| Background and Objectives:Non-small-cell lung cancer (NSCLC) is one of the commom malignant tumor, accounts for the leading cause of cancer deaths worldwide. Malignant pleural effusion (MPE) is a major cause of poor prognosis in patients with NSCLC. Generally, there are many treatment options for MPE, but overall treatment effect is not satisfactory. So, new treatment strategies are needed for this disease.Vascular endothelial growth factor (VEGF-A) plays an important role in in the development process of MPE. VEGF-A binding to VEGF receptor, through a series of mechanisms to promote endothelial cell proliferation, migration, and induce increased capillary permeability, thus lead to the development of the MPE. Therefore, researchers are trying to find new drugs to inhibit the pathological process of MPE by inhibiting VEGF-A expression.Triptolide is a diterpene triepoxide and also is the major pharmacological component extracted from the celastraceae plant-Tripterygium wilfordii Hook. F (TwHF). It turned out to be the most potent bioactive substance in the TwHF extracts. It has been shown to possess potent anti-inflammatory, anti-tumour and antiangiogenic properties. TwHF has been used for centuries in traditional Chinese medicine to treat a variety of immunological disorders, including immune complex nephritis, rheumatoid arthritis and systemic lupus. Triptolide as a chemically active monomer extracted from traditional Chinese medicine have many advantages, such as:cheap, low toxicity, well tolerated in patients. Recently, studies have found that triptolide can inhibit VEGF-A expression in tumors, and inhibit angiogenesis.Therefore, our study intended to investigate the effects of triptolide on LLC cells and MPE nude models in vitro and vivo experiment, and explore its possible mechanisms.Metholds:(1) LLC cells were treated with TL in vitro, MTT and trypan blue staining were performed to detect the apoptosis and proliferation of LLC cells treated by TL. (2) VEGF-A protein expression in LLC cells were detected by Western blotting after 24 h treated with 0,50,100 and 200 nmol/ml TL. (3) Good growth state LLC cells were injected directly into thoracic cavity of nude mice to creat animal model of malignant pleural effusion. The nude mice were randomly divided into two groups, at 3 days after LLC cells injection, the treat group received 0.4mg/kg triptolide injected into right thoracic cavity, while the control group treated with 50^.1 normal saline (NS) which containing 0.01% DMSO. Once every other day. At the 14th day after LLC cells injection, nude mice underwent chest CT examination to observe the formation of pleural effusion. At the 20th day, mice were killed by overdose chloral hydrate. Using 1ml syringe punp pleural effusion, calculate the mean volume of the two group pleural effusions; then, centrifuged pleural effusion, collected the supernatant, adopted Elisa assay to detect the expression levels of VEGF-A. And the sediment was applied to slides, stained with modified Giemsa staining. Next, dissected nude mice, exposed the whole chest, Counting and Weighing the tumor nodules. Finally, tumors were embedded in paraffin and slides were tested by HE staining and immunohistochemical staining to compare the differences in microvessel density (MVD) and the expression of VEGF-A.Results:(1) TL can inhibit LLC cell proliferation through concentration and time dependence, IC50=64.741 nmol/ml. (2) VEGF expression level of the LLC cells reduced after treated by TL, with the concentration of triptolide increased, VEGF-A expression decreased.(3) Chest CT images showed there were MPE in nude pleural. After the mice were killed, we can pump bloody, non-solidified pleural effusions, the mean volume of the TL treat group was 230.83±46.05μl, the control group was350.33±62.35μl (P<0.01) Opening the chest cavity, tumor nodules can be found spreaded over the parietal and visceral pleura surface. The mean tumor number of TL treatment group and control group were12.50±2.26,19.17±2.32, respectively, P<0.01. On 14 days after LLC cells injection, nude mice underwent chest CT examination to observe the formation of pleural effusion. Pleural effusion supernatant ELISA results showed that, the expression of VEGF-A was lower in TL treat group than that in control group (308.50±49.89 ng/ml VS 430.67±34.58 ng/ml, P<0.001). IHC examination of tumor tissues found VEGF-A expression in the TL treatment group vs control group were 2.50±1.05,4.83±2.32, respectively, The difference was statistically significant. MVD reduced from 13.17±2.71 to 8.83±2.04 in two group above, (P<0.05).Conclusions:(1) TL significantly inhibited LLC cells’proliferation and reduces the expression of VEGF-A protein;(2) TL can inhibit the development of MPE, it promised to be an effective drug for treating MPE;(3) TL has effect on MPE may through the mechanism of reducing VEGF-A expression and inhibiting tumor angiogenesis. |
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