Celastrol inhibits laser-induced choroidal neovascularization by decreasing VEGF induced proliferation and migration.

AIM: To evaluate celastrol's effect on choroidal neovascularization (CNV). METHODS: In this study, neovascular formation in vitro (tube formation and aortic ring culture) and in vivo (laser induced neovascular in mice) was treated with celastrol to evaluate this natural compound's impact on CNV. Western blot was applied to explore the possible mechanism for it. For in vitro assay, triplicate for each group was repeated at least three times. For in vivo assay, each group contains 5 mice. RESULTS: Celastrol supressed tube formation and aortic ring sprout neovascularization. In vitro assay exhibited that celastrol inhibiting vascular endothelial growth factor (VEGF)-induced proliferation and migration of human umbilical vein endothelial cells and human choroidal endothelial cells, and by blocking VEGF signaling. Furthermore, intraperitoneal administration of celastrol significantly reduced the area of laser-induced CNV in an in vivo mouse model. By day 14, the area of CNV had decreased by 49.15% and 80.26% in the 0.1 mg/kg celastrol-treated group (n=5) and in the 0.5 mg/kg celastrol treated group (n=5), respectively, compared to the vehicle-treated group (n=5). CONCLUSION: Celastrol inhibits CNV by inhibiting VEGF-induced proliferation and migration of vascular endothelial cells, indicating that celastrol is a potent, natural therapeutic compound for the prevention of CNV.

Expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma and the effect of CIRP downregulation cell proliferation and chemosensitivity to gemcitabine.

The aim of the present study was to investigate the expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma (RCC) and to determine the effects of downregulation of CIRP on cell proliferation and chemosensitivity to gemcitabine. The expression of CIRP was detected by western blot analysis, quantitative polymerase chain reaction and immunohistochemistry (IHC) in 17 RCC and peri-cancerous tissue samples. Subsequently, the RCC 786-0 cell line was selected in order to investigate the function of CIRP using RNA interference (RNAi) technology, which was able to inhibit the expression of CIRP in vitro. Furthermore, the chemosensitivity to gemcitabine of each group [CIRP small interfering RNA (siCIRP), negative control small interfering RNA (siNC) and blank control] was compared. There were marked differences between the RCC and peri-cancerous tissues. IHC demonstrated that the CIRP expression in 13/17 (76.50%) tumor samples was markedly positive compared with that in the peri-cancerous tissues and the most common pathological type was clear cell RCC (92.30%). This observation was further confirmed through western blot analysis of protein expression levels. CIRP downregulation by RNAi in the RCC 786-0 cell line significantly decreased RCC proliferation. Additionally, when RNAi was coupled with gemcitabine treatment, there was a significant increase in apoptosis in the siCIRP group. CIRP was overexpressed in RCC tissues and in the 786-0 cell line. Downregulation of CIRP by siRNA inhibited the proliferation of the 786-0 cell line and enhanced the chemosensitivity of the cells to gemcitabine. Therefore, CIRP downregulation may provide a novel pathway for the treatment of metastatic RCC.

Inhibitory Effects of Hydrogen on Proliferation and Migration of Vascular Smooth Muscle Cells via Down-Regulation of Mitogen/Activated Protein Kinase and Ezrin-Radixin-Moesin Signaling Pathways.

Molecular hydrogen (H2) has recently attracted considerable attention for the prevention of oxidative stress-related vascular diseases. The purpose of this study is to evaluate the effects of hydrogen on proliferation and migration of vascular smooth muscle cells (VSMCs) stimulated by angiotensin II (Ang II) in vitro, and on vascular hypertrophy induced by abdominal aortic coarctation (AAC) in vivo. Hydrogen-rich medium (0.6~0.9 ppm) was added 30 min before 10⁻7 M Ang II administration, then the proliferation and migration index were determined 24 h after Ang II stimulation. Hydrogen gas (99.999%) was given by intraperitoneal injection at the dose of 1 ml/100 g/day consecutively for one week before AAC and lasted for 6 weeks in vivo. Hydrogen inhibited proliferation and migration of VSMCs with Ang II stimulation in vitro, and improved the vascular hypertrophy induced by AAC in vivo. Treatment with hydrogen reduced Ang II- or AAC-induced oxidative stress, which was reflected by diminishing the induction of reactive oxygen species (ROS) in Ang II-stimulated VSMCs, inhibiting the levels of 3-nitrotyrosine (3-NT) in vascular and serum malondialdehyde (MDA). Hydrogen treatment also blocked Ang II-induced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK, c-Jun NH2-terminal kinase (JNK) and the ezrin/radixin/moesin (ERM) in vitro. Taken together, our studies indicate that hydrogen prevents AAC-induced vascular hypertrophy in vivo, and inhibits Ang II-induced proliferation and migration of VSMCs in vitro possibly by targeting ROS-dependent ERK1/2, p38 MAPK, JNK and ERM signaling. It provides the molecular basis of hydrogen on inhibiting the abnormal proliferation and migration of VSMCs and improving vascular remodeling diseases.

Overexpression of CIRP may reduce testicular damage induced by cryptorchidism.

PURPOSE: To investigate the protective effect of overexpression of cold-inducible RNA-binding protein (CIRP) on testicular damage induced by cryptorchidism. METHODS: Male BALB/c mice were made surgically cryptorchid and CIRP gene was transferred into the cryptorchid testis by in vivo electroporation. Seven or ten days after electroporation, the expression of CIRP, p53 and Fas mRNA and protein were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Meanwhile, Histopathological changes were observed by light microscope, and flow cytometry was used to detect testicular cell apoptosis. RESULTS: Testicular weights after transfection with pVAX1-CIRP or pVAX1 were 0.083+/-0.005 g and 0.065+/-0.004 g, respectively, on day 7(P < 0.05) and 0.078+/-0.004 g and 0.052+/-0.007 g, on day 10 (P < 0.05). Testicular cell apoptosis after transfection with pVAX1-CIRP or pVAX1 were 9.8+/-1.1 % and 20.7+/-1.3 %, respectively, on day 7 (P < 0.01) and 10.4+/-0.9 % and 27.5+/-1.2 %, on day 10 (P < 0.01). In addition, the expression of CIRP mRNA and protein in the testes transfected with pVAX1-CIRP were both increased (P < 0.05) at each indicated time point. Meanwhile, the expression of p53 was decreased on day 7 (P < 0.05) and Fas was decreased on day 10(P < 0.05). CONCLUSIONS: Overexpression of CIRP may reduce testicular damage induced by cryptorchidism by down-regulating the levels of p53 and Fas.