The Role Of Mechanosensitive TRPC1 Channel On Neuron Affected By Stretch Force

BACKGROUND Diffuse axonal injury(DAI)is the most common and harmful pathological type of traumatic brain injury(TBI).It occurs when brain is attacked by external force.The brain tissue has a diffuse white matter axonal injury caused by shearing force and pulling force.DAI can be divided into primary and secondary injuries.Primary injury refers to the shearing force and pulling force inside the brain tissue caused by external force on the head.Secondary injury is induced by trauma within hours to tens of days after injury.The local injury of the axon,after a series of complicated pathophysiological processes,gradually develops into axonal rupture,which eventually leads to axonal necrosis and disintegration.Secondary injury is the main cause of poor prognosis of DAI,and the mechanism of injury is still unclear.Clinically,there is no uniform diagnostic criteria and effective treatments for DAI.For forensic scientist,the diagnosis of DAI is also very difficult.Therefore,clarifying the pathogenesis of DAI,and searching for possible sensitive molecular markers,has great potential application value for guiding the forensic identification of DAI.Likewise,it is important for improving the clinical diagnosis and treatment of the disease.The development of DAI involves the transduction between mechanical force signals and biochemical signals.Studies have shown that there are two most likely pathways for force and biochemical signal transduction,one for the force-sensitive receptor pathway and the other for the force-sensitive ion channel pathway.Mechanosensitive transient receptor potential channels are a common force-sensitive ion channel.They are also important pathways for cell mechanical signal transduction.These ion channels are non-selective cation channels that are widely distributed and expressed in almost all tissues and cell types.It has been found that mammalian TRP channels have six subfamilies,namely TRPA,TRPC,TRPM,TRPP,TRPV.Among them,TRPC1 is a member of the TRPC subfamily and is expressed in most cells of the human body.It was found that TRPC1 can mediate the influx of Ca2+,Na+ and other cations when the cell membrane is stretched.The rapid and large Ca2+ overload caused by Ca2+ influx is the vital process of DAI.Ca2+ overload can cause irreversible microfilaments and microtubule structures in the axons,which can cause swelling and fracture of the axons.Ca2+ overload can also cause damage to mitochondria,leading to mitochondrial membrane potential decreasing,permeability transition pores opening and pro-apoptotic factors releasing.In summary,the mechanically sensitive TRPC1 ion channel,which is involved with both force transduction and Ca2+ influx,is likely to play an important role in the development of DAI.Based on the above research progress,this study established a cortical neuron pulling injury model to simulate DAI injury in vitro,and initially explored the regulation of mechanically sensitive TRPC1 channel in tension-induced neuronal injury.OBJECTIVES 1.Establish DAI cell model by pulling the injured cortical neurons,observe the changes of biochemical indexes,such as LDH;2.To investigate the changes of cytoskeleton,mitochondrial membrane potential and intracellular reactive oxygen species in neurons after DAI injury.3.To investigate the role of TRPC1 channel in neuronal injury after DAI.METHODS 1.Primary cultured cortical neurons were identified by immunofluorescence.Then neurons were randomly divided into groups,and the DAI nerve cell pulling injury model was established by using the Ellis method.Previous studies have shown that the silicone membrane deformation distance of 10 mm can cause tensile damage to nerve cells,causing morphological changes of axonal.In the experiment,the culture solution was taken at 0h,4h,12 h,24h and 48 h after injury,and the change of LDH was detected to verify the modeling of the traction damage.2.Primary cultured cortical neurons were divided into drug intervention group and control group according to whether or not TRPC1 inhibitor GsMTx-4 was added.The drug intervention group was subjected to injury modeling after adding inhibitors,and was divided into 0h,4h,12 h,24h and 48 h according to different time points after injury.The deformation distance was unified to 10 mm.The control group was the same as the drug intervention group except that no inhibitor was added.3.Immunofluorescence of microfilament protein and tubulin was used to observe the changes of neuronal cytoskeleton in drug intervention group and control group.4.JC-1 was used to detect the changes of mitochondrial membrane potential in the drug intervention group and the control group.5.DCFH-DA fluorescent probe was used to detect the changes of intracellular reactive oxygen species in the drug intervention group and the control group.RESULTS 1.In this experiment,the deformation distance was unified to 10 mm,and the deformation time was 1s.At 0h after injury,the axonal distortion of some neurons was "S" type,and a small number of neurons axonal fracture.By 48 h,neuronal death was severe,cell vacuolation occurred,the number of axons decreased,the shape was unclear,and some axonal thickening,distortion,fracture,and even disintegration occurred.After the traction injury,the LDH content in the supernatant increased significantly.The LDH content in the supernatant of the control group remained basically stable.2.It was found that the fluorescence intensity of ?-tubulin showed an unstable upward trend after stretching.However,the addition of the TRPC1 antagonist GsMTx-4 to the DAI model did not significantly reverse the change in ?-tubulin expression intensity.The axons of neurons were indicated by F-actin.The axial density of neurons in the control group was lower than that in the drug-treated group,especially in the 12-48 h group.After stretching,the length of the axon in control group became shorter as the elapsed time increased.The trend of the axonal length weakened after intervention with the TRPC1 inhibitor.3.It can be seen that after stretching,the mitochondrial membrane potential of the neurons decreased immediately(0h).With the prolongation of the time after the force,the mitochondrial membrane potential of the neurons recovered to some extent,but did not return to previous level.After adding GsMTx-4 to the neurons,it was found that there was no immediate decrease in mitochondrial membrane potential after the addition,although there was a slight decrease immediately after stretching,but 24 hours later,the mitochondrial membrane potential gradually recovered,and returned to previous level 48 hours later.4.It was found that the active oxygen content increased slightly at 0h after injury,and decreased at 4h,and returned to the pre-injury level at 12 h,then gradually decreased.The change of reactive oxygen species in the drug intervention group was mild.At 0h,the difference between the drug intervention group and the control group was more obvious.CONCLUSIONS 1.The neuronal stretch-induced injury model was established successfully.2.The effect of tension on neurons can lead to delayed axonal rupture,the use of TRPC1 inhibitors can improve the axonal delayed fracture;3.The effect of stretching on neurons can lead to a decrease in mitochondrial membrane potential.The use of TRPC1 inhibitor can improve the mitochondrial membrane potential decrease after injury.