Protective effect of C2 ceramide on dopaminergic neurons in a mouse model of Parkinson's disease / 中国组织工程研究

BACKGROUND:C2 ceramide reduces the formation of Alpha-Synuclein(α-Syn)oligomers as the protein phosphatase 2A agonist,which has an important regulatory effect on cell aging in the central nervous system. OBJECTIVE:To investigate the protective mechanism of C2 ceramide on dopaminergic neurons. METHODS:Twenty-five C57BL/6 mice were randomly divided into control group,model group,C2 ceramide low-,medium-and high-dose groups(n=5 per group).Except for the control group,a mouse model of Parkinson's disease was established by injecting mutant A53T α-Syn oligomers into the left striatum in the other groups.On the 30th day after the striatal injection,three C2 ceramide groups were intragastrically administered with C2 ceramide(1,5,10 μg/g)dissolved in saline at one time,while the control and model groups were administered with the same amount of saline within 30-90 days after modeling,for a total of 60 days.Behavioral changes in each group of mice were observed during this period.On the 90th day after striatal injection,mouse brain tissue was extracted by perfusion under anesthesia,and the changes of dopaminergic neurons in the midbrain substantia nigra were analyzed by immunohistochemical staining.The levels of α-Syn oligomerization and phosphorylation in the midbrain of mice were detected by ELISA,and the changes of enzyme activities related to α-Syn phosphorylation were analyzed. RESULTS AND CONCLUSION:C2 ceramide had an ameliorating effect on Parkinson's disease-like dyskinesia in mice caused by the striatal injection of mutant A53T α-Syn oligomers.High-dose C2 ceramide showed better effects on dyskinesia in mice with Parkinson's disease(P<0.01).The mutant A53T α-Syn oligomers significantly reduced the number of dopaminergic neurons in the substantia nigra of mice(P<0.01),while the number of dopaminergic neurons in the substantia nigra increased significantly in the C2 ceramide high-dose group(P<0.01).The levels of α-Syn oligomers and phosphorylated α-Syn in the brain were significantly reduced in the C2 ceramide high-dose group compared with the model group(P<0.01),while the level of ceramide was increased(P<0.05)and the activity of protein phosphatase 2A was significantly upregulated(P<0.01).To conclude,C2 ceramide can attenuate the neurotoxic effects induced by oligomerized α-Syn by the phosphorylation modification environment of α-Syn in mouse midbrain tissue and protect against the reduction in the number of nigrostriatal dopaminergic neurons in mice,thereby reducing the degree of dyskinesia in Parkinson's disease.

Relaxation Effect of Synthetic Ceramide Analogues in Cat Esophageal Smooth Muscle Cells

Ceramide has emerged as a novel second messenger for intracellular signalling. It is produced from sphingomyelin and is involved in the control of cell differntiation, proliferation, and apoptosis. C2- ceramide, short chain ceramide, plays a role in mediating contraction of cat esophageal smooth muscle cells. We examined the effect of synthesized ceramide analogues on the C2-ceramide and ACh-induced contraction in esophageal smooth muscle cells isolated with collagenase. CY3523, CY3525, or CY3723 inhibited C2-ceramide induced contraction, in a time dependent manne. Each analogue also inhibited the contraction in concentration dependent manners. CY 3523, CY 3525, and CY 3723 had no effect to the contraction induced by PMA. The inhibition with CY3523, CY3525 and CY3723 on the C2- ceramide induced contraction was recovered by PMA. These analogues decreased the density of MAPK bands (p44/42 or p38) in the western blot. These results suggest that ceramide analogues can inhibit C2-ceramide induced contraction via PKC and MAPK dependent pathway.

Growth Inhibitory Effects and Mechanisms of Ceramides in Renal Cell Carcinoma Cells / 대한비뇨기과학회지

PURPOSE: Because metastatic renal cell carcinoma responds to various forms of therapy with low remission rates, safe therapeutic agents is urgently needed. Ceramide is a potent and specific suppressor of cell growth and an inducer of apoptosis via an intracellular mediation of the sphingomyelin cycle. The present study was designed to assess the growth inhibitory effects and their mechanisms of C2-ceramide and C6-ceramide in renal cell carcinoma cells. MATERIALS AND METHODS: A standard microculture tetrazolium(MTT) assay was used to measure the cytotoxicity of C2-ceramide and C6-ceramide in renal cell carcinoma cell line A498. Apoptosis was confirmed by DNA fragmentation assay using agarose gel and TdT-mediated biotin-dUTP nicked-end labelling(TUNEL) technique. C2-ceramide and C6-ceramide were injected to the A498 tumor which was formed after A498 cells were implanted subcutaneously in athymic mice. Growth inhibitory effects of ceramides were examined biweekly. RESULTS: The survival fractions of A498 cells were 92.6+/-6.0, 82.8+/-14.0, 66.4+/-11.3, 41.8+/-9.6 and 24.3+/-6.3% for the concentrations of C2-ceramide 2, 4, 6, 8 and 10microM, repectively. IC50 of C2-ceramide was approximately 6.7microM. The survival fractions of A498 cells were 60.9+/-5.0, 23.4+/-3.0, 8.7+/-2.1, 5.0+/-1.2 and 3.3+/-0.6% for the concentrations of C6-ceramide 2, 4, 6, 8 and 10microM, respectively. IC50 of C6-ceramide was about 2.3microM. There were DNA fragmentations in A498 cells treated with C2-ceramide or C6-ceramide on the agarose gel and apoptotic tumor cells were also identified after treatment of C2-ceramide and C6-ceramide in TUNEL method. In in vivo study using athymic mice, the growth of A498 tumors was significantly suppressed by C2-ceramide and C6-ceramide. In vivo tumor suppressive effect was more prominent with C6-ceramide than with C2-ceramide. There`s no toxicity-related death of ceramide-treated athmic mice for 3 months. CONCLUSIONS: C2-ceramide and C6-ceramide have the growth inhibitory effects in human renal cell carcinoma cell line A498 by apoptosis mechanism in vitro and they have the in vivo tumor suppressive effects in athymic mice. C6-ceramide was more effective than C2-ceramide in both in vitro cytotoxicity test and in vivo animal experiment of growth inhibition. Therefore, ceramides may be used to treat metastatic renal cell carcinoma in the future.