RÉSUMÉ
BACKGROUND:Intervertebral disc degeneration is a condition caused by the combined effects of various factors,such as cellular apoptosis,oxidative stress,and inflammatory responses.Currently,it is believed that the core of this condition lies in the degeneration and apoptosis of nucleus pulposus cells(NPCs).However,the specific pathological mechanisms remain unclear. OBJECTIVE:By investigating the expression of the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/hypoxia-inducible factor 1-alpha(HIF-1α)signaling pathway and the apoptosis of NPCs under different oxygen concentrations,to clarify the biological characteristics of NPCs under varying oxygen levels,and to explore the mechanisms of intervertebral disc degeneration. METHODS:Normal and degenerated NPCs were collected.The second-generation cells were used for imaging identification.The third-generation cells were cultured in the following conditions:37℃,air,100%humidity.Leibovitz's medium containing 100 μmol/L CoCl2 and 10%fetal bovine serum medium containing 100 μmol/L H2O2 were used to create distinct oxygen concentration environments for the third-generation cells,allowing for the investigation of cellular responses and behaviors under normal,low oxygen,and oxidative stress conditions.The third-generation cells were divided into six groups:normal NPCs+hypoxia,normal NPCs+normoxia,normal NPCs+oxidative stress,degenerated NPCs+hypoxia,degenerated NPCs+normoxia,and degenerated NPCs+oxidative stress groups.Cell viability and proliferation were assessed using the cell counting kit-8 method.Cell apoptosis was detected using flow cytometry.RT-PCR and western blot assay were utilized to examine the protein and mRNA expression levels of PI3K,AKT,and HIF-1α. RESULTS AND CONCLUSION:At different oxygen concentrations,the cell proliferation rate of both normal and degenerated NPCs decreased as the oxygen concentration increased.Conversely,the apoptosis rate increased as the oxygen concentration rose(P<0.05).With the normal NPCs+hypoxia group as the control group,the effect of degenerated NPCs on the apoptosis rate was highly significant(P<0.001).Oxygen concentration had a highly significant impact on both NPC proliferation rate and apoptosis rate(P<0.001).The interaction between cell degeneration and oxygen concentration significantly affected both NPC proliferation and apoptosis rates(P<0.05).At different oxygen concentrations,the protein and mRNA expression levels of PI3K,AKT,and HIF-1α in normal NPCs were highest under low oxygen concentration,followed by oxidative stress environment,and lowest under normoxia.In degenerated NPCs,the protein and mRNA expression levels of PI3K,AKT,and HIF-1α decreasd as the oxygen concentration increased.The protein and mRNA expression levels of PI3K and Akt in normal NPCs were significantly higher than those in degenerated NPCs(P<0.05).With the normal NPCs+hypoxia group as the control group,the effects of normal or degenerated NPCs,as well as oxygen concentration,on the protein and mRNA expression of PI3K,AKT,and HIF-1α were highly significant.The interaction between cell degeneration and oxygen concentration also had an extremely significant effect on the protein and mRNA expression of PI3K,AKT,and HIF-1α(P<0.001).To conclude,there is a close correlation between the proliferation and apoptosis of NPCs and both oxygen concentration and the cellular functional state.The PI3K/Akt/HIF-1α signaling pathway exhibits antagonistic regulatory effects under various cellular functional states and different oxygen concentration environments.The mechanism may be associated with the activation of the PI3K/Akt signaling pathway,which consequently transcribes HIF-1α,thus maintaining the balance of reactive oxygen species metabolism.This pathway plays a significant role in inhibiting oxidative stress,antagonizing NPCs apoptosis,and consequently delaying intervertebral disc degeneration.
RÉSUMÉ
BACKGROUND:JNKs and p38 MAPK, as important members of mitogen-activated protein kinase (MAPK), play critical roles in the cell proliferation, differentiation, transformation and apoptosis. OBJECTIVE:To observe the effects of Liuwei Dihuang Pill on the MAPK signaling cascade of nucleus pulposus cells in a rabbit model of intervertebral disc degeneration. METHODS:100 discs samples with endplate cartilages were equivalently randomized into five groups and cultured in the medium containing 10 mg/L tumor necrosis factor-α(TNF-α, 2μL), p38-JNK/SAPK blockers (10μL of 20μmol/L SB203580 and SP600125, 2μL of 10μmol/L TNF-α), 10%Liuwei Dihuang Pill serum, 10 mg/L TNF-α(2μL) plus 10%Liuwei Dihuang Pill serum, respectively. Those cells received no intervention as controls. Samples were collected and detected at 2, 4, 8 and 14 days of culture. RESULTS AND CONCLUSION:Real time-PCR and western blot assay showed that mRNA and protein expressions of JNK and P38 pathways by the nucleus pulposus cells were upregulated with time, while all above indicators were significantly decreased by the Liuwei Dihuang Pill serum. These findings reveal that Liuwei Dihuang Pill can delay the process of disc degeneration by blocking JNK and P38 signaling pathways, thus exerting protective effect on the intervertebral discs.
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BACKGROUND: Liuwei Dihuang Pills (LWPs), a famous Chinese prescription for replenishing the liver and kidney, have achieved good effect in the treatment of backache. However, the mechanism underlying LWPs treating backache with kidney deficiency is unclear. OBJECTIVE: To observe the effect of LWPs on extracellular matrix components of rabbit intervertebral disc degeneration models in vitro, and to explore the effect of LWPs in the prevention and treatment of intervertebral disc degeneration.METHODS: Totally 100 L1-6 intervertebral discs with endplate removed from 20 New Zealand rabbits were randomly divided into control, tumor necrosis factor α (TNF-α), p38-JNK/SAPK blocked, LWPs, and combination groups (n=20 per group). 2 μL of 10 mg/L TNF-α, 10 μL of 20 μmol/L SB203580 (specific inhibitor of p38MAPK) and SP600125 (inhibitor of JNK), 10% LWPs serum, 10% LWPs combined with 2 μL of 10 mg/L TNF-α were added into the medium of the corresponding group. Samples were collected at 2, 4, 6 and 14 days of culture. RESULTS AND CONCLUSION: The addition of TNF-α significantly upregulated the mRNA and protein expression levels of collagen type I, while significantly downregulated the levels of glycosaminoglycan and hyaluronic acid, chondroitin sulfate/keratan sulfate ratio, as well as the protein and mRNA expression levels of proteoglycan and collagen type Ⅱ (P < 0.05). LWPs could partly reverse the changes caused by TNF-α, indicating that LWPs can alleviate intervertebral disc degeneration to a certain extent.
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BACKGROUND:Proteoglycan, chondroitin sulfate and keratan sulfate, and hyaluronic acid are important substances in the maintenance of intervertebral discs of normal structure. They impact the physiological function of intervertebral disc. OBJECTIVE:To observe the influence of Liuwei Dihuang Wan on proteoglycan in rabbit models of intervertebral disc degeneration, and to study the efficacy of Liuwei Dihuang Wan on prevention and treatment of intervertebral disc degeneration. METHODS:80 New Zealand rabbits were randomly divided into Liuwei Dihuang Wan group, model group, sham surgery group and blank control group (n=20 per group). Animal models of intervertebral disc degeneration were established in the model and Liuwei Dihuang Wan groups. In the sham surgery group, surgery via the same approach to expose the lumbar spine was conducted fol owed by layer-by-layer suturing. In the Liuwei Dihuang Wan group, rabbits were given 10 mg/kg Liuwei Dihuang Wan by lavage, once a day. In the model group, sham surgery group and blank control group, rabbits were given an equal volume of physiological saline, once a day, by natural feeding. At 2, 4, 6 and 8 weeks, five rabbits from each group were sacrificed, and intervertebral disc specimens were taken to measure proteoglycan components in the intervertebral disc. RESULTS AND CONCLUSION:With the extension of feeding time, Liuwei Dihuang Wan could increase sugar amino polysaccharide content, chondroitin sulfate/acid ratio of keratin, and hyaluronic acid content in the intervertebral disc degeneration model, and stabilized the proteoglycan content. Thus, it delayed the degeneration of the intervertebral disc to a certain extent.
RÉSUMÉ
10.3969/j.issn.2095-4344.2013.26.015