Trehalose Activates Autophagy To Repair The Nucleus Pulposus Cell Degeneration Caused By Oxidative Stress

Objective(s):Intervertebral disc degeneration(IVDD)is a degenerative pathological change caused by aging,abnormal mechanical stress,inflammation and so on.It could lead to narrowing of the vertebral space,secondary spinal stenosis,and symptoms of nerve compression,such as pain,numbness,weakness and lameness,which seriously affect quality of life and working ability for patients.However,nowadays treatments of the IVDD-related diseases focus on relieving symptoms and do not stop,delay or reverse the progression of the disease.Therefore,it is urgent to find an effective intervention therapy for IVDD.Autophagy plays an important role in a variety of degenerative diseases,and autophagy disorder is also found to be closely related to its pathological process in IVDD.Trehalose is a new autophagy activator,which can inhibit the pathological process of many diseases by activating autophagy.This study aims to explore the role of trehalose on autophagy in nucleus pulposus cells through cell experiments.Further,researchers plan to observe the influence of trehalose pretreatment on TBHP-induced nucleus pulposus cell degeneration,and finally systematically elaborate the impact and molecular mechanism of trehalose in IVDD,clarify the regulatory mechanism of autophagy in IVDD,and provide ideas and directions for the development of precise treatment of IVDD.Methods:(1)Rat primary nucleus pulposus cells were extracted and digested by trypsin combined with type Ⅱ collagenase,and identified by morphological observation and type Ⅱ collagen staining;(2)A vitro model of IVDD was established by using tertbutyl hydrogen peroxide(TBHP)to induce nucleus pulposus cell degeneration,which were divided into control group and TBHP group;(3)The effects of TBHP and trehalose on NP cell viability were determined by MTT method;(4)The effect of TBHP on ECM degradation in IVDD was analyzed by Western blotting;(5)Hoechst33342 and TUNEL reagent were used to verify the effect of TBHP on apoptosis of IVDD cells;(6)ROS probe was used to detect the degree of oxidative stress damage in control group and TBHP group;(7)The influence of trehalose on the autophagy level of NP cells was measured by Western blotting,and the influence of trehalose on the whole process of autophagy of NP cells was determined by combining with autophagy inhibitors;(8)NP cells cultured in vitro were divided into 4 groups: control group,trehalose alone treatment group,TBHP group,trehalose +TBHP group.The changes of autophagy,cell metabolism,oxidative stress,apoptosis and ECM degradation of NP cells were determined by Western blotting,real-time quantitative PCR,immunofluorescence,MTT,ROS assay,Hoechst33342 staining and TUNEL assay;(9)The relationship between the role of trehalose in IVDD and autophagy was detected after the combination of autophagy inhibitor 3-MA.Results:(1)Primary nucleus pulposus cells were successfully extracted from rats and verified by cell morphology and molecular biology methods(type II collagen staining).It was found that both the extracted primary cells and P4 cells grew adherently,mainly in short spindle shape and polygon shape,with full form and clear boundary.At the same time,90% of the primary and P4 generation cells were positive for type II collagen staining,which indicated that the extracted cells were the primary nucleus pulposus cells,and the P4 generation cells grew as well as the primary cells and did not exhibit abnormal differentiation.With 25μM TBHP treatment,the nuclear chromatin condensation was observed after 6 hours,ROS probe green fluorescence was significantly enhanced,matrix protein col2a1 expression was down-regulated,and MMP13 expression was up-regulated,suggesting that TBHP could induce oxidative stress damage,apoptosis and ECM degradation in nucleus pulposus cells,leading to degeneration of nucleus pulposus cells.The pathological changes of IVDD were successfully simulated in vitro.(2)Trehalose(<200m M)had no effect on nucleus pulposus cell activity,and trehalose(10m M for 6h)could reverse TBHP-induced cell viability reduction by cell viability analysis;Compared with the blank group,the expression of autophagy related protein LC3 II was significantly up-regulated in the trehalose treatment group.Meanwhile,LC3 II expression was up-regulated and p62 expression was downregulated after 6h treatment with 10 m M trehalose,which indicated that trehalose had a good autophagy promoting activity in nucleus pulposus cells and the whole autophagy process of nucleus pulposus cells was activated under this condition;Compared with the TBHP group,LC3 II expression was up-regulated and p62 expression was down-regulated in the trehalose +TBHP group,indicating that trehalose could restore the impaired autophagy caused by TBHP.(3)Compared with the TBHP group,trehalose +TBHP group showed that apoptotic cells,ROS probe green fluorescence intensity and MMP13 expression decrease,but col2a1 expression increase.These results indicated that trehalose could inhibit oxidative stress,apoptosis and ECM degradation in IVDD.Compared with trehalose +TBHP group,there were more apoptotic cells,increased ROS,decreased col2a1 expression and increased MMP13 expression in 3-MA+ trehalose +TBHP group,indicating that the protective effect of trehalose for IVDD was weakened after inhibition of autophagy.Conclusion(s):(1)TBHP can induce oxidative stress injury leading to degeneration of nucleus pulposus cells,which successfully mimics the pathological changes of IVDD in vitro;(2)Trehalose can activate the autophagy of nucleus pulposus cells and restore the impaired autophagy of nucleus pulposus cells due to oxidative stress;(3)Trehalose can inhibit nucleus pulposus cell degeneration and alleviate IVDD by activating autophagy to inhibit apoptosis,oxidative stress and ECM degradation.