaling pathway in RF/6A cells,and that inhibition of the JAK2/STAT3 signaling pathway through AG-490 blocks leptin-induced endothelial cells'proliferation, migration, and tube formation.

2.1.7 穩(wěn)定性試驗(yàn) 分別精密吸取“2.1.3”項(xiàng)制備的同一姜黃供試品溶液（1號(hào)）10 μL，分別在0、2、4、6、8、12、18、24 h按“2.1.1”項(xiàng)下色譜條件測(cè)定，姜黃樣品中姜黃素、去甲氧基姜黃、雙去甲氧基姜黃素的峰面積RSD分別為0.8%、1.4%、2.0%，結(jié)果表明供試品溶液在24 h內(nèi)穩(wěn)定。

更多的時(shí)候因?yàn)椤皞鹘y(tǒng)”發(fā)酵，引來(lái)了許多盜墓賊或梁上君子，我也不例外，剝離了傳統(tǒng)與現(xiàn)代與生活，模糊了審美的視線(xiàn)，使美的發(fā)現(xiàn)愈來(lái)愈難。于是，困惑中我在畫(huà)外追問(wèn)：前人的生活與現(xiàn)代人的生活？我的靈魂因此而漂泊，在漂泊中發(fā)現(xiàn)，生活帶來(lái)最初的沖動(dòng)是源于心動(dòng)，是無(wú)法抵御和抑制的激情奔騰。我的畫(huà)是從這里開(kāi)始，以驗(yàn)證著一個(gè)為個(gè)性化的漂泊靈魂尋找歸宿和充滿(mǎn)著執(zhí)著與真正的人生的追戀。

The Matrigel tube formation assay revealed that the number of tubular structures that developed in control, 50, 100, and 200 ng/mL leptin groups was 38.6±2, 50.1±3.5, 67.7±5.2, and 84±5.6,respectively (

=68.7,

=0.001). These findings suggest that leptin can promote RF/6A cell tube formation (Figure 3).

Data are analyzed using SPSS 20.0 software and given as the mean±standard deviation. Comparison of the means between groups was analyzed by single-factor ANOVA,while the LSD method was used to compare two groups.

＜0.05 was considered to be statistically significant.

RESULTS

AG-490 is a tyrosine kinase inhibitor that inhibits STAT-3 activation by selectively blocking JAK2. Thus, JAK/STAT-3 activation is inhibited using AG-490 selectively. To verify the inhibitory effect of AG-490 on the JAK/STAT signaling pathway, cells were separated into control, 100 ng/mL leptin,and AG-490 (100 ng/mL leptin+10 μmol/L AG-490) groups.We found that AG-490 could inhibit leptin-induced JAK2 and STAT3 phosphorylation (P-JAK2:

=63.29,

=0.001;P-STAT3:

=70.68,

=0.001; Figure 4).

Migration of endothelial cells is essential during early angiogenesis.To determine the effect of leptin on angiogenesis, we used the Transwell assay to measure the cell migration ability of RF/6A cells. We found that leptin led to an obvious increase in the migration of endothelial cells after 24h significantly.The number of migrating cells in the control, 50, 100, and 200 ng/mL leptin groups were 43±4.5, 65±6.5, 83.5±7.2, and 112.1±7.6, respectively (

=145.8,

=0.001; Figure 2).

Cells were lysed and then total protein was extracted. Samples were separated by SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. Overnight at 4

C, membranes were incubated with the following primary antibodies: GAPDH (1:1000), JAK2 (1:1000), p-JAK2 (1:1000),STAT3 (1:1000), and p-STAT3 (1:2000). The JAK2 and p-STAT3 antibodies were purchased from Cell Signaling Technologies (USA), the p-JAK2 antibody was obtained from Abcam (UK), the STAT3 antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd. (China) and the GAPDH antibody was purchased from Hangzhou Xianzhi Biological Co., Ltd. (China). After washing, membranes were incubated with the corresponding secondary HRP-conjugated antibodies at 37

C for 2h. Protein bands were analyzed by BandScan.

To determine the effect of leptin on the JAK/STAT signaling pathway, we used Western blot analyses to detect the protein expression levels of JAK2, p-JAK2, STAT3, and p-STAT3.It was found that incubation with leptin increased the phosphorylation levels of JAK2 and STAT3 were significantly increased in RF/6A cells significantly (P-JAK2:

=47.33,

=0.001; P-STAT3:

=111.86,

=0.001).

The effects of different concentrations of leptin on cell proliferation were examined using the CCK8 assay. As shown in Figure 1, varying concentrations of leptin (20, 50, 100, and 200 ng/mL) led to a significant dose-dependent increase in RF/6A proliferation after 24h (

＜0.05).

The role of the JAK/STAT signaling pathway on leptin-induced cell proliferation was examined using AG-490, the selective JAK/STAT signaling pathway inhibitor. The effects of the following treatment groups: control,100 ng/mL leptin, and AG-490 (100 ng/mL leptin+10 μmol/L AG-490) on cell proliferation were assessed using the CCK8 assay. We found that AG-490 could significantly inhibit the proliferation of RF/6A cells induced by 100 ng/mL leptin(

=131.99;

=0.001; Figure 5).

To determine the role of the JAK/STAT signaling pathway on leptin-induced cell migration, the Transwell assay was used to assess the levels of migration in control,100 ng/mL leptin, and AG-490 (100 ng/mL leptin+10 μmol/L AG-490)-treated cells. We found that inhibition of the JAK/STAT pathway by AG-490 significantly inhibited the migration of RF/6A cells induced by 100 ng/mL leptin (

=150.31;

=0.001;Figure 6).

RF/6A cells were purchased from the Cell Bank of Typical Cell Culture Preservation Committee, Chinese Academy of Sciences. Cell culture consumables were purchased from Biyuntian Biotechnology Company (China). RF/6A cells growing in good condition were digested by 0.25% trypsin, collected and centrifuged at 1000 rpm for 5min, and then moistened with phosphate buffered saline (PBS) twice to remove the residual serum.According to different treatment, RF/6A cells were randomly divided into four groups: control, 50, 100, and 200 ng/mL leptin groups. In leptin treatment group, different concentrations of leptin (0, 50, 100, and 200 ng/mL) were added to the culture medium for culture. Recombinant human leptin was purchased from Peprotech (USA). In leptin+AG-490 treatment group,leptin (100 ng/mL) and AG-490 (10 μmol/L) were added to the cell culture medium. AG-490 inhibitor was purchased from AbMole (USA). RF/6A cells were treated for 24h, then assayed as outlined below.

DISCUSSION

Treated RF/6A cells were digested with 0.25% trypsin, collected and diluted(1×10

cells/mL). Medium containing 10% FBS (800 μL) was placed into a 24-well plate containing a Transwell chamber.One hour later, 200 μL of cell suspension was added to the upper section of the Transwell chamber and incubated at 37

C in an atmosphere of 5% CO

for 24h. Cells were washed with PBS and fixed in 10% methanol for 30min after the Transwell chamber was removed. The membrane was stained with 5%crystal violet at room temperature for 20min. After washing with PBS, samples were observed and photographed under the inverted microscope (Nikon, ECLIPSE Ts2).

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The JAK/STAT pathway was initially regarded as a transmitter of interferon signaling on cells

. The JAK family is comprised of non-receptor tyrosine protein kinases and includes JAK1,JAK2, JAK3, and Tyk2. The JAK family has a C-terminal tyrosine kinase domain, while the N-terminus plays a regulatory role when JAK couples with the receptor protein. The JAK/STAT signaling pathway relays signals from the membrane to the nucleus,

tyrosine phosphorylation, leading to the activation and nuclear translocation of STATs

. Activation of the upstream JAK2 kinase by cytokines or growth factors results in tyrosine phosphorylation and activation of STAT3,which in turn regulates the transcription of target genes and mediates diverse biological effects including cell proliferation,differentiation, and apoptosis

. The activation of STATs is transient in normal cells, lasting only a few minutes or hours.In tumor cells, abnormal and persistent activation of STAT3 is closely associated with the biological behavior of the tumor and its pathogenesis

.

隨著我國(guó)經(jīng)濟(jì)的不斷發(fā)展，人們的生活水平逐漸提高，如今水果市場(chǎng)的銷(xiāo)量有了非常大的提升。在這樣的情況下，提升葡萄產(chǎn)量、提早葡萄上市時(shí)間對(duì)于提升果農(nóng)的經(jīng)濟(jì)收益有著非常大的幫助。通過(guò)應(yīng)用大棚進(jìn)行促成栽培，合理的對(duì)早中晚品種進(jìn)行搭配，可以使葡萄更早的上市，延長(zhǎng)葡萄的銷(xiāo)售期。因此對(duì)于葡萄大棚促成栽培技術(shù)的研究對(duì)于我國(guó)葡萄種植有著非常重要的意義。本文通過(guò)對(duì)葡萄大棚促成栽培技術(shù)的技術(shù)要點(diǎn)進(jìn)行了分析，希望可以為果農(nóng)和研究人員提供一些參考。……