Effects Of RNF146 On Radiation Spinal Cord Injury In Rats And Mechanism Research

Radioactive spinal cord injury is irreversible serious complications in tumor radiotherapy.In recent years,with the wide application of radiation in the comprehensive treatment of tumor,the number of cases of radiation spinal cord injury?RSCI?have marked raised^[1].While early SCI appear only in a flat paralysis,but often late development for completely paralyzed,especially cervical spinal injury,then this causes a variety of serious complications^[1].Because the pathogenesis is not clear,there is currently no effective treatments.Now,high blood pressure,low blood pressure,diabetes,spinal deformity,vascular disease and malaria history were supposed to be the risk factors of the disease,many chemotherapy drugs neurotoxicity can also lead to SCI after radiation therapy^[1].Therefore,to clarify the molecular mechanisms of SCI of rats caused by ionizing radiation,help to make a clear definition of the pathogeny of radiation injury of spinal cord.The radiation dose of the normal spinal cord should be not more than 50Gy,and the radiation dose more than 60Gy can easily cause the spinal radiation injury^[2].Some researchers found that the radiation tolerance dose of normal cervical spinal cord was69.4 Gy^[3].The lesion of the spinal cord in patients with radiation is usually caused by demyelination,necrosis of white matter area and infiltration of a large number of inflammatory cells^[4][5].In the early stage of SCI,there is an acute response to the body headache and dizziness caused by the edema of nerve cells^[6][7].At the same time,the number of astrocytes and microglial cells increased,participating in the nutrition and repair of the damaged part;Next,the phagocytes mass activation release IL-1 and TNF-?;Finally,the neuronal vacuolar degeneration,bleeding necrosis in the lesion site,and the formation of neuroglial scar^[1][4].Radiation injury causes the injury of neurons,glial cells and extracellular matrix in the spinal cord tissue^[8][9].However,in the progress of SCI,spinal neurons are the main target cells for radiation injury^[10].It is great significance to explore the causes and mechanism of spinal cord neurons after radiation.In the previous study,we found that there is an E3 ubiquitin ligase expressing in mouse spinal cord^[11].Rnf146 is newly found a kind of endogenous neural protective genes,and its encoding product is RING finger protein 146 with E3 ligase activity^[12].In the face of external stimuli such as ultraviolet radiation,the body's DNA is inevitably damaged.So timely DNA damage repair is very important for maintaining the integrity of the genome^[13].RNF146 as a DNA damage response protein,and the sequence is highly conservative^[11].RNF146 could specifically recognize PARsylation-AIF,inducing its ubiquitination degradation,controling AIF tanslocated into nuclear and inhibitting Parthanatos,further protecting the nervous system^[14][15].Because RNF146 ubiquitin ligase activity depends on the concentration of poly adenosine diphosphate ribose?PAR?in the intracellular^[16],RNF146 function object is usually some PAR modified protein.We have tested some molecules mentioned in the literat ure which appered PAR chemical modification and telated to cell proliferation,apoptosis and autophagy,such as shaft protein?Axin?^[17],MIKI^[18],BRCA1^[19],SH3BP2^[20]and PTEN^[21].It was exciting to see that the tumor suppressor gene PTEN was up-regulated in the radiation spinal cord tissue.PTEN has protein phosphatase and lipid phosphatase activity,through catalyzing three phosphoinositide?PI3P?dephosphorylation to phosphoinositide?PI2P?to against phosphatidylinositol 3-kinase?PI3K?-3,further to inhibit the activity of Akt.Moreover,it could cause the stagnation of the cell cycle,induce apoptosis and inhibit cell adhesion and migration^[22].Is the increase of the tumor suppressor gene PTEN the cause apoptosis of the anterior horn motor neurons in RSCI rats?Is the rise of PTEN related to RNF146?Does the ubiquitin ligase RNF146 in the motor neurons of the spinal cord have regulatory effect on PTEN?What is the molecular mechanism.To solve above problems,this study intende to consider the regulation of RNF146 to PTEN as the breakthrough point.We integrated use a variety of experimental techniques,in terms of animal and cell,to explore what the control plays the role in motor neuron apoptosis.Then it will provide new targets and new ideas for clinical prevention of RSCI.Part?Effects of X-ray on rat spinal cord injury and expression of RNF146 and PTENObjective To observe the influence of X-ray radiation on normal rat spinal cord,number of anterior horn motor neurons and RNF146 expression.Methods 60 adult male SD rats were randomly divided into three groups?Blank,IR 24 h and IR 4 8h?,each group of 20 rats.In IR groups,rats were exposed under 40 Gy X-ray irradiation.Then spinal cords were taken respectively in 24 h,48 h after irradiation.There are three continue paraffin sections for HE staining,Nissl staining and immunohistochemical staining,further to observe spinal cord anterior horn motor neurons form change after X ray radiation.Tunel staining was used to evaluate motor neuron apoptosis after X ray radiation.Immunohistochemical staining and immunofluorescence staining were respectively used to detect the location and expression of RNF146 and PTEN in spinal motor neurons.Western blot evaluated the expression of RNF146 and PTEN in rat spinal cord.Results HE staining results showed that 24 h after X ray radiation,the spinal cord anterior horn motor neurons have obvious cell shrinkage,number of glial cells was significantly reduced at the same time;48 h after radiation,visible motor neuron cell hyperchromatic,pyknosis,appear even cell cavitation,glial cells was also significantly reduced.Nissl staining of 24 h group showed neurons appear shrivel,deep dyeing in the cytoplasm;In 48 h group,neurons damage is more serious,appearing cell cavity;Number of glial cells after radiation significantly less than the control group at the same time.The results consistent with the results of HE staining.Neu N immunohistochemical staining showed cells suffered serious damage were spinal cord anterior horn motor neurons.TUNEL staining after prompt radiation,both 24h and 48h group,neurons appeared significant DNA damage,and it means cell apoptosis.Immunohistochemical staining and immunofluorescence staining showed that RNF146 located in the cytoplasm of rat spinal cord motor neurons and the expression of RNF146 decreased significantly both 24 h and 48 h group;Instead,the expression of PTEN obviously increased and PTEN also located in neuronal cytoplasm.Western blot analysis found that the expression of RNF146 decreased significantly,while the expression of PTEN increased significantly;Those results are consistent with immunohistochemical staining.Conclusion Rats spinal cord anterior horn motor neurons after 40 Gy high-dose radiation were happened a serious injury,and there has been a marked apoptosis neurons;After radiation,the level of RNF146 significantly was lower,and the expression of PTEN increased significantly.Part? The molecular mechanism of NCS induced damage to HT22 cellsObjective In this study,HT22 cells were used as cell models to study the apoptosis of neuronal cells induced by X-ray irradiation and the molecular mechanism of expression of PTEN regulating by RNF146.In order to avoid the uncertainties in the process of cell radiation model construction,X-ray radiation was simulated with NCS in this study.Methods We digested the well-developed HT22,inoculated into the culture plate,and then added respectively 0,300,600,1000,2000,3000 ng/ml concentrations of NCS.To test the optimal dose of radiation damage model,The effect of NCS on the proliferation rate of HT22 cells was detected by CCK8 cell activity test.The DNA amage and apoptosis of HT22 radiation damage model were detected by TUNEL staining and Hoechst staining respectively.The expression of RNF146 and PTEN protein was detected by Western blot.The experiment was divided into blank control group?Con?,Vehicle group,LV-RNF146 group.The Vehicle group and LV-RNF146 group were given NCS simulant treatment.RNF146 was up-regulated in HT22 cells to detect the change of PTEN,then further to analyze the regulation mechanism of RNF146 on PTEN.Results The NCS concentration of 1000 ng/ml was the optimal concentration for building HT22 cell radiation damage model.TUNEL and Hoechst staining showed that the cell had obvious DNA fracture,which further explained that the cell had significant apoptosis.At the same time,Western blot results showed that the expression of RNF146 protein was significantly decreased in HT22 cells,while the expression level of PTEN was significantly increased.In HT22 cells up-regulated with RNF146,the expression of PTEN was significantly lower than that in the control group.It was revealed that RNF146 could regulate the expression of PTEN in the course of simulating HT22 cells by NCS.Further,CO-IP and ubiquification tests confirmed that RNF146 can affect the process of cell radiation damage by regulating the ubiquification of PTEN proteinsConclusion Radiation can induce apoptosis of HT22 cells.In this process,RNF146 plays an important role in regulating the ubiquification of PTEN.