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ObjectiveTo explore the mechanism of Zhuangyao Tongluo Formula（壮腰通络方，ZTF） in delaying intervertebral disc degeneration. MethodsM1 macrophages were induced from THP-1 cells using LPS， IFN-γ and PMA. The induced M1 macrophages were then co-cultured with nucleus pulposus cells in a transwell system. Fetal bovine serum was used as the control serum， and the effects of different concentrations （5%， 10%， 15%， 20%） of serum from rats treated with ZTF on the activity of M1 macrophages and nucleus pulposus cells were analyzed using MTT assay. Experiment 1 was established， including the nucleus pulposus cell control group， M1 macrophage control group， nucleus pulposus cell + ZTF group， nucleus pulposus cell + TNF control group， nucleus pulposus cell + TNF + ZTF group， co-culture group， and co-culture + ZTF group. The levels of IL-1β， and IL-18 in the culture supernatant were detected using ELISA. The mRNA expression of IL-1β and IL-18 in nucleus pulposus cells was detected using qPCR. Additionally， the expression of GSDMD protein in nucleus pulposus cells was detected using cell immunofluorescence. In experiment 2， co-culture groups were constructed using TNF-α overexpression （OE） or empty vector （EV） plasmids， including co-culture group， TNF-EV + co-culture group， TNF-EV co-culture group + ZTF， co-culture + ZTF group， TNF-OE co-culture group + ZTF， and TNF-OE + co-culture group. The mRNA and protein expression of TNF-α in M1 cells in each group were detected using qPCR and WB. ResultsThe ZTF with 10% serum was selected for subsequent experiments. The results of experiment 1 showed that compared to the control group of nucleus pulposus cells， there was no statistically significant difference in the levels of IL-1β， IL-18， mRNA， and GSDMD expression in the nucleus pulposus cells + ZTF group （P＞0.05）. However， the TNF-α + co-culture group showed a significant increase in IL-1β， IL-18 levels， mRNA， and GSDMD expression （P＜0.01）. When compared to the co-culture group， the ZTF+ co-culture group showed a significant decrease in IL-1β， IL-18 levels， mRNA， and GSDMD expression （P＜0.01）. The results of experiment 2 showed that there was no statistically significant difference in TNF-α mRNA and protein expression between the empty vector plasmids + co-culture group and the co-culture group （P＞0.05）. Compared to the empty vector + co-culture group， the expression of TNF-α mRNA and protein was significantly reduced in the empty vector co-culture + ZTF group （P＜0.01）. Compared to the co-culture group and the empty vector + co-culture group， the expression of TNF-α mRNA and protein was significantly reduced in the co-culture + ZTF group （P＜0.01）. Compared to the co-culture + ZTF group， the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture + ZTF group （P＜0.01）. Compared to the overexpression vector co-culture + ZTF group， the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture group （P＜0.01）. ConclusionZTF serum can inhibit the TNF-α-induced apoptosis of nucleus pulposus cells and delay lumbar disc degeneration by reducing the expression of TNF-α in M1 macrophages.
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This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.
Subject(s)
Humans , NF-kappa B/metabolism , Interleukin-10 , Nucleus Pulposus/metabolism , Tumor Necrosis Factor-alpha/metabolism , Aggrecans/metabolism , Toll-Like Receptor 4/metabolism , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Inflammatory microenvironment of nucleus pulposus cells is an important factor to promote nucleus pulposus degeneration. Inflammatory factors amplify the cascade of inflammation by activating the nuclear factor-κB signaling pathway to form a vicious cycle and accelerate the process of nucleus pulposus degeneration. It could delay the progression of intervertebral disc degeneration by inhibiting the activity of nuclear factor-κB signaling pathway. OBJECTIVE: To investigate the mechanism of Pheretima extract on nucleus pulposus cell degeneration. METHODS: Pheretima active ingredients were extracted by cold dipping. The cell counting kit-8 method was used to detect the effects of 0, 25, 50, 100, 200, 400 mg/L Pheretima extract on rat nucleus pulposus cell activity, and then suitable drug concentrations were chosen for following research. Tumor necrosis factor-α (TNF-α) was used to stimulate nucleus pulposus cells to induce the nucleus pulposus cell degeneration model. Pheretima extracts (50, 100, 200 mg/L) were used to treat the cell model. Western blot was used to detect the expression of Aggrecan, Collagen II, TNF-α and interleukin-6. RT-qPCR was used to detect mRNA expression of TNF-α and interleukin-6. Immunofluorescence was used to detect the nuclear expression of P65 in the nucleus pulposus cells. RESULTS AND CONCLUSION: Compared with the blank group (0 mg/L), 400 mg/L Pheretima extract inhibited the activity of nucleus pulposus cells, and 50, 100, 200 mg/L extracts of Pheretima were used for subsequent experimental research. Compared with the normal group, the expression of Aggrecan and Collagen II in the model group was reduced, the protein and mRNA expression levels of TNF-α and interleukin-6 were up-regulated, and P65 expression increased in the nucleus. Compared with the model group, Pheretima extract could increase the expression of Aggrecan and Collagen II, down-regulate the protein and mRNA levels of TNF-α and interleukin-6, and reduce P65 expression in the nucleus. To conclude, Pheretima extract can reduce the expression of inflammatory factors, increase the synthesis of extracellular matrix and inhibit nuclear factor-κB pathway activity. Pheretima extract may ameliorate nucleus pulposus cell degeneration in the intervertebral disc via the nuclear factor-κB signaling pathway.
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BACKGROUND: Compressive stress can change the morphology and activity of cells, but whether the morphology and activity of nucleus pulposus cells change under hydrostatic pressure still needs further study. OBJECTIVE: To study the morphology and activity of human nucleus pulposus cells in vitro. METHODS: The human nucleus pulposus cells were separated, cultured and passed on for three generations, and pressurized for 2, 4 and 6 hours under the hydrostatic pressure of 0.3, 1, and 3 MPa. Then, the morphological changes and growth of the cells before and after pressurization were observed by inverted phase contrast microscope. Transmission electron microscope was used to observe the ultrastructural changes and differences of the cells. Cell counting kit-8 was used to detect the proliferation activity, morphology and activity of the cells under different hydrostatic pressures. RESULTS AND CONCLUSION: (1) Cell culture and passage: The growth curves of the first, second and third generations of human nucleus pulposus cells were S-shaped, and the cells proliferated fastest in a straight line from 3 to 7 days. The protuberances of the 5th and 6th generation cells were long shuttle shaped, grew slowly and degenerated. (2) Cell morphology: the human nucleus pulposus cells were shrunk under hydrostatic pressures of 0.3, 1, 3 MPa. At 0.3 and 1 MPa, the cells became slightly smaller and the morphology was basically complete; at 3 MPa, the cells were most obviously shrunk and the morphology was incomplete. The results showed that when the human nucleus pulposus cells were pressurized for 2, 4 and 6 hours under 0.3, 1 and 3 MPa hydrostatic pressures, the change of cell morphology was the most obvious under 3 MPa hydrostatic pressure, but there was no obvious change under the same hydrostatic pressure for different time. (3) Cell viability: Under 0.3 MPa hydrostatic pressure, the proliferation rate of human nucleus pulposus cells first increased and then decreased with the increase of time, and the cell proliferation rate reached the peak at 4 hours. Under 1 and 3MPa hydrostatic pressures, the proliferation rate of the cells gradually decreased with the increase of time, and the cell proliferation rate under 1 MPa hydrostatic pressure was significantly higher than that under 3 MPa hydrostatic pressure at the same action time (P < 0.05). These findings indicate that proper hydrostatic pressure stimulation helps to promote the proliferation of human nucleus pulposus cells, and long-term improperly high hydrostatic pressure stimulation can reduce the proliferation rate of human nucleus pulposus cells, leading to the occurrence of intervertebral disc degeneration.
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Lumbar disc herniation is a common disease characterized by the degeneration of intervertebral discs (IVDs), accompanied by imbalance of metabolic and inflammatory homeostasis. Current studies establish that IVD degeneration is induced by increased apoptosis of nucleus pulposus (NP) cells. However, the underlying mechanisms of NP cell survival/apoptosis are not well elucidated. Here, we reveal a novel mechanism by which mTORC1 signaling controls NP cell survival through regulating metabolic homeostasis. We demonstrated that hyperactivated mTORC1 activity induced by inflammatory cytokines engenders the apoptosis of NP cells, whereas pharmacological inhibition of mTORC1 activity promotes NP cell survival. Using an integrative approach spanning metabolomics and biochemical approaches, we showed that mTORC1 activation enhanced glucose metabolism and lactic acid production, and therefore caused NP cell apoptosis. Our study identified mTORC1 in NP cells as a novel target for IVD degeneration, and provided potential strategies for clinical intervention of lumbar disc herniation.
Subject(s)
Humans , Intervertebral Disc Degeneration/drug therapy , Nucleus Pulposus , Apoptosis , Mechanistic Target of Rapamycin Complex 1 , Inflammation/drug therapyABSTRACT
BACKGROUND: The relationship between nuclear factor (NF)-kB signaling pathway and intervertebral disc degeneration is a hotspot in the field of orthopedics. In-depth investigation on various signaling pathways in the intervertebral disc contributes to understanding the mechanism of intervertebral disc degeneration. OBJECTIVE: To investigate the regulation of the serum containing Yishen Huoxue Tongluo Recipe on the NF-kB signal pathway of human intervertebral disc nucleus pulposus cells under different hydrostatic pressures, attempting to explore the possible therapeutic mechanism and target of Yishen Huoxue Tongluo Recipe in the treatment of degenerative diseases of the intervertebral disc from the perspective of molecular biology. METHODS: Passage 3 Human intervertebral disc nucleus pulposus cells were divided into eight groups and were cultured in the drug-containing serum of Yishen Huoxue Tongluo Recipe. The cells were intervened for 2, 4, and 6 hours under different hydrostatic loading conditions (0.3,1, and 3 MPa). The morphology and growth of nucleus pulposus cells were observed by an inverted phase contrast microscope. Ultrastructural changes of intervertebral disc nucleus pulposus cells were observed by a transmission electron microscopy. Cell counting kit-8 method was used to detect the proliferation activity of nucleus pulposus cells. Annexin V-FITC/Propidium Iodide apoptotic kit was used to double-stain nucleus pulposus cells to detect the cell apoptotic rate. Western blot method was used to detect the changes of NF-kB p65, Collagen II, ADAMTS-4, MMP-13 and Caspase-3 in nucleus pulposus cells. RESULTS AND CONCLUSION: (1) Under the same pressure and time, the morphology and growth of nucleus pulposus cells in the pressure+drug-containing serum groups were better than those in the normal pressure group and the simple pressure groups. Among them, the 0.3 and 1 MPa pressure+drug-containing serum groups had more intact cell morphology and better cell growth than the 3 MPa pressure+ drug-containing serum group. (2) The proliferative activity of nucleus pulposus cells was higher in the pressure+drug-containing serum group, and there was a significant difference between the 0.3 MPa pressure+drug-containing serum group and the 0.3 MPa simple pressure group (P < 0.05). (3) The apoptotic rate of nucleus pulposus cells in the pressure+drug-containing serum group was lower than that in the normal pressure group and simple pressure intervention group (P < 0.05). (4) The expression of Collagen II and Caspase-3 increased in the pressure+drug-containing serum group, while the expression of NF-kappa B p65, ADAMTS-4 and MMP-13 decreased in the pressure+drug-containing serum group (P < 0.05). To conclude, Yishen Huoxue Tongluo Recipe can increase cell activity, reduce cell apoptosis and effectively delay the degeneration of nucleus pulposus cells. Its mechanism is likely to promote the expression of Collagen II and Caspase-3 through the NF-kB signaling pathway of nucleus pulposus cells of intervertebral disc, and inhibit the expression of NF-kB p65, ADAMTS-4 and MMP-13.
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BACKGROUND: Capparis spinosa total alkaloids (CSTA) have certain effects on cell growth and extracellular matrix synthesis. The aging and apoptosis of nucleus pulposus cells are one of the main pathologies of intervertebral disc degeneration. Therefore, it is assumed that CSTA may have certain effect on the degeneration of the intervertebral disc. OBJECTIVE: To study the effect of CSTA on intervertebral disc degeneration rat model and nucleus pulposus cells. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into sham, model, CSTA-H, and CSTA-L groups with eight in each group. Rat models of intervertebral disc degeneration were made in the model group, CSTA-L group and CSTA-H group. The CSTA-L and CSTA-H groups were given intragastric administration of CSTA 225 mg/kg/d and 450 mg/kg/d for 4 weeks respectively. Hematoxylin-eosin staining was used to observe the pathological changes of the intervertebral disc. Immunohistochemistry and western blot were used to detect the expression of type II collagen and aggrecan. Nucleus pulposus cells from the intervertebral disc of another two Sprague-Dawley rats were separated, cultured and divided into a control group, an oxygen-glucose deprivation (OGD) group and an administration group (OGD+CSTA 10 mg/L). After being cultured for 24 hours, the morphology of nucleus pulposus cells was observed, the cell proliferation ability was detected by cell counting kit-8, the cell apoptosis was detected by flow cytometry, and the expression of type II collagen and aggrecan were detected by western blot. RESULTS AND CONCLUSION: (1) Compared with the sham group, the intervertebral disc tissue of the model group showed fiber ring fissures, aggregation and shrinking of nucleus pulposus cells, and different improvements were found in the CSTA-L and CSTA-H groups. (2) The expression levels of type II collagen and aggrecan in the model group were significantly lower than those in the sham, CSTA-L and CSTA-H groups (P < 0.05). (3) Compared with the control group, the cells in the OGD group showed irregular morphology and death status, whereas the cell morphology in the administration group was improved. (4) Compared with the control group, nucleus pulposus cells in the OGD group showed lower proliferation, higher apoptotic rate, and lower levels of type II collagen and aggrecan (P < 0.05). Compared with the OGD group, nucleus pulposus cells in the administration group showed faster proliferation, lower apoptotic rate, and higher levels of type II collagen and aggrecan. To conclude, CSTA can improve intervertebral disc degeneration by promoting proliferation and inhibiting apoptosis of nucleus pulposus cells as well as inhibiting the degradation of extracellular matrix.
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BACKGROUND: Cyclic RNA plasmacytoma variant translocation 1 (circPVT1) is involved in the senescence of fibroblasts, but the relationship of circPVT1 with nucleus pulposus senescence and its mechanism are still unclear. OBJECTIVE: To investigate the expression of circPVT1 in nucleus pulposus cell senescence and to explore its possible mechanism. METHODS: Human nucleus pulposus cells were cultured in vitro, and the senescence of nucleus pulposus cells was induced by ionizing radiation (5 Gy, 6 days). The expression of circPVT1 and let-7 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). CircPVT1 siRNA and anti-let-7 were transfected into normal nucleus pulposus cells, which were divided into control group, si-NC+anti-NC group, si-circPVT1+anti-NC group, si-NC+anti-let-7 group, and si-circPVT1+anti-let-7 group. The expressions of circPVT1 and let-7 mRNA were detected by qRT-PCR. Cell counting kit-8 assay was used to detect the inhibition of cell proliferation. Plate cell clone formation assay was used to detect colony formation. Cell senescence was detected by SA-β-gal staining. The expressions of p21, p27, let-7 target high mobility group protein A2 (HMGA2) and KRAS were detected by western blot assay. Double luciferase activity assay was used to verify the relationship between let-7 and target regulation of HMGA2 and KRAS. RESULTS AND CONCLUSION: (1) Compared with normal nucleus pulposus cells, the expression of circPVT1 was decreased, while let-7 expression and the positive rate of SA-β-gal staining were increased in the irradiated cells (P < 0.05). (2) Compared with the control group and si-NC+anti-NC group, the si-circPVT1+anti-NC group appeared to have decreased expression of circPVT1 mRNA, HMGA2 and KRAS proteins and number of clones formed as well as increased let-7 mRNA expression, p21, p27 protein expression, cell inhibition rate and positive rate of SA-β-gal staining (P < 0.05). However, opposite changes were found in the si-NC+anti-let-7 group in relative to the control group (P < 0.05). (3) The expression of circPVT1 mRNA, clone formation, and expressions of HMGA2 and KRAS proteins in the si-circPVT1+anti-let-7 group were higher than those in the si-circPVT1+anti-NC group, and lower than those in the si-NC+anti-let-7 group. Let-7 mRNA expression, cell inhibition rate, positive rate of SA-β-gal staining, and expressions of p21 and p27 proteins in the si-circPVT1+anti-let-7 group were lower than those in the si-circPVT1+anti-NC group, and higher than those in the si-NC+anti-let-7 group (P < 0.05). Double luciferase activity assay showed that HMGA2 and KRAS were the targets of let-7. These findings indicate that inhibition of circPVT1 can inhibit the aging of nucleus pulposus cells. The mechanism may be through binding let-7 to inhibit the targeting of HMGA2 and KRAS proteins.
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BACKGROUND: Previous studies have found that continuous loading pressure can induce intervertebral disc degeneration by reducing the number of nucleus pulposus cells and reducing the expression of extracellular matrix in the nucleus pulposus. However, its mechanism of action is unclear. OBJECTIVE: To study the effect of continuous loading pressure on apoptosis and Wnt/β-catenin signaling pathway in nucleus pulposus cells. METHODS: The in vitro models of rabbit spinal motion segment under loading pressure were established and divided into control group (no treatment), pressure group (3 kg continuous loading pressure) and observation group (3 kg continuous loading pressure + 20 μmol/L SB216763). After 3 days of intervention, the pathological changes of nucleus pulposus cells were detected by hematoxylin-eosin staining, and the expression of caspase-3 and GSK-3β protein in nucleus pulposus tissue was detected by western blot assay.
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Objective: To investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro. Methods: The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO 2, 20%O 2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO 2, 1%O 2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups. Results: HE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A ( P0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B ( P<0.05). Conclusion: Hypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
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Objective@#To explore the expression of the A2A adenosine receptor and the inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in human degenerative nucleus pulposus (NP) cells after they have been treated with a pulsed electromagnetic field (PEMF).@*Methods@#Human degenerative NP cells were cultured in vitro and treated using an 0.8mT PEMF with a pulse frequency of 50Hz. The pulse width was 150μs and the exposure time was 30min, repeated 5 times at 12 hour intervals. The expression of the A2A adenosine receptor in NP cells was determined using western blotting and reverse transcription polymerase chain reactions. The expression of the inflammatory cytokines IL-1β and TNF-α were detected using enzyme-linked immunosorbent assays (ELISA). The human degenerative NP cells were also treated with an antagonist and agonist of the A2A adenosine receptor, and the expression of IL-1β and TNF-α were also determined using ELISA.@*Results@#After the PEMF treatment the expression of the A2A adenosine receptor increased significantly, while the expression of IL-1β and TNF-α decreased significantly. However, the A2A adenosine receptor antagonist reversed the inhibitory effect of the PEMF on the expression of IL-1β and TNF-α, while the agonist played an opposite role.@*Conclusion@#A PEMF can significantly inhibit the expression of IL-1β and TNF-α in human degenerative NP cells, which could be related to up-regulation of the expression of the A2A adenosine receptor in those cells.
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Objective Overexpressed inflammatory factors play an important role in the process of intervertebral disc degeneration. This study aimed to investigate the effect of iguratimod on the expression of inflammatory factors in degenerative intervertebral disc cells. Methods Sixty 8-12 weeks old SD rats were equally randomized into a compression (the tail compressed by external fixation) and a non-compression control group. The nucleus pulposus cells (NPC) of the degenerated intervertebral disc were isolated and treated with iguratimod at the concentrations of 0, 0.3, 3, 10, 20, and 30 μg/mL, followed by measurement of the contents of inflammatory factors and matrix metalloproteinases (MMP) secreted from the NPCs and determination of the effects of different concentrations of iguratimod on the expressions of inflammation-related genes in the NPCs by RT-PCR. Results After treatment with iguratimod at 3, 10, 20, and 30 μg/mL, the expression levels of IL-6 in the NPCs were (204.18 ± 6.96), (122.73 ± 9.38), (97.87 ± 7.81), and (86.31 ± 8.57) pg/mL, respectively, and those of TNF-α were (202.46 ± 7.84), (132.52 ± 11.4), (101.26 ± 10.38), and (96.89 ± 9.60) pg/mL, respectively, all decreased significantly in a concentration-dependent manner (P < 0.05). Meanwhile, the contents of MMP-2, MMP-3 and MMP-9 in the iguratimod-treated NPCs also showed remarkable concentration-dependent decreases (P < 0.05). Conclusion Iguratimod can effectively inhibit the expression of inflammatory factors in nucleus pulposus cells and block the progression of inflammatory response, which has provided a new idea for the treatment of degenerative intervertebral disc disease.
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Objective: To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism.
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Methods: Ten patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot.
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AIM:To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells(HNPCs).METHODS:Cultured HNPCs were subjected to oxygen-glucose deprivation(OGD)to mimic the micro-environment of degenerative HNPCs.The morphological changes of the cells in control group and OGD group were observed under optical microscope.The cells were treated with ginsenoside Rg 1 at concentrations of 25,50 and 100 μmol/L.The expression of collagen II and aggrecan at mRNA and protein levels was determined by real -time PCR and Western blot analysis.The cell viability was measured by CCK-8 assay.The mRNA level of Ki67 was detected by real-time PCR.The apoptosis was analyzed by flow cytometry.The activity of caspase-3 was measured by a caspase-3 kit.The ex-pression of Wnt/β-catenin pathway-related proteins was determined by Western blot.Furthermore,the expression of Wnt/β-catenin pathway-related proteins,the cell viability and apoptosis,and the expression of extracellular matrix synthesis pro-teins were assessed after the cells were co-treated with LiCl and 100 μmol/L ginsenoside Rg1.RESULTS:Normal HNPCs attached on the cell culture plate faster, and were almost round with rich cytoplasm.However, the cell adherence was slower,and the cells were long fusiform with decreased cytoplasm after OGD treatment,indicating that the model of degen-erative HNPCs was successfully established.Compared with normal HNPCs,the expression of collagen II and aggrecan at mRNA and protein levels was decreased in OGD group(P<0.05),which was then increased after the cells were treated with ginsenoside Rg1 at 25,50 and 100 μmol/L(P<0.05).Compared with normal HNPCs,the cell viability and Ki67 expression were decreased in OGD group(P<0.05), which were increased after treatment with ginsenoside Rg 1(P<0.05).Meanwhile,the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells(P<0.05), which were decreased after treatment with ginsenoside Rg 1(P<0.05).In addition,the activation of Wnt/β-catenin path-way was also inhibited by ginsenoside Rg 1 treatment at dose of 100 μmol/L(P<0.05).LiCl,a Wnt/β-catenin pathway agonist,obviously decreased the protective effects of ginenoside Rg 1 on OGD-induced cells(P<0.05),indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg 1 on degenerative HNPCs.CONCLUSION:Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt /β-cate-nin pathway.This study will provide a new idea for prevention and treatment of degenerative HNPCs.
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Objective Normal nucleus pulposus cells (nNPCs) can induce the differentiation of mesenchymal stem cells (MSCs).However, the inductive effect of degeneration nucleus pul-posus cells ( dNPCs ) on MSCs has not been determined .This study aimed to compare nNPCs with dNPCs in inducing MSCs into nucleus pulposuslike cells. Methods MSCs (cell line) were co-cultured with NPCs (cell line/isolated from patients with intervertebral disc degeneration) in 3D in vitro.The cells were divided into 5 groups:nNPCs control group, dNPCs control group, MSCs control group, nNPCs-MSCs group (MSCs induced by nNPCs), dNPCs-MSCs group (MSCs induced by dNPCs).After co-cultured for 7 days, the expression level of aggrecan (ACAN), type II collagen (COL-2), SOX-9, matrix metalloproteinase-1 (MMP-1) and MMP-3 in all the 5 groups'cellswere detected by RT-PCR and Western-blot. Results The RT-PCR results showed the expression of ACAN , COL-2 and SOX-9 in nNPCs-MSCs group was higher than that in dNPCs control group and dNPCs -MSCs group (all P<0.05).The expression of MMP-1 and MMP-3 in nNPCs-MSCs group and dNPCs-MSCs group was lower than that in dNPCs control group ( all P<0.01) , but there was no difference when compared with nNPCs control group and MSCs control group (all P>0.05).Western-blot analysis showed that the expression of ACAN , COL-2 and SOX-9 in nNPCs-MSCs group was higher than that in dNPCs control group and dNPCs-MSCs group (all P<0.05).The expression of MMP-1 and MMP-3 in nNPCs-MSCs group and dNPCs-MSCs group was lower than that in nNPCs control group and dNPCs control group ( all P<0.01) , but there was no difference compared with MSCs control group ( all P>0.05) . Conclusion Both nNPCs and dNPCs could induce the differentiation of MSCs into nucleus pulposus cells .After co-cultured with nNPCs, the expression level of ACAN, COL-2 and SOX-9 of MSCs was similar to that of nNPCs, which was superior to the induc-tion of MSCs by dNPCs under the same culture conditions .
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Objective To find the relationship between nuclear factor kappB (NF-κB) activation and cell apoptosis.Methods Degenerative human nucleus pulposus cells were cultured in vitro.Useing CCK-8 to observe the proliferative effect of LPS on the nucleus pulposus cells in vitro, in the concentration of 100, 200, 500 and 1 000 μg/mL and choose the most apropriate concentration.The experiment was divided into blank control group, LPS(500 μg/mL)groups, and NF-κB inhibitor(BAY11-7082)plus LPS(500 μg/mL)group,Annexin V-FITC/PI flow cytometry and Hoechest33258 staining was used to analyze apoptosis.The expression of cleaved caspase-3,PARP,P65,P-P65 proteins were detected by Western blot respectively.Results When LPS was 500 μg/mL, the cell vitality was obviously declined.Compared with the blank control group, cell apoptosis rate of the LPS group is increasing (P<0.05), and the expression of P-P65,cleaved caspase-3, cleaved PARP were alsohigher (P< 0.05).Compared with the LPS group, cell apoptosis rate of the NF-κB inhibitor plus LPS group is significantly lower (P<0.05) and the expression of P-P65,cleaved caspase-3,cleaved PARP were also lower (P< 0.05).Conclusions NF-κB signaling pathway may be associated with the nucleus pulposus cell apoptosis in disc degeneration.
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Objective To investigate the role of SDF-1/CXCR4 axis on the apoptosis of human degenerative nucleus pulposus cells (NPCs) and its potential molecular mechanism .Methods The intervertebral disces tissues from clinical discectomy were divided into normal group and intervertebral disc degeneration ( IVD) group according to Pfirrmann classification.The different expression of SDF 1 and CXCR4 in human IVDs was tested by immunohistochemistry, quantify polymerase chain reaction (q-PCR) and Western blot.The primary degenerative NPCs were primary cultured.The generation Ⅲ~Ⅴ NPCs was treated with 10 ng/mL SDF-1, in the presence of or in the absence of CXCR4 siRNA transfection and 20 μmol/L NF-κB inhibitor (pyrrolidine dithiocar bamate,PDTC).The transfection efficiency and target protein of signal pathway were verified by Western blot , the apoptosis of NPCs were tested by Annexin V /PI, the nucleus transferlocation of P65 from NF-κB were tested by immunofluorescent method.Results SDF-1and CXCR4 were both expressed in all donor tissues, however, there was a significantly increased in the degenerative IVDs .The apoptosis of degenerative NPCs was expedited by SDF -1 stimulation,which was significantly suppressed by CXCR 4 silencing by siRNA (P<0.05).Furthermore, with SDF-1 stimulation,the expressions of phosphorylated P 65 was significantly increased and the P65 perssad transferred to the nucleuses,which could be suppressed by the NF-κB inhibitor, PDTC(P<0.05).Conclusions The expression levels of SDF-1 and CXCR4 are increased in degenerative NP tissue.The SDF-1/CXCR4 axis is considered to induce apoptotic of human degenerative NPCs via the NF-κB signaling pathway.
ABSTRACT
Intervertebral disc degeneration (IDD) is one of the main causes of low back pain—a common clinical problem. Alterations of the phenotype, reduction of survival time, decline of metabolic activity, and decrease of extracellular matrix of nucleus pulposus (NP) cells are thought associated with IDD. The intervertebral disc is the largest avascular tissue in the body, with the most distinctive characteristic being low oxygen tension. Hypoxia-inducible factor (I III'') is a transcriptional factor induced by the hypoxia condition to initiate a scries of cellular responses, accommodating the hypoxia environment. HIF can start transcription of target genes by binding the hypoxia response element and may play an important role in the pathological process of IDD and serve as a crucial target for stopping progression or curing IDD. Here we summarized the roles of HIF in regulating metabolism activity of NP cells, including expression of HIF in NP cells, and regulatory roles of HIF in phenotype, survival, metabolism and extracellular matrix accumulation of NP cells.
ABSTRACT
This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.