The Role And Mechanism Of 17?-estradiol On Alleviating Apoptosis And Enhancing Matrix Biosynthesis Of Nucleus Pulposus Cells Under A High Glucose Condition

An epidemiological study has showed that the incidence of disc degeneration in diabetes mellitus(DM)patients is higher than that in non-DM patients.Emerging studies have proved that DM is a potential risk factor of disc degeneration.Previously,several studies have demonstrated that high glucose imparts detrimental effects to disc nucleus pulposus(NP)cells' biology through oxidative injury,such as increasing cellular apoptosis and decreasing NP matrix production.Recently,17?-estradiol(17?-E2)has been broadly proven not only for its improving insulin resistance but also for its function of suppressing cell apoptosis.However,the association between 17?-E2 and high glucose induced apoptosis of NP cells has not been reported.So,in the present study,we mainly aimed to investigate the role and mechanism of 17?-E2 in regulating NP cell apoptosis and NP matrix production under the high glucose condition.The estrogen receptor(ER)? inhibitor PHTPP and ER ? activator ERB041 were used to investigate the role of ER p.Additionally,intracellular reactive oxygen species(ROS)content was also evaluated.Part ? Isolation,culture and identification of the nucleus pulposus cells from rat intervertebral discObjective:The nucleus pulposus cells(NPCs)were isolated and extracted from rat intervertebral disc in vitro.NPCs were cultured in monolayer and determined characteristics by morphology,NP marker gene and matrix protein expression.Methods:NPCs were isolated by trysin plus type ? collagenase digestion in vitro and cultured with DMEM/F12 medium containing 10%(v/v)fetal bovine serum and 1%(v/v)penicillin-streptomycin under a standard condition.The phenotype of NPCs was characterized by morphology under an inverted microscope.Inmunocytochemical staining was performed to investigate the protein expression of aggrecan,type ? collagen and Sry-type HMG box 9(SOX9).We also evaluated the mRNA expression of CA12,FOXF1 and PTN by quantitative real-time PCR and agarose electrophoresis.Results:NPCs were isolated,eultured and identified in vitro successfully,which were observed in good grovvth status,showing long spindle shape under inverted microscope.These NPCs could positively express type ? collagen,aggrecan and SOX9.Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes.Conclusion:NPCs phenotype was successful identified with morphology,NP marker gene,key matrix proteins and the factor SOX9.Part ? The effects of 17?-estradiol on the cellular apoptosis and matrix biosynthesis of nucleus pulposus cells induced by high glucose in rat nucleus pujposus cellstranscriptionObjective:To investigate the role of 17?-E2 on influencing the cellular apoptosis and matrix biosynthesis of rat NPCs induced by high glucose,and whether it is mediated via ER p.Methods:Because subcultivation can lead to senescence of NPCs in vitro,the passage 2(P2)NPCs were used in the research,which were assigned into four groups:HG group(high glucose group,0.2 M high glucose concentration),HG+17E2 group(high glucose+17?-E2 group,0.2 M high glucose with 10-7 M 17?-E2),HG+17E2+ERB041 group(high glucose+17?-E2+ERB041 group,0.2 M high glucose with 10-7 M 17?-E2 and 10 ?M ER ? activator ERB041),HG+17E2+PHTPP group(high glucose+17?-E2+PHTPP group,0.2 M high glucose with-1-7 M 17?-E2 and 1 ?M ER p inhibitor PHTPP).The P2 NPCs were incubated with the above culture medium respectively for 72 h before each assay.Flow cytometry was used to measure the apoptotic incidence.Gene expression of apoptosis-related molecules(Bcl-2,Bax and Caspase-3)was evaluated by the real-time PCR assay.Protein expression of apoptosis-related molecules(Cleaved caspase-3 and Cleaved PARP)was evaluated by the Western blot assay.Gene expression of matrix molecules(aggrecan and collagen ?)and immunocytochemical analysis of matrix proteins(aggrecan and collagen ?)were used to analyze NP matrix production.Results:Compared with the control group,17?-E2 significantly decreased NP cell apoptosis ratio,down-regulated gene expression of pro-apoptosis molecules(Bax and Caspase-3)but up-regulated gene expression of anti-apoptosis molecule(Bcl-2),decreased protein expression of apoptosis markers(Cleaved caspase-3 and Cleaved PARP),up-regulated gene expression of matrix macromolecules(aggrecan and collagen ?),and increased protein deposition of these macromolecules.When the ER ? function was inhibited by PHTPP,these results were partly reversed compared with the NPCs treated with 17?-E2.Additionally,these effects of 17?-E2 not only on NP cell apoptosis but also on protein deposition and gene expression of the matrix macromolecules were further enhanced when the ER ? function was activated by ERB041.Conclusions:Compared with the control NPCs,17?-E2 decreased NP cell apoptosis ratio,down-regulated gene expression of Bax and Caspase-3,up-regulated gene expression of Bcl-2,decreased protein expression of Cleaved Caspase-3 and Cleaved PARP,and increased expression of matrix molecules(aggrecan and collagen ?).Further analysis showed that ER p inhibition partly reversed these effects of 17?-E2 whereas ER ?activation further promoted its effects.Part ? 17?-Estradiol inhibits high glucose-induced oxidative stress in rat nucleus pulposus cellsObjective:To investigate the role of 17?-E2 on influencing the oxidative damage of rat NPCs induced by high glucose,and whether it is mediated via ER ?.Methods:The NPCs were isolated and extracted from rat intervertebral disc,and cultured.Because subcultivation can lead to senescence of NPCs in vitro,the passage 2(P2)NPCs were used in the research,which were assigned into four groups:HG group(high glucose group,0.2 M high glucose concentration),HG+17E2 group(high glucose+17?-E2 group,0.2 M high glucose with 10-7 M 17?-E2),HG+17E2+ERB041 group(high glucose+17?-E2+ERB041 group,0.2 M high glucose with 10-7 M 17?-E2 and 10 ?M ER? activator ERB041),HG+17E2+PHTPP group(high glucose+17?-E2+PHTPP group,0.2 M high glucose with 10-7 M 17?-E2 and 1 ?M ER ? inhibitor PHTPP).The P2 NPCs were incubated with the above culture medium respectively for 72 h before each assay.Because it has been established that high glucose can deliver detrimental effects to NPCs through inducing an oxidative injury,we analyzed the intracellular ROS content to evaluate oxidative damage among these groups,which was measured by the method of DCFH-DA staining with a ROS detection kit.Results:Compared with the control group,17?-E2 significantly decreased intracellular ROS content.However,inhibitor PHTPP and activator ERB041 partly reversed and further enhanced the effects of 17?-E2 on ROS content,respectively.These results indicate that 17?-E2/ER ? interaction is helpful to attenuate oxidative damage under a high glucose condition.Conclusions:Compared with the control NPCs,17?-E2 decrease oxidative damage of NPCs via ER ? under a high glucose condition.