The Protective Effect And Its Mechanism Of Mild Hypothermia On Photoreceptors Against Injury

Background:Photoreceptors are a type of neuron in the retina that process and transmit visual information.Its function is to convert light signal into bioelectrical signal to form vision.When the energy of cells is not enough or the cells receive intense light energy,photoreceptors undergo irreversible damage,which may cause loss of visual function and even lead to blind.That seriously affects the health and life quality of the patient.Therefore,it is great theoretical and practical to study the mechanism of photoreceptor cell damage and its prevention methods.The hypothermia treatment is a method of improving the therapeutic effect of a disease by maintaining the patient’s body temperature at a specific temperature physically or chemically.In the treatment of neurological diseases,there are many researches of hypothermia treatment in stroke,craniocerebral injury,elevated intracranial pressure,subarachnoid hemorrhage,spinal cord injury,hepatic encephalopathy and neonatal perinatal encephalopathy.Researches have also shown that hypothermia can reduce brain neuron damage,inhibit endothelial cell and blood-brain barrier dysfunction,and the protective mechanism may be related to inhibition of apoptosis.Therefore,this study intends to explore the protective effects of hypothermia in glucose deprivation and light damage,discuss the possible mechanism of hypothermia treatment,and initially observe the protection of hypothermia in light-induced retinal damage.Purpose:1.Determine the protective effect of hypothermia in GD-induced and light-induced photoreceptor cell damage.2.Explore the molecular mechanism of hypothermia in GD-induced and light-induced photoreceptor cell damage by over-expression/inhibition of Cirbp.3.Observe the hypothermia protection in light-induced animal model.Method:1.Explore the mild hypothermia protection in GD-induced 661 w cells Photoreceptors were cultured in glucose-free medium and divided into normal temperature group(37 ° C)and hypothermia group(32 ° C).MTT assay was used to detect the cell viability of each experimental group in different time.Hoechest/PI staining,flow cytometry,ROS staining,mitochondrial membrane potential detecting,Western Blot and other methods were used to explore the apoptosis of each group.2.Explore the mild hypothermia protection in light-induced 661 w cells Cells were cultured under the light exposure.And the cells were divided into a normal temperature group(37 ° C)and a hypothermia group(32 ° C).Hoechest/PI staing,ROS staining,Western Blot and other methods were used to explore the apoptosis of each group.3.Explore the protective mechanism of hypothermia on 661 w cells(1)Cirbp was over-expressed or inhibited in 661 w cells by lentivirus.(2)Cirbp-661 w or sh Cirbp-661 w cells were cultured in the glucose-free medium or under the light exposure.The effect of Cirbp on the protection of hypothermia were detected by Hoechest/PI ROS staining and Western Blot.4.Explore the hypothermia protection on the retina In light damage animals,hypothermia protection on the retina was examined by observing the thickness of the outer nuclear layer of the retina and the ERG changes of the retina.Proteins were detected by Western Blot.Result:1.661 w cells were cultured in glucose-free medium,the cell viability decreased significantly within 24 hours.Compared with the normal temperature group,the hypothermia group significantly improve cell viability at 8h and 16 h in glucose-free culture,but there is no significant difference between the two groups at 24 h.At 16 h,the apoptotic cells in the hypothermia group were significantly reduced,the production of ROS was decreased,and the mitochondrial membrane potential was decreased.Compared with the normal temperature group,the Caspase-dependent apoptosis-related proteins BAX,Caspase-3 and Caspase-9 decreased,and the anti-apoptosis-related proteins Bcl-2 increased,while the Cirbp was increased.2.Under light exposure with hypothermia,the apoptotic cells were significantly reduced,the generation of ROS was decreased compared with the normal temperature group.PARP-1-dependent apoptosis-related protein PARP-1 decreased in the hypothermia group,while the Cirbp increased.3.Over-expressing Cirbp in GD cells showed that apoptotic cells decreased,ROS production decreased,and Caspase-3,BAX decreased,but Bcl-2 increased at room temperature.Under light exposure,apoptotic cells and ROS were also reduced compared with the control group,while PARP-1 decreased.4.Inhibiting Cirbp in GD cells showed that apoptotic cells increased,ROS production increased,and Caspase-3,BAX increased,but Bcl-2 decreased at room temperature.Under light exposure,apoptotic cells and ROS production were also increased compared with the control group,while PARP-1 increased.5.The retinal thickness of the light damage group with hypothermia was significantly thicker than that of light damage group,and cell morphology and the structure were clear.Retinal function detectd by ERG was also superior to the light damage group.PARP-1 was also significantly lower than that of the light damage group.Conclusion:In this study,661 w was used as the research object,and glucose deprivation and light damage were used to explore the protection and possible mechanisms of hypothermia.The results showed that hypothermia up-regulated the expression of Cirbp,inhibited the production of ROS,and inhibited Caspase-dependent apoptosis induced by glucose deprivation,as well as PARP-1 dependent apoptosis induced by light damage.The expression of Cirbp is closely related to the hypothermia protection.In addition,mild hypothermia can alleviate the light-induce retinal damage and improve the retinal function.