Regulation Of Human Degenerative Intervertebral Disc Nucleus Pulposus Cells Autophagy By SIRT1

PART ⅠISOLATION, VERIFICATION OF HUMAN INTERVERTEBRAL DISC NUCLEUS PULPOSUS TISSUEAND CULTURE OF NUCLEUS PULPOSUS CELLSObjective To master technique of isolation and culture of human intervertebral disc (IVD) nucleus pulposus (NP) cells.Methods Immunohistochemistry of Collagen Ⅱ and transmission electron microscope(TEM) were used to verify the human NP samples from lumbar vertebral fracture in young adults and intervertebral disc degenerative disease in elderly patients. NP samples were digested at 37 ℃ in 0.25% tyrisin for 30 minutes, following,3-4 hours in 2% type Ⅱ collagenase. Then the monolayer NP cells were cultured in DMEM/F12 containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37℃ in a humidified atmosphere containing 5% CO2. Then, the verification of human NP cells were performed by immunocytochemistry detection using Collagen Ⅱ antibody.Results Human NP tissues were isolated and verified by Immunohistochemistry staining using Collagen II antibody and TEM. Human nucleus NP were cultured successfully and verified according to its characterization under microscope and immunocytochemistry staining using Collagen Ⅱ antibody.Conclusion Human IVD NP tissues and cells were verified and cultured successfully.PART ⅡTHE EXPRESSION AND SIGNIFICANCE OF AUTOPHAGY ACTIVITY IN HUMAN NORMAL AND DEGENERATIVE INTERVERTEBRAL DSIC NUCLEUS PULPOSUS CELLSObjective To clarify the expression difference of autophagy activity and SIRT1 between normal and degenerative human NP cells.Methods The number of autophagosome was detected by TEM. The gene and protein expression of SIRT1, LC3 and Beclin-1 in normal and degenerative NP cells were investigated by real-time RT-PCR and Western Blot. The protein expression of SIRT1, Collagen Ⅱ and Caspase3 in normal and degenerative NP cells were investigated by western blot. TUNEL technology was used to detect the difference of apoptosis rate of NP cells between normal and degenerative NP cells.Results The autophagosome number, the mRNA level of LC3 and Beclin-1, the protein expression of LC3-Ⅱ/Ⅰ and Beclin-1, decreased in degenerative NP cells(P< 0.05). The incidence of apoptotic cell in degenerative NP was significantly greater compared with that in normal NP(P<0.05).Conclusion In degenerative NP, the expression of SIRT1 decreased, the autophagy activity reduced and the apoptosis rate of NP cells increased. There is a forward relationship between SIRT1 and autophagy activity, an inverse relationship between SIRT1 and cellular apoptosis and an inverse relationship between autophagy activity and cellular apoptosis in human NP cells.PART ⅢREGULATION OF HUMAN DEGENERATIVE INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS AUTOPHAGY BY SIRT1 AND ITS SIGNAL TRANSDUCTION MECHANISMObjective To investigate whether SIRT1 could regulate autophagy of degenerative NP cells and its molecular mechanism.Methods Resveratrol, nicotinamide and SIRT1-siRNA were used to treat degenerative NP cells and the protein expression of SIRT1, LC3 Ⅱ/Ⅰ and Beclin-1 were detected by western blot. The flow cytometry was used to detect the apoptosis rate of NP cells. After autophagy inhibition by bafilomycin A, the protein expression of LC3 Ⅱ/Ⅰ and cleaved caspase3 were detected by western blot and the apoptosis rate of NP cells was detected by flow cytometry. Finally, the expression of Akt and Erkl/2 were detected by western blot after treatment of LY294002 and PD98059 in NP cells.Results Western blot results revealed that LC3-II/I was significant higher in NP cells with treatment of both resveratrol and bafilomycin A than those treated with resveratrol alone (P<0.05), and it was also higher in NP cells with treatment of both resveratrol and bafilomycin A than those treated with bafilomycin A alone but without difference in statistics(P<0.05). Meanwhile, the expression of cleaved Caspase3 protein increased in NP cells with bafilomycin A in combination with or without resveratrol, as compared with those treated with resveratrol alone. Further results of flow cytometry also showed that the apoptotic incidence increased significantly in NP cells with bafilomycin A pretreatment independently whether resveratrol was added or not. Western blot results also showed that LY294002 could inhibit autophagy by suppressing Akt phosphorylation but resveratrol could partly reverse the effect of LY294002. PD98059 could partly inhibit autophagy by suppressing phosphorylation of Erkl/2 and resveratrol failed to override this effect.Conclusion SIRT1 inhibits human degenerative NP cells apoptosis via promotion of autophagy through the Erkl/2 pathway.