How to enhance the ability of mesenchymal stem cells to alleviate intervertebral disc degeneration

Intervertebral disc(ID)degeneration(IDD)is one of the main causes of chronic low back pain,and degenerative lesions are usually caused by an imbalance between catabolic and anabolic processes in the ID.The environment in which the ID is located is harsh,with almost no vascular distribution within the disc,and the nutrient supply relies mainly on the diffusion of oxygen and nutrients from the blood vessels located under the endplate.The stability of its internal environment also plays an important role in preventing IDD.The main feature of disc degeneration is a decrease in the number of cells.Mesenchymal stem cells have been used in the treatment of disc lesions due to their ability to differentiate into nucleus pulposus cells in a nonspecific anti-inflammatory manner.The main purpose is to promote their regeneration.The current aim of stem cell therapy is to replace the aged and metamorphosed cells in the ID and to increase the content of the extracellular matrix.The treatment of disc degeneration with stem cells has achieved good efficacy,and the current challenge is how to improve this efficacy.Here,we reviewed current treatments for disc degeneration and summarize studies on stem cell vesicles,enhancement of therapeutic effects when stem cells are mixed with related substances,and improvements in the efficacy of stem cell therapy by adjuvants under adverse conditions.We reviewed the new approaches and ideas for stem cell treatment of disc degeneration in order to contribute to the development of new therapeutic approaches to meet current challenges.

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

Nucleus Pulposus Cells from Calcified Discs Promote the Degradation of the Extracellular Matrix through Upregulation of the GATA3 Expression

Objective Disc calcification is strongly associated with disc degeneration;however,the underlying mechanisms driving its pathogenesis are poorly understood.This study aimed to provide a gene expression profile of nucleus pulposus cells(NPCs)from calcified discs,and clarify the potential mechanism in disc degeneration.Methods Primary NPCs were isolated from calcified and control discs(CAL-NPC and CON-NPC),respectively.The proliferation and extracellular matrix(ECM)metabolism capacities of the cells were evaluated using MTT and Western blotting,respectively.RNA sequencing was used to identify differentially expressed genes(DEGs)in the CAL-NPCs.The biological functions of the DEGs were analyzed using the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases.The transcription factor database and Cytoscape software were used to construct the transcription factor-DEGs regulatory network.The role of the verified transcription factor in NPC proliferation and ECM metabolism was also investigated.Results The CAL-NPCs exhibited a lower proliferation rate and higher ECM degradation capacity than the CON-NPCs.In total,375 DEGs were identified in the CAL-NPCs.The GO and KEGG analyses showed that the DEGs were primarily involved in the regulation of ribonuclease activity and NF-kappa B and p53 signaling pathways.GATA-binding protein 3(GATA3)with the highest verified levels was selected for further studies.Overexpression of GATA3 in the CON-NPCs significantly inhibited their proliferation and promoted their ECM degradation function,while the knockdown of GATA3 in the CAL-NPCs resulted in the opposite phenotypes.Conclusion This study provided a comprehensive gene expression profile of the NPCs from the calcified discs and supported that GATA3 could be a potential target for reversing calcification-associated disc degeneration.

An esterase-responsive ibuprofen nano-micelle pre-modified embryo derived nucleus pulposus progenitor cells promote the regeneration of intervertebral disc degeneration

Stem cell-based transplantation is a promising therapeutic approach for intervertebral disc degeneration(IDD).Current limitations of stem cells include with their insufficient cell source,poor proliferation capacity,low nucleus pulposus(NP)-specific differentiation potential,and inability to avoid pyroptosis caused by the acidic IDD microenvironment after transplantation.To address these challenges,embryo-derived long-term expandable nucleus pulposus progenitor cells(NPPCs)and esterase-responsive ibuprofen nano-micelles(PEG-PIB)were prepared for synergistic transplantation.In this study,we propose a biomaterial pre-modification cell strategy;the PEG-PIB were endocytosed to pre-modify the NPPCs with adaptability in harsh IDD microenvironment through inhibiting pyroptosis.The results indicated that the PEG-PIB pre-modified NPPCs exhibited inhibition of pyroptosis in vitro;their further synergistic transplantation yielded effective functional recovery,histological regeneration,and inhibition of pyroptosis during IDD regeneration.Herein,we offer a novel biomaterial pre-modification cell strategy for synergistic transplantation with promising therapeutic effects in IDD regeneration.

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy

BACKGROUND To date,there has been no effective treatment for intervertebral disc degeneration(IDD).Nucleus pulposus-derived mesenchymal stem cells(NPMSCs)showed encouraging results in IDD treatment,but the overexpression of reactive oxygen species(ROS)impaired the endogenous repair abilities of NPMSCs.6-gingerol(6-GIN)is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.AIM To investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.METHODS The cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN.ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis.Matrix metalloproteinase(MMP)was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay.TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate.Additionally,autophagy-related proteins(Beclin-1,LC-3,and p62),apoptosisassociated proteins(Bcl-2,Bax,and caspase-3),and PI3K/Akt signaling pathwayrelated proteins(PI3K and Akt)were evaluated by Western blot analysis.Autophagosomes were detected by transmission electron microscopy in NPMSCs.LC-3 was also detected by immunofluorescence.The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction(RT-PCR),and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.RESULTS 6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs,decreased hydrogen peroxide-induced intracellular ROS levels,and inhibited cell apoptosis.6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression.The MMP,Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide.6-GIN treatment promoted extracellular matrix(ECM)expression by reducing the oxidative stress injury-induced increase in MMP-13 expression.6-GIN activated autophagy by increasing the expression of autophagy-related markers(Beclin-1 and LC-3)and decreasing the expression of p62.Autophagosomes were visualized by transmission electron microscopy.Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux.The PI3K/Akt pathway was also found to be activated by 6-GIN.6-GIN inhibited NPMSC apoptosis and ECM degeneration,in which autophagy and the PI3K/Akt pathway were involved.CONCLUSION 6-GIN efficiently decreases ROS levels,attenuates hydrogen peroxide-induced NPMSCs apoptosis,and protects the ECM from degeneration.6-GIN is a promising candidate for treating IDD.

Early efficacy of endoscopic translaminar and intervertebral foraminal approaches in the treatment of lumbar disc herniation

Objective:To investigate the early efficacy of two approaches for lumbar disc herniation under spinal endoscopy.Methods:45 cases of lumbar disc herniation were divided into interlaminar approach(27 cases)and intervertebral foramen approach(18 cases)according to different surgical approaches.Postoperative pain visual analogue scale(VAS)was used.Japanese Orthopaedic Association(JOA)lumbar spine score(JOA)and modified Macnab criteria were used to evaluate the postoperative outcome.Results:(1)VAS score.There is no interaction effect between the access mode and the time factor(F=0.620,P=0.603).There were statistically significant differences in pain VAS scores between preoperative and postoperative time points,that is,there was a time effect(F=2157.488,P=0.000).The overall VAS scores of the two groups were compared,and the difference was not statistically significant,that is,there was no grouping effect(F=2.610,P=0.114).The VAS score of pain in both groups decreased with time,and the differences between the two groups were not statistically significant before surgery,at discharge,1 month after surgery and 3 months after surgery(t=0.067,P=0.947;t=1.415,P=0.164;t=0.564,P=0.575;t=0.442,P=0.660);JOA score.There is no interaction effect between the access mode and the time factor(F=1.296,P=0.280).The difference of JOA score between preoperative and postoperative time points was statistically significant,that is,there was a time effect(F=1464.830,P=0.000).JOA scores of the two groups showed an increasing trend with time,and the differences between the two groups were not statistically significant before surgery,at discharge,1 month after surgery and 3 months after surgery(t=0.067,P=0.947;t=1.415,P=0.164;t=0.564,P=0.575;t=0.442,P=0.660);(2)The improved Macnab standard was used to evaluate the excellent and good rate at 3 months after surgery.In the interlaminar group,12 cases were excellent,13 cases were good and 2 cases were fair.The excellent and good rate was 92.6%.In the intervertebral foramen group,7 cases were excellent,10 cases were good and 1 case was fair.The excellent and good rate was 94.4%.The overall excellent and good rate of the two groups was 93.3%.Conclusion:Both approaches can achieve satisfactory efficacy in the treatment of lumbar intervertebral disc herniation,which is worthy of clinical application.However,for beginners,l5-s1 lumbar disc herniation is more suitable for intervertebral disc approach,so as to achieve satisfactory efficacy.

Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells

BACKGROUND Intervertebral disc(IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus(NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α(SDF-1α) and its receptor CX-C chemokine receptor type 4(CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIM To investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells(NPSCs).METHODS We performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups.Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTS SDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Realtime RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1αnot only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement.Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSION These findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Transplantation of gene-modified nucleus pulposus cells reverses rabbit intervertebral disc degeneration

Background Intervertebral disc degeneration is the main cause of low back pain. The purpose of this study was to explore potential methods for reversing the degeneration of lumbar intervertebral discs by transplantation of gene-modified nucleus pulposus cells into rabbit degenerative lumbar intervertebral discs after transfecting rabbit nucleus pulposus cells with adeno-associated virus 2 （AAV2）-mediated connective tissue growth factor （CTGF） and tissue inhibitor of metalloproteinases 1 （TIMP1） genes in vitro. Methods Computer tomography （CT）-guided percutaneous annulus fibrosus injury was performed to build degenerative lumbar intervertebral disc models in 60 New Zealand white rabbits, rAAV2-CTGF-IRES-TIMPI-transfected rabbit nucleus pulposus cells were transplanted into degenerative lumbar intervertebral discs （transplantation group）, phosphate-buffered saline （PBS） was injected into degenerative lumbar intervertebral discs （degeneration control group） and normal lumbar intervertebral discs served as a blank control group. After 6, 10 and 14 weeks, the disc height index （DHI） and signal intensity in intervertebral discs were observed by X-ray and magnetic resonance imaging （MRI） analysis The expression of CTGF and TIMP1 in nucleus pulposus tissue was determined by Western blotting analysis, the synthesis efficiency of proteoglycan was determined by a 35S-sulfate incorporation assay, and the mRNA expression of type II collagen and proteoglycan was detected by RT-PCR. Results MRI confirmed that degenerative intervertebral discs appeared two weeks after percutaneous puncture. Transgenic nucleus pulposus cell transplantation could retard the rapid deterioration of the DHI. MRI indicated that degenerative intervertebral discs were relieved in the transplantation group compared with the degeneration control group. The expression of collagen II mRNA and proteoglycan mRNA was significantly higher in the transplantation group and the blank control group compared with the degeneration control group （P 〈0.05）. Conclusions CT-guided percutaneous puncture can successfully build rabbit degenerative intervertebral disc models. Both CTGF and TIMPl-transfected cell transplantation helps to maintain disc height, and promotes the biosynthesis of tvDe II collaQen and proteoalvcan in intervertebral discs, reversinq the de（：ieneration of intervertebral discs.

Autologous nucleus pulposus transplantation to lumbar 5 dorsal root ganglion after epineurium discission in rats： a modified model of non-compressive lumbar herniated intervertebral disc

Background Nucleus pulposus of intervertebral discs has proinflammatory characteristics that play a key role in neuropathic pain in lumbar herniated intervertebral disc. One of the most commonly used animal models （the traditional model） of non-compressive lumbar herniated intervertebral disc is created by L4-L5 hemilaminectomy and the application of autologous nucleus pulposus to cover the left L4 and L5 nerve roots in rats. However, such procedures have the disadvantages of excessive trauma and low success rate. We proposed a modified model of non-compressive lumbar herniated intervertebral disc in which only the left L5 dorsal root ganglion is exposed and transplanted with autologous nucleus pulposus following incision of epineurium. We aimed to compare the modified model with the traditional one with regard to trauma and success rate. Methods Thirty Sprague-Dawley male rats were randomized into three groups： sham operation group （n=6）, traditional group （n=12）, and modified group （n=12）. The amount of blood loss and operative time for each group were analyzed. The paw withdrawal threshold of the left hind limb to mechanical stimuli and paw withdrawal latency to heat stimuli were examined from the day before surgery to day 35 after surgery. Results Compared with the traditional group, the modified group had shorter operative time, smaller amount of blood loss, and higher success rate （91.7% versus 58.3%, P 〈0.05）. There was no decrease in paw withdrawal latency in any group. The sham operation group had no decrease in postoperative paw withdrawal threshold, whereas the modified and traditional groups had significant reduction in paw withdrawal threshold after surgery （mechanical hyperalgesia）. Conclusions Transplantation of nucleus pulposus onto the L5 dorsal root ganglion following incision of epineurium in rats established an improved animal model of non-compressive lumbar herniated intervertebral disc with less trauma and more stable pain ethology.

Comparison of microendoscopic discectomy and open discectomy for single-segment lumbar disc herniation

BACKGROUND Lumbar disc herniation is a common disease.Endoscopic treatment may have more advantages than traditional surgery.AIM To compare the clinical efficacy and safety of microendoscopic discectomy(MED)and open discectomy with lamina nucleus enucleation in the treatment of singlesegment lumbar intervertebral disc herniation.METHODS Ninety-six patients who were operated at our hospital were selected for this study.Patients with single-segment lumbar disc herniation were admitted to the hospital from March 2018 to March 2019 and were randomly divided into the observation group and the control group with 48 cases in each group.The former group underwent lumbar discectomy and the latter underwent laparotomy and nucleus pulpectomy.Surgical effects were compared between the two groups.RESULTS In terms of surgical indicators,the observation group had a longer operation time,shorter postoperative bedtime and hospital stay,less intraoperative blood loss,and smaller incision length than the control group(P<0.05).The excellent recovery rate did not differ significantly between the observation group(93.75%)and the control group(91.67%).Visual analogue scale pain scores were significantly lower in the observation group than in the control group at 1 d,3 d,1 mo,and 6 mo after surgery(P<0.05).The incidence of complications was significantly lower in the observation group than in the control group(6.25%vs 22.92%,P<0.05).CONCLUSION Both MED and open discectomy can effectively improve single-segment lumbar disc herniation,but MED is associated with less trauma,less bleeding,and a lower incidence of complications.

Barriers to mesenchymal stromal cells for low back pain

Intervertebral disc degeneration is the main cause of low back pain.In the past 20 years,the injection of mesenchymal stromal cells(MSCs)into the nucleus pulposus of the degenerative disc has become the main approach for the treatment of low back pain.Despite the progress made in this field,there are still many barriers to overcome.First,intervertebral disc is a highly complex loadbearing composite tissue composed of annulus fibrosus,nucleus pulposus and cartilaginous endplates.Any structural damage will change its overall biomechanical function,thereby causing progressive degeneration of the entire intervertebral disc.Therefore,MSC-based treatment strategies should not only target the degenerated nucleus pulposus but also include degenerated annulus fibrosus or cartilaginous endplates.Second,to date,there has been relatively little research on the basic biology of annulus fibrosus and cartilaginous endplates,although their pathological changes such as annular tears or fissures,Modic changes,or Schmorl's nodes are more commonly associated with low back pain.Given the high complexity of the structure and composition of the annulus fibrosus and cartilaginous endplates,it remains an open question whether any regeneration techniques are available to achieve their restorative regeneration.Finally,due to the harsh microenvironment of the degenerated intervertebral disc,the delivered MSCs die quickly.Taken together,current MSC-based regenerative medicine therapies to regenerate the entire disc complex by targeting the degenerated nucleus pulposus alone are unlikely to be successful.

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.

青藤碱可有效抑制白细胞介素1β介导的髓核细胞凋亡

背景:椎间盘退变是导致脊柱退行性疾病的基础,然而目前尚无有效的治疗药物。目的:探讨青藤碱是否可以抑制白细胞介素1β诱导的髓核细胞凋亡及其分子机制。方法:采用胰酶联合Ⅱ型胶原酶消化法体外培养大鼠髓核细胞,并绘制细胞生长曲线,采用CCK-8法筛选合适的青藤碱药物浓度。将髓核细胞分为对照组、青藤碱组、白细胞介素1β组、青藤碱+白细胞介素1β组、锌原卟啉(血红素氧合酶1抑制剂)组、锌原卟啉+青藤碱组、锌原卟啉+白细胞介素1β组、青藤碱+锌原卟啉+白细胞介素1β组。分别检测各组髓核细胞增殖活性、活性氧含量、凋亡率及血红素氧合酶1的表达情况。结果与结论:①体外培养的大鼠髓核细胞呈现多角形、三角形、短楔形等形态,其呈现“S”型曲线生长,接种第1-3天生长缓慢,第4-6天生长迅速,第七八天生长速度缓慢,进入“平台期”,细胞数量不再增加;②当青藤碱的浓度≤80μmol/L时,髓核细胞的增殖活性不会受到显著影响(P>0.05);③白细胞介素1β可以显著降低髓核细胞的增殖活性,增加活性氧含量,导致细胞凋亡(P<0.01);④当采用青藤碱干预后,不仅可以促进血红素氧合酶1的表达(P<0.05),而且可以抑制白细胞介素1β诱导的髓核细胞增殖活性降低、活性氧含量和凋亡率增加(P<0.05),其作用可被锌原卟啉逆转(P<0.01)。

YAP通过促进自噬抑制髓核细胞胞外基质分解代谢

目的探讨YAP对椎间盘髓核细胞的作用及其可能机制。方法收集重庆医科大学附属第一医院2021年3月至2022年7月接受腰椎手术患者的相对正常和退变的椎间盘组织,采用免疫组化和Western blot检测YAP在人椎间盘髓核组织中的表达差异。体外培养人原代髓核细胞,通过IL-1β处理诱导髓核细胞退变,将实验分为对照组、IL-1β组、IL-1β+LV-YAP组、IL-1β+YAP-siRNA组和IL-1β+LV-YAP+3-MA组。采用Western blot检测细胞外基质分解代谢及自噬水平变化。最后建立大鼠椎间盘退变模型,利用MRI、阿利新蓝染色及免疫组化检测YAP和LC3的表达及椎间盘退变情况。结果YAP在退变椎间盘组织的表达水平明显低于相对正常的椎间盘组织(P<0.05)。体外细胞实验结果显示,IL-1β+LV-YAP组显著提高IL-1β诱导的髓核细胞中CollagenⅡ、Aggrecan和LC3-Ⅱ的蛋白表达(P<0.05),降低MMP-3和MMP-13的蛋白表达(P<0.05),而IL-1β+YAP-siRNA组则表现出完全相反的作用。而予以自噬抑制剂3-MA预处理后明显降低IL-1β+LV-YAP组中GFP-LC3阳性颗粒数量和CollagenⅡ、Aggrecan、LC3-Ⅱ蛋白表达(P<0.05),且提高MMP-3和MMP-13的蛋白表达(P<0.05)。进一步在体内构建大鼠椎间盘退变模型,YAP过表达促进LC3表达和抑制椎间盘退变。结论YAP过表达通过促进人髓核细胞自噬抑制细胞外基质降解,从而延缓椎间盘退变。

低氧对炎性环境下髓核细胞凋亡的作用及机制

目的探讨低氧对炎性环境下髓核细胞凋亡的影响及作用机制。方法体外培养大鼠原代髓核细胞,采用低氧小室进行低氧干预,采用促炎因子肿瘤坏死因子-α(TNF-α)刺激髓核细胞模拟炎性环境。通过流式细胞术及TUNEL染色检测髓核细胞凋亡情况,通过蛋白质印迹法检测凋亡相关蛋白表达水平,明确低氧对髓核细胞凋亡的影响;随后通过荧光定量PCR及酶联免疫吸附试验评估炎性环境下髓核细胞内炎性反应情况,并通过蛋白质印迹法分析髓核细胞内NLRP3炎性小体激活水平;最后采用NLRP3炎性小体激动剂QS-21,通过回复实验明确NLRP3炎性小体在低氧调控髓核细胞凋亡中的作用。结果低氧对正常环境下髓核细胞凋亡无明显影响,但能抑制炎性环境下髓核细胞凋亡。在炎性环境下,髓核细胞炎性细胞因子白细胞介素(IL)-1β、IL-8、IL-18的表达及分泌增加,细胞内NLRP3炎性小体激活,而低氧干预能缓解上述改变。回复实验表明,NLRP3炎性小体激动剂QS-21可削弱低氧对炎性环境下髓核细胞凋亡的抑制作用。结论低氧通过抑制NLRP3炎性小体激活缓解炎性环境下髓核细胞凋亡。

丁香苷抑制大鼠椎间盘退变

背景:椎间盘退变是由于椎间盘内部髓核和纤维环组织发生损伤和退化导致的椎间盘结构和功能发生变化,目前尚无有效的治疗药物。目的:探讨丁香苷抑制大鼠椎间盘退变的作用。方法:取10只雄性SD大鼠,将每只大鼠的尾椎Co_(4)/Co_(5)椎间盘设为模型组、Co_(5)/Co_(6)椎间盘设为丁香苷组、Co_(6)/Co_(7)椎间盘设为对照组,对照组不进行任何处理,模型组、丁香苷组采用微型穿刺针进行纤维环全层穿刺建立椎间盘退变模型,造模后即刻,模型组、丁香苷组椎间盘分别注射2.5μL的生理盐水、丁香苷溶液(5μmol/L)。注射4周后取材,采用苏木精-伊红和番红O-固绿染色观察大鼠椎间盘退变程度,免疫组化染色分析大鼠椎间盘组织内Ⅱ型胶原、聚集蛋白聚糖及基质金属蛋白酶3,13的表达。结果与结论:①苏木精-伊红染色显示,模型组椎间盘高度降低,软骨终板变薄且有裂隙出现,纤维环结构紊乱且出现裂隙,髓核消失;丁香苷组椎间盘高度正常或略低于对照组,软骨终板退变程度较模型组轻,纤维环排列较模型组相对规整且无裂隙,髓核部分皱缩。②番红O-固绿染色显示,模型组椎间盘软骨终板出现缺损且软骨钙化层变薄,出现明显退变;丁香苷组椎间盘软骨终板结构形态有一定程度恢复。③免疫组化染色显示,与对照组比较,模型组椎间盘软骨组织内Ⅱ型胶原、聚集蛋白聚糖的表达降低(P<0.0001),基质金属蛋白酶3,13的表达升高(P<0.0001);与模型组比较,丁香苷组椎间盘软骨组织内Ⅱ型胶原、聚集蛋白聚糖的表达升高(P<0.001,P<0.0001),基质金属蛋白酶3,13的表达降低(P<0.001,P<0.0001)。④结果表明,丁香苷可通过抑制基质金属蛋白酶3,13的表达、提高Ⅱ型胶原和聚集蛋白聚糖的表达来改善椎间盘的结构和功能,预防和减缓椎间盘退变过程。

负载丹酚酸B甲基丙烯酰化明胶水凝胶对椎间盘退变的作用

背景:丹酚酸B可抑制H2O2诱导的细胞损伤,有效清除过量的活性氧,发挥抗氧化特性,已被应用于许多疾病的治疗研究中。但是,有关丹酚酸B在椎间盘退变中作用及其作用机制的研究相对较少。目的:以甲基丙烯酰化明胶水凝胶为载体,通过体外细胞实验与动物体内实验观察丹酚酸B对氧化应激诱导的椎间盘退变的作用以及作用机制。方法:制备负载丹酚酸B的甲基丙烯酰化明胶水凝胶(载药水凝胶)。①体外细胞实验:分离提取成年SD大鼠腰椎髓核细胞,选择第3代髓核细胞,分组处理:A组加入完全培养基;B组加入含H2O2的完全培养基;C组接种于未载药水凝胶上,加入含H2O2的完全培养基;D组接种于载药水凝胶上,加入含H2O2的完全培养基;E组接种于载药水凝胶上,加入含H2O2与TLR4信号通路抑制剂的完全培养基,检测细胞增殖、氧化应激、炎症反应、细胞基质相关蛋白的基因表达以及TLR4/核因子kB信号通路蛋白表达。②动物体内实验:将60只成年SD大鼠随机分为正常组、针刺组、针刺+丹酚酸组、针刺+水凝胶组、针刺+载药水凝胶组,每组12只,后4组采用针刺建立椎间盘退变模型后依次注射生理盐水、丹酚酸B溶液、未载药水凝胶、载药水凝胶治疗,术后4周分别进行影像学检测、病理组织形态观察。结果与结论:①体外细胞实验:与A组比较,B组细胞增殖下降,氧化应激反应及炎症反应增强,细胞外基质降解酶(基质金属蛋白酶3、基质金属蛋白酶13、ADAMTS4、ADAMTS5)的表达增加(P<0.05),细胞外基质(Ⅱ型胶原、蛋白聚糖)的合成减少(P<0.05),TLR4/核因子kB信号通路蛋白表达增加(P<0.05);与B组比较,D、E组细胞增殖升高,氧化应激反应及炎症反应减弱,细胞外基质降解酶的表达减少(P<0.05),细胞外基质的合成增加(P<0.05),TLR4/核因子kB信号通路蛋白表达减少(P<0.05),其中以E组效果更显著。②动物体内实验:术后4周,针刺+载药水凝胶组退变椎间盘的椎间盘高度指数、MRI指数以及椎间盘病理组织学评分均较针刺组有了显著改善,而单纯注射水凝胶或是丹酚酸B溶液虽也可一定程度地改善椎间盘退变情况,但均不及注射载药水凝胶组。③结果表明,负载丹酚酸B的甲基丙烯酰化明胶水凝胶可抑制退变椎间盘组织中的氧化应激反应与炎症反应,抑制细胞外基质的降解,缓解椎间盘退变的进程,该作用可能通过抑制TLR4/核因子kB信号通路来完成的。

腰椎间盘髓核组织工程研究进展

腰椎间盘退行性病变(退变)是脊柱外科常见多发疾病之一,临床以腰痛、下肢麻木和大小便功能障碍等为主要症状,其发生发展由多个因素共同决定,但目前病理生理学和细胞生物学机制尚未完全清晰。髓核组织工程是结合生物组织学和材料科学治疗疾病的一种新兴生物疗法,可有效治疗腰椎间盘退变。临床医师正确认识髓核组织工程和腰椎间盘退变之间的复杂关系,了解髓核组织工程在腰椎间盘退变中的应用及其机制,有利于临床上腰椎间盘退变治疗、腰椎间盘退变治疗后康复和人群腰椎间盘退变预防工作开展。

中药单体促髓核细胞自噬缓解椎间盘退变的研究进展

椎间盘退变是临床常见疾病,髓核与髓核细胞是椎间盘中主要的病变组织与细胞类型。髓核细胞受病理因素影响而加速衰老或出现代谢障碍时,髓核稳态被破坏,这导致了椎间盘退变的发生发展。自噬是细胞在病理环境下降解受损细胞器与异常蛋白质以维持正常生理功能的途径之一,能促进细胞自我调节以抵御致病因素影响。椎间盘退变时,髓核细胞处于应力失衡与代谢障碍的异常环境中,促进髓核细胞自噬可清除有害代谢产物累积、延缓细胞老化,这有助于维持髓核与椎间盘的健康生理状态。随着中医药治疗椎间盘退变疾病相关研究的不断深入,大量提取自传统中草药的单体成分被发现可以促进髓核细胞自噬以缓解椎间盘退变。根据最新的研究进展,讨论了髓核细胞的自噬与椎间盘退变的关联,共获取了14种在促进髓核细胞自噬以缓解椎间盘退变领域展现出潜力的中药单体,并将其作用机制归纳为以下4种:促自噬抑制髓核细胞凋亡、促自噬拮抗髓核细胞氧化应激、促自噬抑制髓核细胞外基质降解和促自噬促进髓核细胞外基质大分子合成,以期为中药单体调节髓核细胞自噬从而缓解椎间盘退变的研究提供新的思路与参考。

中药单体介导相关信号通路治疗椎间盘退行性变研究现状

背景:椎间盘退行性变是由一系列复杂的分子机制引起的椎间盘衰老、损伤,最终导致严重临床症状的一种病理性变化。中药具有价格低廉、无成瘾性、作用靶点多、毒副作用少、易被患者接受的特点,在椎间盘退行性变治疗中具有独特的优势。目的:综述中药单体干预相关信号通路治疗椎间盘退行性变的最新研究成果,阐析中药单体对椎间盘退行性变的作用机制,为未来的基础研究和临床治疗提供新的途径和理论依据。方法:第一作者通过中国知网、PubMed、维普、万方数据库查阅2018年1月至2023年2月国内外相关文献,检索词为“椎间盘,信号通路”“intervertebral disc,signal”。通过初步筛查文献标题及摘要,排除不符合的文献,最终选取72篇文献进行综述分析。结果与结论:中药单体可以调节多种经典信号通路,如Wnt/β-catenin、PI3K/Akt、mTOR、NF-κB、MAPK等,通过调节氧化应激调整细胞内促/抗凋亡蛋白的表达,刺激细胞自噬功能,减轻细胞炎性因子刺激,提高细胞外基质标志物的表达,减少基质降解酶的产成,维持细胞外基质的合成稳定,并诱导髓核间充质干细胞向髓核细胞分化,促进细胞内源性修复与重建,控制髓核细胞凋亡和衰老,提高髓核细胞的活性,从而改善椎间盘内部微环境,维持椎间盘正常生理功能,延缓椎间盘退行性变。