| Background:Medical ozone treatment has been widely used to protrusion of lumbar intervertebral disc and osteoarthritis for its convenience.However,some studies found that overdose or wrong operation cause the imbalance of oxygen/anti-oxygen system when we use medical ozone to treat protrusion of lumbar intervertebral disc.It is vulnerable that the nervous system is sensitive to the imbalance of oxygen/anti-oxygen system when we treat progression of lumbar intervertebral disc with a high concentration of medical ozone.Nevertheless,it’s indistinct how the mechanism of medical ozone results in the nervous system injury.But,it must have relative with its strong oxidizing property.Li’s study shows that adjusting the release of Ca2+and CaMKII/MAPK pathway by activating endoplasmic reticulum stress damaged the nervous system.Both Autophagy and the pathway of Nuclear factor erythroid-2-related factor 2(NRF2)-antioxidant response element(ARE)are inevitable for body to antioxidants.As usual,Kelch-like ECH-associated protein 1(KEAP1)link to NRF2 which reduces NRF2 translating to nuclear to enhance the antioxidant ability.P62 is the key of the autophagy.On the other hand,P62 disturbs the combination of KEAP1 and NRF2 with a competitive method to free and stable NRF2 and promote NRF2 translate into nuclear hence the level of antioxidant.Besides,NRF2 has the power to activate P62.A positive feedback formed between NRF2 and P62 to keep the balance of the oxidation/anti-oxidation system,together.In addition,autophagy and NRF2-ARE pathways have an important influence on the form,damage,repair and degeneration of the nervous system.Tert-butylhydroquinone(tBHQ),an activator of NRF2,has been used to explore the function of NRF2 on anti-inflammation,anti-oxidant,autophagy and nervous protection.However,there is lack of study on how tBHQ interferes with nervous injury caused by overdose of medical ozone.This study plan to activate NRF2 through tBHQ to further explore the influence of P62-NRF2-ARE on the nervous injury caused by overdose of medical ozone.Methods①Extract and cultivate of spinal neurons,then,7th day’s cells cultivated used for experiment;②Authenticate spinal neurons with MAP2 stained by Immunofluorescence;③Deal with cell with the concentration of 0、10、20、30、40μg/ml medical ozone,CCK8 were used to detect the vitality of cells to choose an appropriate concentration for experiment.④Divided cells into control group(Control group),30μg/ml medical ozone group(O3-30 group),40μg/ml medical ozone group(O3-40 group),40μg/ml medical ozone+40μmol/l tBHQ group(T40 group)and 40μmol/l tBHQ group(TO).RT-qPCR and Western Blot were used to detect the changes of NRF2,HO1,P62,LC3 in the level of mRNA and protein.⑤Immunofluorescence was used to detect the different expressions of NRF2 in nuclear among these groups.⑥Normal/Apoptotic/Necrotic Cell Detection Kit(AO/EB)was used to detect the rate of live,Apoptotic and necrotic cells.SPSS(IBM Corp.Version 23.0.Armonk,NY:IBM Corp)was used for statistical analysis.The data was expressed as the means ± SDs.The statistical significance of the difference between treatment groups was measured using a one-way ANOVA,followed by the Tukey’ test for multiple comparisons.P<0.05 was considered statistically significant.All measurements were carried out at least three times.Results1.The purity of spinal neuron over 95%which satisfied the requirement of experiment.2.We chose 40μg/ml medical ozone for the experiment concentration due to the result of CCK8 showing that the activity of cells reduced to 71%after 40μg/ml medical ozone was added.3.RT-qPCR showed that the change of P62 and LC3Ⅱ in mRNA level of O3-40 group have no statistical significance(P<0.05)compared with the control group.Nevertheless,in the T0 group,the mRNA level of NRF2 and HO1 have statistical significance compared with the control group(P<0.05)which shows that tBHQ has activated NRF2 pathway.Besides,the mRNA level of HO1 in O3-40 groups is lower than O3-30 groups(P<0.05)and the NRF2 in T40 group is increased than O3-40 groups(P<0.05).4.Western Blot:The protein level of NRF2,HO-1,P62 and LC3II in O3-30 groups is higher than control group,T40 group reversed the decrease of NRF2,HO-1,P62 and LC3Ⅱin protein level caused by 40μg/ml medical ozone.5.Immunofluorescence was used to detect the expression changes of NRF2 in nuclear.Its result shows 40μg/ml medical ozone decreases the expression of NRF2 in nuclear compared with 30μg/ml medical ozone.However,the T40 group reversed the decrease taking place in 03-40 groups.6.The T40 group reversed the necrosis rate increase and the live cells decreased in O3-40 groups.Conclusion:The mechanism of Primary spinal cord neurons injury caused by 40μg/ml medical ozone in vitro damages the pathway of P62/NRF2/ARE to interrupt NRF2 transfer into nuclear.What’s more,tBHQ activates both autophagy and NRF2 pathway to protect Primary spinal cord neurons from damage caused by 40μg/ml medical ozone in vitro. |
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