Acidic Environment Induces Pyroptosis of NLRP3 Inflammatory Vesicle-Mediated Nucleus Pulposus Cells Through Up-Regulation of POSTN Expression

SUMMARY: Intervertebral disc degeneration (IVDD) is induced by nucleus pulposus (NP) dysfunction as a result of massive loss of NP cells. It has been reported that the acidic microenvironment of the intervertebral disc (IVD) can induce NP cell pyroptosis, and that up-regulation of periostin (POSTN) expression has a negative effect on NP cell survival. However, the relationship between the acidic environment, POSTN expression level and NP cell pyroptosis is unclear. Therefore, the aim of this study was to explore the relationship between acidic environment and POSTN expression level in NP cells, as well as the effect of POSTN in acidic environment on NP cell pyroptosis. NP cells were obtained from the lumbar vertebrae of Sprague Dawley (SD) male rats. These cells were divided into normal and acidic groups according to whether they were exposed to 6 mM lactic acid solution. And NP cells in the acidic group were additionally divided into three groups: (1) Blank group: no transfection; (2) NC group: cells transfected with empty vector plasmid; (3) sh-POSTN group: cells transfected with sh-POSTN plasmid to knock down the expression level of POSTN. Quantitative real-time PCR (qRT-PCR) and western blot was performed to assess the expression of POSTN at the mRNAand protein levels. CCK8 was used to evaluate cell survival. Western blot, in addition, was performed to examine acid-sensing ion channels (ASIC)-related proteins. And pyroptosis was detected by ELISA and western blot. The expression level of POSTN was significantly increased in NP cells in acidic environment. Knockdown of POSTN expression promoted the survival of NP cells in acidic environment and reduced the protein levels of ASIC3 and ASIC1a in NP cells. Moreover, knockdown of POSTN expression decreased the pyroptosis proportion of NP cells and the levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The levels of pyroptosis-related proteins NLRP3, ASC, cleaved-Caspase-1, and cleaved-GSDMD were also affected by the decreased POSTN expression. The extracellular acidic environment created by lactic acid solution activated NLRP3 inflammatory vesicle-induced caspase-1 to get involved in NP cell pyroptosis by up-regulating POSTN expression.

La degeneración del disco intervertebral (DDIV) es inducida por una disfunción del núcleo pulposo (NP) como resultado de una pérdida masiva de células NP. Se ha informado que el microambiente ácido del disco intervertebral (DIV) puede inducir la piroptosis de las células NP y que la regulación positiva de la expresión de periostina (POSTN) tiene un efecto negativo en la supervivencia de las células NP. Sin embargo, la relación entre el ambiente ácido, el nivel de expresión de POSTN y la piroptosis de las células NP es poco clara. Por lo tanto, el objetivo de este estudio fue explorar la relación entre el ambiente ácido y el nivel de expresión de POSTN en células NP, así como el efecto de POSTN en ambiente ácido sobre la piroptosis de las células NP. Las células NP se obtuvieron de las vertebras lumbares de ratas macho Sprague Dawley (SD). Estas células se dividieron en grupos normales y ácidos según se expusieron a una solución de ácido láctico 6 mM. Las células NP en el grupo ácido se dividieron adicionalmente en tres grupos: (1) Grupo en blanco: sin transfección; (2) grupo NC: células transfectadas con plásmido vector vacío; (3) grupo sh-POSTN: células transfectadas con plásmido sh-POSTN para reducir el nivel de expresión de POSTN. Se realizó una PCR cuantitativa en tiempo real (qRT-PCR) y una transferencia Western para evaluar la expresión de POSTN en los niveles de ARNm y proteína. Se utilizó CCK8 para evaluar la supervivencia celular. Además, se realizó una transferencia Western para examinar las proteínas relacionadas con los canales iónicos sensibles al ácido (ASIC). La piroptosis se detectó mediante ELISA y Western blot. El nivel de expresión de POSTN aumentó significativamente en células NP en ambiente ácido. La eliminación de la expresión de POSTN promovió la supervivencia de las células NP en un ambiente ácido y redujo los niveles de proteína de ASIC3 y ASIC1a en las células NP. Además, la eliminación de la expresión de POSTN disminuyó la proporción de piroptosis de las células NP y los niveles de citocinas proinflamatorias interleucina (IL) - 1β e IL-18. Los niveles de proteínas relacionadas con la piroptosis NLRP3, ASC, Caspasa-1 escindida y GSDMD escindida también se vieron afectados por la disminución de la expresión de POSTN. El ambiente ácido extracelular creado por la solución de ácido láctico activó la caspasa-1 inducida por vesículas inflamatorias NLRP3 para involucrarse en la piroptosis de las células NP mediante la regulación positiva de la expresión de POSTN.

Nimbolide targeting SIRT1 mitigates intervertebral disc degeneration by reprogramming cholesterol metabolism and inhibiting inflammatory signaling

Inflammation, abnormal cholesterol metabolism, and macrophage infiltration are involved in the destruction of the extracellular matrix of the nucleus pulposus (NP), culminating in intervertebral disc degeneration (IDD). Whether nimbolide (Nim), a natural extract, can alleviate IDD is unclear. In this study, we demonstrated that Nim promotes cholesterol efflux and inhibits the activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by activating sirtuin 1 (SIRT1) in nucleus pulposus cells (NPCs) during inflammation. Thus, Nim balanced matrix anabolism and catabolism of NPCs. However, the inhibition of SIRT1 significantly attenuated the effects of Nim. We also found that Nim promoted the expression of SIRT1 in RAW 264.7, which enhanced the proportion of M2 macrophages by facilitating cholesterol homeostasis reprogramming and impeded M1-like macrophages polarization by blocking the activation of inflammatory signaling. Based on these results, Nim can improve the microenvironment and facilitate matrix metabolism equilibrium in NPCs. Furthermore, in vivo treatment with Nim delayed IDD progression by boosting SIRT1 expression, modulating macrophage polarization and preserving the extracellular matrix. In conclusion, Nim may represent a novel therapeutic strategy for treating IDD.

Effect of Zhuangyao Tongluo Formula（壮腰通络方） Containing Serum on TNF-α-mediated Vicious Circle of Pyroptosis of Nucleus Pulposus Cells / 中医杂志

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.

Studies on the differentiation of human urine derived stem cells into nucleus pulposus-like cells induced by human nucleus pulposus cell exosomes / 中华骨科杂志

Objective:To investigate the effects of exosomes of human nucleus pulposus cells (NPCs) on the differentiation of urine derived stem cells (USCs) into nucleus pulposus-like cells.Methods:USCs and NPCs were isolated and cultured in vitro. The exosomes of NPCs were extracted and detected by Western-blot. USCs cytoplasm was transfected with GFP lentivirus, while nucleus was transfected with DAPI dye. The NPCs exosomes were transfected with PKH26 dye. After co-incubation for 12 h, USCs and NPCs exosomes were observed by macroscopy. USCs differentiation was induced by NPCs exosomes and non-contact co-culture methods. The relative expression of marker gene mRNA of nucleus pulposus cells in each group and the absorbance at 450 nm wavelength were detected.Results:The isolated USCs had the ability to differentiate into osteocytes, adipocytes and chondrocytes with high expression of marker CD29 (99.57%), CD44 (97.46%) and CD73 (97.71%) and with low expression of negative proteins CD31 (0.59%) and CD45 (0.19%). The isolated NPCs highly expressed nuclear pulposus cell marker COL2A1, ACAN and SOX-9. The exosomes extracted from NPCs showed high expression of exosome marker CD63, CD81 and Tsg101. After 12 h co-incubation, NPCs exosomes fused with USCs membrane and appeared in the cytoplasm of USCs. At 3, 5 and 7 days of co-culture, the absorbance value of USCs cells in exosome group (0.44±0.004, 0.76±0.004, 0.82±0.006) was higher than that in co-culture group (0.39±0.022, 0.63±0.035, 0.69±0.012) ( P<0.05). The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (1.80±0.31, 3.50±0.21, 5.35±0.31, 7.46±0.12), COL2A1 (1.43±0.15, 4.33±0.23, 6.89±0.22, 8.11±0.31), SOX-9 (2.21±0.13, 3.13±0.11, 3.96± 0.14, 4.52±0.26) and HIF-1α (1.45±0.16, 2.14±0.21, 4.31±0.41, 4.01±0.25) in exosomes group were significantly higher than those in the control group ( P<0.05) at the 3rd, 7th, 14th and 21st days. The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (5.69±0.21, 6.69±0.13), COL2A1 (6.33±0.17, 7.89±0.15), SOX-9 (4.19±0.29, 4.38±0.12), HIF-1α (4.49±0.32, 4.96±0.26) in exosomes group were significantly higher than those ACAN (3.69±0.35, 5.13±0.23), COL2A1 (3.40±0.16, 6.79±0.19), SOX-9 (2.26±0.32, 3.69±0.26), HIF-1α (2.39±0.11, 3.96±0.13) in non-contact co-culture group ( P<0.05) at the 14th and 21st days. Conclusion:Human nucleus pulposus exosomes could induce differentiation of human USCs into nucleus pulposus-like cells in vitro. Compared with non-contact co-culture, exosomes have higher induction efficiency and can better maintain the proliferation activity of nucleus pulposus-like cells

Pheretima extract ameliorates nucleus pulposus cell degeneration in the intervertebral disc by inhibiting nuclear factor-kappa B pathway / 中国组织工程研究

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.

Morphology and activity of human nucleus pulposus cells under different hydrostatic pressures / 中国组织工程研究

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.

Intervertebral disc tissue engineering based on polymer materials: Research focus and hot spots / 中国组织工程研究

BACKGROUND: In recent years, the rapid development of tissue engineering technology provides a new idea for the treatment of intervertebral disc degeneration; that is, biomaterials are used to reconstruct the damaged intervertebral disc structure. OBJECTIVE: To summarize polymer materials which are applicable for intervertebraltissue engineering construction, and overview the advantages, disadvantages and application progress of each material. METHODS: We searched related articles from inception to February 2020 in PubMed, Web of Science and CNKI databases with “polymer, intervertebral disc, tissue engineering, nucleus pulposus, annulus fibrosus” as English and Chinese key words. Initially 189 related articles were searched, and 109 eligible articles were included in final analysis according to inclusion and exclusion criteria. RESULTS AND CONCLUSION: Intervertebral disc is composed of inner soft nucleus pulposus and outer stiff annulus fibrosus. Accordingly, it requires two components with different structures and functions to reconstruct a complete intervertebral disc with tissue engineering method. Chitosan, alginate and hyaluronic acid are considered as optimal materials for nucleus pulposus construction because of their appropriate swelling character and ability of inducing cells to secrete nucleus pulposus matrix. Silk fibroin, collagen, polyethylene glycol, polylactic acid, polyglycolic acid and polycaprolactone with high mechanical strength are suitable for annulus fibrosus construction to bear high loading burden. By further surface modification, these synthesis scaffolds wound show a better cellular compatibility and promote tissue integrity after in vivo implantation.

Pharmacological inhibition of mTORC1 activity protects against inflammation-induced apoptosis of nucleus pulposus cells

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.

Vértebra limbus, rara causa de dolor lumbar en jóvenes / Limbus vertebra as a rare cause of lumbar pain in young people

RESUMEN La vértebra limbus es una rara condición poco descrita en la literatura médica. Se caracteriza por una herniación marginal intracorporal del núcleo pulposo que resulta en la separación de un fragmento óseo de forma triangular. Usualmente se presenta en niños o adolescentes, principalmente en la columna lumbar, y ocasiona dolor que, en la mayoría de los casos, mejora con el tratamiento con antiinflamatorios.

A B S T R A C T The limbus vertebra is a rare condition poorly described in the medical literature. Marginal intrabody herniation of the nucleus pulposus resulting in the separation of a triangular bone fragment. It usually occurs in children or adolescents mainly in the lumbar spine and causes pain that in most cases improves with treatment with anti-inflammatories.

Research progress of hydrogel used for regeneration of nucleus pulposus in intervertebral disc degeneration / 中国修复重建外科杂志

Objective: To summarize the research progress of hydrogels for the regeneration and repair of degenerative intervertebral disc and to investigate the potential of hydrogels in clinical application. Methods: The related literature about the role of hydrogels in intervertebral disc degeneration especially for nucleus pulposus was reviewed and analyzed. Results: Hydrogels share similar properties with nucleus pulposus, and it plays an important role in the regeneration and repair of degenerative intervertebral disc, which can be mainly applied in nucleus pulposus prosthesis, hydrogel-based cell therapy, non-cellular therapy, and tissue engineering repair. Conclusion: Hydrogels are widely used in the regeneration and repair of intervertebral disc, which provides a potential treatment for intervertebral disc degeneration.

Expression and correlation analysis of hypoxia inducible factor 1α and autophagy related molecules in rat nucleus pulposus cells under hypoxia / 中国修复重建外科杂志

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.

Effects of lentivirus-mediated insulin-like growth factor 1 and platelet derived growth factor genes on nucleus pulposus tissue of human degenerated intervertebral disc / 中国修复重建外科杂志

Objective: To observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP. Methods: Human degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot. Results: HE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group ( P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group ( t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A ( P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant ( P<0.05). Conclusion: Both IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.

Research progression in the role and clinical value of circRNAs in intervertebral disc degeneration / 解放军医学杂志

Degenerative intervertebral disc disease (DDD) is a kind of spinal disease characterized by intervertebral disc degeneration, which is the most common reason of low back pain. In recent years, the incidence rate of DDD is increasing year by year, and the diseased population shows a younger tendency. Therefore, it is very important to find new effective molecular targets for treating this disease. As a new type of endogenous noncoding RNA, circular RNAs are widely distributed in the human cells. A large number of studies have shown that, circular RNAs are ubiquitous in human cells and play a key role in the pathological process of many diseases. This review summarizes the research status of circular RNAs in the pathogenesis of DDD, and discusses the potential clinical value,to provide a theoretical basis for the diagnosis and treatment of DDD.

New insight intocell transplantation for repairing intervertebral discs / 中国组织工程研究

BACKGROUND: Current treatment of disc degeneration is limited to palliative care or active surgical intervention, but neither can it slow or reverse disease progression, nor is the long-term efficacy satisfactory. In recent years, with the continuous development of tissue engineering and regenerative medicine, the use of cell therapy to repair degenerative intervertebral discs has attracted more and more researchers’ attention. OBJECTIVE: To summarize the current status of cell transplantation in the treatment of intervertebral disc degeneration, and to provide theoretical basis and experimental basis for future research. METHODS: The relevant articles were retrieved from the CNKI, WanFang and PubMed databases by computer. The search time was set from January 2001 to January 2019. The keywords were “intervertebral disc degeneration, cell therapy, stem cells, cell transplantation” in Chinese and English, respectively. Articles related to stem cell treatment of intervertebral disc degeneration, especially focusing on the latest experimental and clinical research results, were included. RESULTS AND CONCLUSION: Degeneration of the intervertebral disc is mainly caused by the decreased vitality and quantity of nucleus pulposus cells, the reduction of proteoglycan synthesis, the dehydration of nucleus pulposus and the increase of metabolic waste. Continuous explorations on the therapeutic mechanism by which cell transplantation promotes nucleus pulposus regeneration cannot only provide a more detailed understanding of the pathogenesis and repair mechanism of intervertebral disc degeneration, but also give new strategies for the prevention and cell therapy of other related diseases.

Proliferative activity of nucleus pulposus cells in human intervertebral disc and intervention with yishen huoxue tongluo recipe / 中国组织工程研究

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.

Effect of capparis spinosa total alkaloid on proliferation and apoptosis of nucleus pulposus cells in an intervertebral disc degeneration rat model / 中国组织工程研究

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.

Research progress of intervertebral disc repair with decellularized matrix materials / 中国组织工程研究

BACKGROUND: With the development of tissue engineering, the repair and regeneration of disc becomes possible. Decellularized extracellular matrix is an important means for disc regeneration. OBJECTIVE: To review and summarize the processing, quality control and application of decellularized matrix materials applied in intervertebral disc regeneration in recent years and put forward the prospect. METHODS: PubMed, Web of Science and CNKI databases were searched for the articles concerning decellularized methods and decellularized matrix repairing intervertebral disc with the search terms of “intervertebral disc, decellularization, extracellular matrix, scaffold material, tissue engineering” in English and Chinese, respectively. After screening based on the inclusion and exclusion criteria, the articles with high relevance were included for review. RESULTS AND CONCLUSION: The decellularized tissue-engineered intervertebral disc aims to maintain the physiologically relevant bioactivators to a great extent, improve mechanical properties and biocompatibility, and reduce immunogenicity. The decellularized matrix material can simulate the microenvironment of the extracellular matrix in the intervertebral disc. As a cell carrier, it can well induce the differentiation of seed cells, which has achieved certain progress in the repair of intervertebral discs. However, further studies need to address the following issues: proper porosity of decellularized matrix materials, immunological rejection, implant ways in vivo and repair effect.

Expression of cyclic RNA plasmacytoma variant translocation 1 in senescence of nucleus pulposus cells and its regulation mechanism / 中国组织工程研究

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.

Observation of herniated cervical intervertebral disc volume based on quantitative volume measurement “monitoring” based on PACS software / 中国组织工程研究

BACKGROUND: Herniated cervical intervertebral disc volume measurement is an important parameter for quantitative evaluation of cervical disc degeneration, but it faces a lot of problems such as different measurement standards and the undefined measurement error range. OBJECTIVE: To investigate the accuracy of PACS software in measuring cervical disc volume, provide reliable measurement methods and accurate data support for clinical observation and research on cervical disc volume change and degeneration. METHODS: The error rate was obtained by repeated measurements of the normal saline with a known volume of 5.0 mL by means of PACS software. With reference to this error rate, volume changes of cervical disc herniation before and after cervical microendoscopic laminoplasty were “monitored” and analyzed in 30 cases. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (approval No. 2019-KY-274) on September 26, 2019. RESULTS AND CONCLUSION: (1) For the measurement of normal saline with known volume, it was found that the error rate of measurement by PACS software was ±5%, suggesting that the measurement of cervical disc volume by PACS software is a simple and accurate method. (2) After cervical microendoscopic laminoplasty, there were 70 patients with reduced cervical disc volume reduction absorption rate of 5%-100%, and the absorption ratio was 76.1% (70/92). The volume increased by 11, but the increase was not more than 5% in the patients with cervical disc herniation after treatment. (3) The spontaneous disappearance or reduction of the herniated cervical disc after cervical microendoscopic laminoplasty was as early as 7 days, and the longest was 76 months. (4) The effects were excellent in 11 cases, good in 15 cases, and fair in 4 cases. The excellent and good rate was 86.7%.

Research progress and development space of microRNA in the process of intervertebral disc degeneration / 中国组织工程研究

BACKGROUND: Full-understanding of the mechanism of microRNA (miRNA) in the process of intervertebral disc degeneration at the cellular and molecular levels can provides new idea for the early prevention or treatment of a series of spinal diseases to intervertebral disc degeneration. OBJECTIVE: To summarize the research status of the role of miRNA in the cause and mechanism of intervertebral disc degeneration. METHODS: A computed-based online retrieval of PubMed, Wanfang and CNKI databases was conducted with the keywords of “miRNA, intervertebral disc degeneration, extracellular matrix, apoptosis, autophagy, cartilage endplate, nucleus pulposus, fibrous ring” in English and Chinese, respectively. Finally 58 eligible articles were included for review. RESULTS AND CONCLUSION: The role of miRNA in intervertebral disc degeneration has been widely studied, and some of the specific mechanisms have been verified. Most of the studies are limited to the nucleus pulposus, and there are few reports on cartilage endplate and annulus fibrosus. With the in-depth study of miRNA, there is still much space for clinical research.