Effects Of Taurine On The Apoptosis And Differentiation Of Neuroepithelial Cells Treated With Hyperthermia

Objective and Significance Maternal hyperthermia significantly increases the risk of congenital malformations in human and animal models. Neural tube defects(NTDs) are among the most common and pernicious congenital malformations associated with maternal hyperthermia. They place an enormous emotional and economic burden on the population. Hyperthermia is a common environmental agent, and many diseases can cause fever, so it is hard for pregnant women to ward off its effect. Therefore, it possesses important significance for reducing congenital malformation to devise strategies aimed at reducing the NTDs occurrence.Previous studies have shown that neuroepithelial apoptosis is responsible for NTDs. For example, hyperglycemia effectively enhanced oxygen free radical formation and neuroepithelial apoptosis, which can result in maternal diabetes-induced NTDs. Hyperthermia can promote cell apoptosis in the neuroepithelial and mesenchyme cells of embryo, decrease viable count, induce neural tube closure delay or no closure. Thereby, neural tube defect, such as anencephalia, exencephalia, meningocele are formed. Therefore, if we can decrease the apoptosis of neuroepithelium, the NTDs will be reducible.Taurine (2-aminoethanesulfonic acid) is an almost ubiquitous cellular constituent throughout the animal kingdom. It is also one of the most abundant free amino acids in the brain. The concentration of taurine in the central nervous system (CNS) is just lower than that of glutamate. Taurine possesses a number of cytoprotective properties through its actions as a neurotransmitter, neuromodulator, osmoregulator, modulator of intracellular calcium homeostasis, anti-oxidant, membrane stabilizer, and anti-inflammation factor. Under cell-damaging conditions, the release of taurine is increased. The increase in the extracellular levels of taurine in cell-damaging conditions may be an important endogenous protective mechanism. Taurine has been reported to reduce glutamate-induced neurotoxicity, and improve the recovery of neuronal functions after cerebral hypoxia. Moreover, taurine-containing neurons in hippocampus are more resistant to the damage induced by ischemia than other neurons.Whether taurine has protective effect on hyperthermia-induced neuroepithelial apoptosis is still unknown. Therefore, we proposed hypothesis that taurine can rival neuroepithelial apoptosis induced by hyperthermia. In this experiment we observed the protective effect of taurine on proliferation, apoptosis and differentiation of neuroepithelium after being treated with hyperthermia, and explain the conceivable mechanism, and finally, aim to provide a new experimental evidence for the precaution and therapy of neural tube defects.Methods:1. Observing the apoptosis of neuroepithelial cells induced by hyperthermia The mice were killed on 12.5th day of pregnancy. Neuroepithelial cells suspension were isolated with stereoscopic microscope. After mechanical blowing, they were gained in 1% serum medium, and identified by Nestin immurofluorescance staining. Nuroepithelial cells were divided randomly into 4 groups: control group and 39℃, 40℃, 41℃hyperthermia groups. Cell viability and apoptosis were determined by MTT assay and DAPI staining at 12 h and 24 h after hyperthmia.2. Observing the effect of taurine on neuroepithelial cells proliferation and apoptosis after hyperthemia Nuroepithelial cells were divided randomly into 7 groups: control group, hyperthermia group and 2 mM, 6 mM, 10 mM, 15 mM, 20 mM taurine groups. Cell viability and apoptosis were determined at 12 h and 24 h after hyperthermia by MTT assay and DAPI staining. Immunofluorescence staining was employed to explore the expression of Caspase-3.3. Observing the effect of taurine on the differentiation of neuroepithelial cells treated with hyperthermia The neuroepithelial cells were cultured in 10% serum medium. Immunofluorescence staining was used to test the expression of MAP-2 and GFAP in cultured cells on the 5th day after being treated with hyperthermia.Results:1 .Morphological characteristics The cells were spherical, or elliptical, and dispersed in culture medium with obvious boundary. Nestin immurofluorescence staining showed more than 95% cells were strongly positive, which revealed that most of the cells were neuroepithelial cells.2. The results of MTT assay showed that hyperthermia can depress cell viability, and DAPI staining showed hyperthermia can induce neuroepithelial cell apoptosis. It was an optimal condition to be treated with 40℃for 30 min for the cellular model of neuroepithelial cell apoptosis induced by hyperthermia.3. The cell viability of 6 mM and 10 mM taurine groups were obviously higher than that of hyperthermia group. The apoptosis rate of cells in hyperthermia group had largely increased from 6 h after hyperthermia; the apoptosis rates in 6mM and 10 mM taurine groups decreased from 12 h after hyperthermia compared with that of hyperthermia group. The changes of Caspase-3 expression in various groups were similar to those of apoptosis rate.4. On the 5th day of being cultrued in 10% serum medium, cells in control group extended axons and neurites, which form an extensive network. In the hyperthermia group, the neurites were shortened or lost, and the survival cells decreased in number, and the networks of cell processes were sparse. The states in 6 mM and 10 mM taurine groups were well improved, the networks were thicker than that in hyperthermia group. The immurofluorescence staining showed that MAP-2 positive rate in control group was 37.74%, while that in hyperthermia group was 29.68%. The MAP-2 positive rate in 6 mM and 10 mM taurine group was significantly higher than that of the hyperthermia group, but had no obvious difference to the control group. The GFAP positive rate had no significant difference among the four groups. Conclusion:1. Hyperthermia could induce neuroepithelial cells apoptosis. Being treated with 40℃for 30 min was an optimal condition for the cellular model of neuroepithelial cell apoptosis induced by hyperthermia.2. 6 mM～10 mM taurine could obviously increase the cell viability and inhibit the apoptosis induced by hyperthermia. The inhibitation of the cell apoptosis in the taurine groups might be relavent to the low expression of Caspase-3.3. 6 mM～10 mM taurine may promote neuroepithelial cells which were treated with hyperthermia differentiate into neurons.The results of this study revealed that taurine had a protective effect on neuroepithelial cells apoptosis induced by hyperthermia, and might promote neuroepithelial cells differentiate into neurons after hyperthermia. Therefore, it provided theoretical and experimental proof for precaution and cure of NTDs induced by hyperthermia.