Study On The Signal Pathway And Related Mechanism Of Estrogens Inhibiting Apoptosis Induced By Interleukin-1 Beta In Rat NPCs

Part One Effect of Estrogen on interleukin-1 induced apoptosis in rat nucleus pulposus cells via mTOR / caspase-3 pathwayPurpose: Intervertebral disk degenerative(IVDD)is the main pathological basis for spinal degenerative diseases.Abnormal apoptosis of nucleus pulposus cells is the main cause of intervertebral disc degeneration.It has been reported that estrogen plays an anti-apoptosis role by activating protein kinase B(PKB/Akt).In our previous study,17β-estradiol(E2)was found to protect rats NPCs from apoptosis induced by interleukin-1β(IL-1β)through the PI3K/Akt signaling pathway.However,the downstream signal path of PI3K/Akt is not yet clear.mTOR is an important protein in the downstream pathway of PI3K/Akt.The aim of this study was to investigate whether the downstream signaling pathway mTOR of PI3k/Akt pathway is associated with estrogen inhibition of NPCs apoptosis in rats.Method: In this study,NPCs apoptosis induced by interleukin-1 beta was used as a model.In estrogen inhibition of NPCs apoptosis,we were application of related pathway inhibitor(Rapamycin)and observed the relationship between the relative activation levels of mTOR signaling pathway protein and the incidence of early apoptosis of NPCs and cell activity.The CCK-8 method determined the change trend of cell proliferation rate of NPCs between experimental and control groups at different time points and found optimal processing time.The MTS method is used to verify.The cell adhesion test was used to detect the adhesion ability of NPCs and type II collagen in each group.The incidence of early apoptosis of NPCs in each group was determined by Annexus V/PI double staining method.The protein and its phosphorylation expression levels of mTOR was determined by the Westernbolt experiment.The activation level of Caspase3 enzyme in each group was verified by the experiment of Caspase3 ase activity.The expression of cleaved caspase-3 protein was used for verification.Results: Estrogen inhibits the reduction of cell proliferation,cell activity,and adhesion caused by IL-1β cell toxicity,and reduces the early apoptosis rate of IL-1β-induced NPCs.In addition,in the Westernbolt experiment,E2 can also increase the relative expression of phosphorylated mTOR,while reducing the Caspaste3 enzyme activity and the protein expression level of cleaved caspase-3.The addition of mTOR pathway inhibitor,rapamycin,effectively reduced the cytoprotective effect of 17β-estradiol and increased the early apoptosis rate of NPCs(9.85 % ± 0.38 %).Furthermore,the relative expression of phosphorylated mTOR was reduced,and the activity of caspaste3 enzyme and the expression of cleaved caspase-3 were increased.Conclusion: The apoptosis of NPCs induced by interleukin 1β was inhibited by estrogen via mTOR/caspase-3 signaling pathway;Part Two Effect of Estrogen on interleukin-1 induced apoptosis in rat nucleus pulposus cells via GSK-3β/ caspase-3Purpose: Intervertebral disk degenerative(IVDD)is the main pathological basis for spinal degenerative diseases.Abnormal apoptosis of nucleus pulposus cells is the main cause of intervertebral disc degeneration.It has been reported that estrogen plays an anti-apoptosis role by activating protein kinase B(PKB/Akt).In our previous study,17β-estradiol(E2)was found to protect rats NPCs from apoptosis induced by interleukin-1β(IL-1β)through the PI3K/Akt signaling pathway.However,the downstream signal path of PI3K/Akt is not yet clear.GSK-3β is an important protein in the downstream pathway of PI3K/Akt.The purpose of this study was to investigate whether the downstream signaling pathway GSK-3β of PI3k/Akt pathway is associated with estrogen inhibition of NPCs apoptosis in rats.Method: In this study,NPCs apoptosis induced by interleukin-1 beta was used as a model.In estrogen inhibition of NPCs apoptosis,we were application of related pathway inhibitors(SC75741)and observed the relationship between the relative activation levels of GSK-3β signaling pathway protein and the incidence of early apoptosis of NPCs and cell activity.The CCK-8 method determined the change trend of cell proliferation rate of NPCs between experimental and control groups at different time points and found optimal processing time.The MTS method is used to verify.The cell adhesion test was used to detect the adhesion ability of NPCs and type II collagen in each group.The incidence of early apoptosis of NPCs in each group was determined by Annexus V/PI double staining method.The protein and its phosphorylation expression levels of GSK-3β were determined by the Westernbolt experiment.The activation level of Caspase3 enzyme in each group was verified by the experiment of Caspase3 ase activity.Results: Estrogen inhibits the reduction of cell proliferation,cell activity,and adhesion caused by IL-1β cell toxicity,and reduces the early apoptosis rate of IL-1β-induced NPCs.Furthermore,IL-1β increased the relative expression of phosphorylated GSK-3β in Western blot.The addition of a signal pathway GSK-3β inhibitor,SB216763,does not attenuate the cytoprotective effect of 17β-estradiol.At the same time,the early apoptosis rate of NPCs was also reduced(7.32 % ± 0.47 %),and the activity of caspaste3 enzymes was also decreased.Conclusions: 1.The signaling pathway GSK-3β/caspase-3 is not involved in estrogen-induced apoptosis of NPCs induced by interleukin-1β;2.GSK-3β signaling pathway may play a role in interleukin-1-induced apoptosis of NPCs.Part Three Effect of Estrogen on interleukin-1 induced apoptosis in rat nucleus pulposus cells via NF-κB / caspase-3Purpose: Intervertebral disk degenerative(IVDD)is the main pathological basis for spinal degenerative diseases.Abnormal apoptosis of nucleus pulposus cells is the main cause of intervertebral disc degeneration.It has been reported that estrogen plays an anti-apoptosis role by activating protein kinase B(PKB/Akt).In our previous study,17β-estradiol(E2)was found to protect rats NPCs from apoptosis induced by interleukin-1β(IL-1β)through the PI3K/Akt signaling pathway.However,the downstream signal path of PI3K/Akt is not yet clear.NF-κB is an important protein in the downstream pathway of PI3K/Akt.The aim of this study was to investigate whether the downstream signaling pathway NF-κB of PI3k/Akt pathway is associated with estrogen inhibition of NPCs apoptosis in rats.Method: In this study,NPCs apoptosis induced by interleukin-1 beta was used as a model.In estrogen inhibition of NPCs apoptosis,we were application of related pathway inhibitor(SC75741)and observed the relationship between the relative activation levels of NF-κB(P65)signaling pathway proteins and the incidence of early apoptosis of NPCs and cell activity.The CCK-8 method determined the change trend of cell proliferation rate of NPCs between experimental and control groups at different time points and found optimal processing time.The MTS method is used to verify.The cell adhesion test was used to detect the adhesion ability of NPCs and type II collagen in each group.The incidence of early apoptosis of NPCs in each group was determined by Annexus V/PI double staining method.The protein and its phosphorylation expression levels of NF-κB(P65)were determined by the Westernbolt experiment.The activation level of Caspase3 enzyme in each group was verified by the experiment of Caspase3 ase activity.Results: Estrogen inhibits the reduction of cell proliferation,cell activity,and adhesion caused by IL-1β cell toxicity,and reduces the early apoptosis rate of IL-1β-induced NPCs.The addition of a signal pathway NF-κB inhibitor,SC75741,can not inhibit the cytoprotective effect of 17β-estradiol.At the same time,the early apoptosis rate(7.76 % ± 0.48 %)and caspaste3 enzyme activity of medulla cells were reduced.However,with the addition of estrogen and pathway inhibitor,there was no corresponding change in the relative expression level of phosphorylated P65.Conclusion: The signaling pathways NF-κB / caspase-3 did not participate in estrogen inhibiting apoptosis of NPCs induced by Interleukin-1β.