Low Doses Of Low-temperature Plasma Promote The Proliferation Of MC3T3-E1 Cells By PI3K/Akt Axis

Objective: The speed of bone healing in fracture patients after treatment is a clinical problem to be solved.Under the premise of ensuring the correct reconstruction of bone tissue,patients with fractures should get out of bed as soon as possible.In this way,it can not only reduce the psychological and economic burden of the patient,but also restore the relaxation and contraction activities of the muscles,tendons,ligaments and other soft tissues of the affected limb,so that the patient can perform functional exercise and rehabilitation treatment at an early stage,and promote the blood circulation of the affected limb.Osteoblast proliferation is a very important stage in the process of bone formation and plays a key role in the process of bone remodeling.Low temperature plasma(LTP)has received extensive attention in the field of biology in the last decade.Previous results have demonstrated that low temperature plasma promotes cell proliferation when irradiating cells for a short time.This project is to explore the effect and molecular mechanism of LTP on mouse embryonic osteoblast precursor cells.Methods: First,six dishes were used to culture mouse embryonic osteoblast precursor cells,so that the cell confluence of each dish reaches about 80-90%.Cells in each dish were treated using a DBD-discharged plasma device.When processing cells,the airtight device that generates plasma needs to be filled with working gas.The working gas we use is helium,and each airtight container is inflated for 3 minutes,and the air in the device is emptied as much as possible.Cells were washed twice with sterile PBS and 5 ml of fresh culture medium added before each treatment with low temperature plasma.The control group was not treated,that is,the 0s group.The experimental groups were treated with low temperature plasma for 5s,10 s,15s,20 s and 25 s,respectively.Next,the cell proliferation was detected by MTT assay.ROS and RNS kits were used to detect both concentrations in cell culture media,respectively.Detection of intracellular ROS levels using DCFH-DA probes.The distribution of LTP-treated cells at different times of cell cycle was detected by flow cytometry.The expression of various proteins on the PI3K/Akt axis was detected by western blotting.Results: The results of MTT assay indicated that when the cells were irradiated for a short time,low temperature plasma can promote the proliferation of MC3T3-E1 cells,and the cell viability reached a peak at 10 s,while high-dose low-temperature plasma inhibited cell growth.After LTP treatment,the concentrations of ROS and RNS in culture medium increased with the increase of treatment time.The level of intracellular ROS also increased with increasing time of LTP treatment.The results of cell cycle revealed that low-dose(LTP treatment for 10s)LTP could promote the rapid entry of cells in G0/G1 phase into S phase,increase the percentage of cells in S phase.The results of Western blotting demonstrated that the protein expression on the PI3K/Akt axis was enhanced when LTP was treated for 10 s compared with the control group,but this enhanced effect was weakened after NAC was added.LTP may activate related protein expression on the PI3K/Akt axis via ROS.Conclusion: Low temperature plasma can activate the expression of related proteins on the PI3K/Akt signaling pathway through intracellular ROS at low doses,and promote the proliferation of MC3T3-E1 cells.