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 nucl...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.展开更多
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 mesenchym...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.展开更多
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 overexpress...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.展开更多
Transcription factor Brachyury,a protein containing 435 amino acids,has been widely investigated and reported in notochord differentiation and nucleus pulposus development.The crucial functions and underlying mechanis...Transcription factor Brachyury,a protein containing 435 amino acids,has been widely investigated and reported in notochord differentiation and nucleus pulposus development.The crucial functions and underlying mechanisms by Brachyury are discussed in this paper,which suggests Brachyury can be developed into a potential novel target for the therapy of intervertebral disc degeneration.展开更多
BACKGROUND In degenerative intervertebral disc(IVD),an unfavorable IVD environment leads to increased senescence of nucleus pulposus(NP)-derived mesenchymal stem cells(NPMSCs)and the inability to complete the differen...BACKGROUND In degenerative intervertebral disc(IVD),an unfavorable IVD environment leads to increased senescence of nucleus pulposus(NP)-derived mesenchymal stem cells(NPMSCs)and the inability to complete the differentiation from NPMSCs to NP cells,leading to further aggravation of IVD degeneration(IDD).Urolithin A(UA)has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress.AIM To explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism.METHODS In vitro,we harvested NPMSCs from rat tails,and divided NPMSCs into four groups:the control group,H2O2 group,H2O2+UA group,and H2O2+UA+SR-18292 group.Senescence-associatedβ-Galactosidase(SA-β-Gal)activity,cell cycle,cell proliferation ability,and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α(SIRT1/PGC-1α)pathway-related proteins and mRNA were used to evaluate the protective effects of UA.In vivo,an animal model of IDD was constructed,and Xrays,magnetic resonance imaging,and histological analysis were used to assess whether UA could alleviate IDD in vivo.RESULTS We found that H2O2 can cause NPMSCs senescence changes,such as cell cycle arrest,reduced cell proliferation ability,increased SA-β-Gal activity,and increased expression of senescence-related proteins and mRNA.After UA pretreatment,the abovementioned senescence indicators were significantly alleviated.To further demonstrate the mechanism of UA,we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1αpathway that regulates mitochondrial function.UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1αpathway.In vivo,we found that UA treatment alleviated an animal model of IDD by assessing the disc height index,Pfirrmann grade and the histological score.CONCLUSION In summary,UA could activate the SIRT1/PGC-1αsignaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.展开更多
Objective:To discuss the effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38 MARK phosphorylation in autologous nucleus pulposus transplantation model of rats.Methods:A tota...Objective:To discuss the effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38 MARK phosphorylation in autologous nucleus pulposus transplantation model of rats.Methods:A total of 60 SD rats were randomly divided into the blank group,model group and Yaobitong capsule group,with 20 rats in each group.The animal model of autologous nucleus pulposus transplantation around the lumbar nerve root was built.Three days after the modeling,rats were given the drugs for the first time,while rats in the model group were given the equivalent normal saline.After 30 d of continuous administration,samples were collected from rats.HE staining was performed on the dorsal root ganglion of L4 and L5 spinal cord of rats in each group and the expression of p38 MARK phosphorylation was measured.All data were treated with the statistical analysis.Results:The histological examination showed that the histomorphology of dorsal root ganglion in the Yaobitong capsule group was more significantly improved than the one in the model group,while the results of western blot showed that Yaobitong capsule could significandy inhibit the level of p38 MAPK phosphorylation of dorsal root ganglion cells.Conclusions:Yaobitong capsule can improve the symptoms and nerve radiculopathy of autologous nucleus pulposus transplantation of rats and its mechanism may be associated with its inhibiting effect on the level of p38 MAPK phosphorylation.展开更多
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 contrib...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.展开更多
To find a new source of seed cells for constructing tissue-engineered intervertebral disc,nucleus pulposus(NP) cells and mesenchymal stem cells(MSCs) were isolated from New Zealand white rabbits.The nucleus pulposus c...To find a new source of seed cells for constructing tissue-engineered intervertebral disc,nucleus pulposus(NP) cells and mesenchymal stem cells(MSCs) were isolated from New Zealand white rabbits.The nucleus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact.Morphological changes were observed every 12 h.Semi-quantitaive reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9,aggreacan and type Ⅱ collagen every 24 h after the co-culture.MSCs treated with direct contact rounded up and presented a ring-like appearance.The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h.No significant change was found after coculture without direct contact.Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.展开更多
This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 ad...This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type Ⅱ collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at G l phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G 1 phase and failed to enter S phase. The expression of type Ⅱ collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.展开更多
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,l...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.展开更多
Objective: We have reported the presence of discogenic visceral pain secondary to anterior herniation of the lumbar disc. The aim of this study was to observe the inflammatory response of the sympathetic trunk to an a...Objective: We have reported the presence of discogenic visceral pain secondary to anterior herniation of the lumbar disc. The aim of this study was to observe the inflammatory response of the sympathetic trunk to an autologous degenerative nucleus pulposus(NP) injection under fluoroscopy. Methods: A total of 72 rats were used. In 24 rats,the autologous NP suspension was injected into the right sympathetic trunk. Next,food intake and body weight of each rat were monitored for 14 days. Fourteen days after the injection,the right lumbar sympathetic trunk was harvested for histological assessment,and protein levels of IL-1 β,IL-6,and TNF-α were quantified via ELISA. Results: In the NP-treated group,endoneural hyperemia and intensive infiltration of inflammatory cells could be identified in sections of the sympathetic trunk by the hematoxylin and eosin(H&E) stain. Meanwhile,elevated concentrations of IL-1 β,IL-6,and TNF-α were determined in the sympathetic trunk of the NP group as compared to that of the na?ve and control groups,which indicated the development of an inflammatory response. Furthermore,food intake and body weight of rats in the NP group decreased significantly. Conclusions: The results indicated that an inflammatory response in the sympathetic trunk can be caused by anterior herniation of the lumbar disc,which can generate a sympathetic inflammatory pain response.展开更多
OBJECTIVE: To investigate the analgesic effect of Gua Sha and its underlying mechanism in rats with noncompressive lumbar disk herniation induced by autologous nucleus pulposus.METHODS: A rat model of noncompressive l...OBJECTIVE: To investigate the analgesic effect of Gua Sha and its underlying mechanism in rats with noncompressive lumbar disk herniation induced by autologous nucleus pulposus.METHODS: A rat model of noncompressive lumbar disk herniation was established and rats were randomly divided into model group, sham group, and Gua Sha group(24 in each group). Gua Sha was performed from the 5 th day after the surgery, once every other day, 3 times for a course of treatment,and totally 3 courses. The thermal withdrawal latency was evaluated using the intelligent hot plate one day before the surgery, and on days 4(the day before the treatment), 10(the end of the firstcourse), 16(the end of the second course) and 22(the end of the third course). On days 4, 10, 16 and22, six rats in each group were picked randomly and their blood samples were drawn to assess the expression of interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-alpha(TNF-α).RESULTS: Compared to rats in the sham group, the application of nucleus pulposus to right L5 dorsal root ganglion induced prolonged thermal hyperalgesia, and up-regulated the expression of IL-1β,IL-6 and TNF-α in serum(P < 0.01). The therapy of Gua Sha attenuated thermal hyperalgesia potently,inhibited the expression of IL-1β, IL-6 and TNF-α in a time-dependent manner(P < 0.01). There were no significant differences in the thermal withdrawal latency and the expression of inflammatory cytokines between the sham and Gua Sha groups at the end of the treatment(P > 0.01).CONCLUSION: The current study showed that Gua Sha might alleviate thermal hyperalgesia in rats with lumbar disc herniation induced by autologous nucleus pulposus via inhibiting the expression of proinflammatory cytokins.展开更多
There are many documented sex differences in the clinical course,symptom expression profile,and treatment response of Parkinson’s disease,creating additional challenges for patient management.Although subthalamic nuc...There are many documented sex differences in the clinical course,symptom expression profile,and treatment response of Parkinson’s disease,creating additional challenges for patient management.Although subthalamic nucleus deep brain stimulation is an established therapy for Parkinson’s disease,the effects of sex on treatment outcome are still unclear.The aim of this retrospective observational study,was to examine sex differences in motor symptoms,nonmotor symptoms,and quality of life after subthalamic nucleus deep brain stimulation.Outcome measures were evaluated at 1 and 12 months post-operation in 90 patients with Parkinson’s disease undergoing subthalamic nucleus deep brain stimulation aged 63.00±8.01 years(55 men and 35 women).Outcomes of clinical evaluations were compared between sexes via a Student’s t-test and within sex via a paired-sample t-test,and generalized linear models were established to identify factors associated with treatment efficacy and intensity for each sex.We found that subthalamic nucleus deep brain stimulation could improve motor symptoms in men but not women in the on-medication condition at 1 and 12 months post-operation.Restless legs syndrome was alleviated to a greater extent in men than in women.Women demonstrated poorer quality of life at baseline and achieved less improvement of quality of life than men after subthalamic nucleus deep brain stimulation.Furthermore,Hoehn-Yahr stage was positively correlated with the treatment response in men,while levodopa equivalent dose at 12 months post-operation was negatively correlated with motor improvement in women.In conclusion,women received less benefit from subthalamic nucleus deep brain stimulation than men in terms of motor symptoms,non-motor symptoms,and quality of life.We found sex-specific factors,i.e.,Hoehn-Yahr stage and levodopa equivalent dose,that were related to motor improvements.These findings may help to guide subthalamic nucleus deep brain stimulation patient selection,prognosis,and stimulation programming for optimal therapeutic efficacy in Parkinson’s disease.展开更多
基金funded by the Youth Research Fund of the Peking Union Medical College Hospital(No.pumch201911708).
文摘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.
基金Supported by the National Natural Science Foundation of China,No.82172462,No.81972136the Traditional Chinese Medicine Science and Technology Development Plan Project of Jiangsu Province,No.YB2020085Cross Cooperation Project of Northern Jiangsu People’s Hospital,No.SBJC21014.
文摘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.
基金Supported by National Natural Science Foundation of China,No.81972136National Natural Science Foundation for Young Scholars of China,No.81401830+3 种基金Guangxi Natural Science Foundation General Project,No.2018JJA14775Young Medical Scholars Major Program of Jiangsu Province,No.QNRC2016342Innovation Team Project of Jiangsu Province,No.CXTDB2017004and Key Funding Project of Maternal and Child Health Research of Jiangsu Province,No.F201801.
文摘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.
基金This work was supported by the Gusu Health Talent Project of Suzhou(Grant No.GSWS2020056)“333”Talent Project of Jiangsu Province(Grant No.BRA2017057).
文摘Transcription factor Brachyury,a protein containing 435 amino acids,has been widely investigated and reported in notochord differentiation and nucleus pulposus development.The crucial functions and underlying mechanisms by Brachyury are discussed in this paper,which suggests Brachyury can be developed into a potential novel target for the therapy of intervertebral disc degeneration.
基金National Natural Science Foundation of China,No.81972136Young Medical Scholars Major Program of Jiangsu Province,No.QNRC2016342+1 种基金Key Funding Project of Maternal and Child Health Research of Jiangsu Province,No.F201801and Highlevel Health Professionals"Six projects"Top-notch Talent Research Program of Jiangsu Province,No.LGY2019035.
文摘BACKGROUND In degenerative intervertebral disc(IVD),an unfavorable IVD environment leads to increased senescence of nucleus pulposus(NP)-derived mesenchymal stem cells(NPMSCs)and the inability to complete the differentiation from NPMSCs to NP cells,leading to further aggravation of IVD degeneration(IDD).Urolithin A(UA)has been proven to have obvious effects in delaying cell senescence and resisting oxidative stress.AIM To explore whether UA can alleviate NPMSCs senescence and to elucidate the underlying mechanism.METHODS In vitro,we harvested NPMSCs from rat tails,and divided NPMSCs into four groups:the control group,H2O2 group,H2O2+UA group,and H2O2+UA+SR-18292 group.Senescence-associatedβ-Galactosidase(SA-β-Gal)activity,cell cycle,cell proliferation ability,and the expression of senescence-related and silent information regulator of transcription 1/PPAR gamma coactivator-1α(SIRT1/PGC-1α)pathway-related proteins and mRNA were used to evaluate the protective effects of UA.In vivo,an animal model of IDD was constructed,and Xrays,magnetic resonance imaging,and histological analysis were used to assess whether UA could alleviate IDD in vivo.RESULTS We found that H2O2 can cause NPMSCs senescence changes,such as cell cycle arrest,reduced cell proliferation ability,increased SA-β-Gal activity,and increased expression of senescence-related proteins and mRNA.After UA pretreatment,the abovementioned senescence indicators were significantly alleviated.To further demonstrate the mechanism of UA,we evaluated the mitochondrial membrane potential and the SIRT1/PGC-1αpathway that regulates mitochondrial function.UA protected mitochondrial function and delayed NPMSCs senescence by activating the SIRT1/PGC-1αpathway.In vivo,we found that UA treatment alleviated an animal model of IDD by assessing the disc height index,Pfirrmann grade and the histological score.CONCLUSION In summary,UA could activate the SIRT1/PGC-1αsignaling pathway to protect mitochondrial function and alleviate cell senescence and IDD in vivo and vitro.
基金supported by Shandong Natural Science Fund(No.:Y2008C147)
文摘Objective:To discuss the effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38 MARK phosphorylation in autologous nucleus pulposus transplantation model of rats.Methods:A total of 60 SD rats were randomly divided into the blank group,model group and Yaobitong capsule group,with 20 rats in each group.The animal model of autologous nucleus pulposus transplantation around the lumbar nerve root was built.Three days after the modeling,rats were given the drugs for the first time,while rats in the model group were given the equivalent normal saline.After 30 d of continuous administration,samples were collected from rats.HE staining was performed on the dorsal root ganglion of L4 and L5 spinal cord of rats in each group and the expression of p38 MARK phosphorylation was measured.All data were treated with the statistical analysis.Results:The histological examination showed that the histomorphology of dorsal root ganglion in the Yaobitong capsule group was more significantly improved than the one in the model group,while the results of western blot showed that Yaobitong capsule could significandy inhibit the level of p38 MAPK phosphorylation of dorsal root ganglion cells.Conclusions:Yaobitong capsule can improve the symptoms and nerve radiculopathy of autologous nucleus pulposus transplantation of rats and its mechanism may be associated with its inhibiting effect on the level of p38 MAPK phosphorylation.
基金the National Natural Science Foundation of China,No.81772399
文摘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.
基金a grant from the National Natural Sciences Foundation of China (No. 30772206)
文摘To find a new source of seed cells for constructing tissue-engineered intervertebral disc,nucleus pulposus(NP) cells and mesenchymal stem cells(MSCs) were isolated from New Zealand white rabbits.The nucleus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact.Morphological changes were observed every 12 h.Semi-quantitaive reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9,aggreacan and type Ⅱ collagen every 24 h after the co-culture.MSCs treated with direct contact rounded up and presented a ring-like appearance.The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h.No significant change was found after coculture without direct contact.Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.
文摘This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type Ⅱ collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at G l phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G 1 phase and failed to enter S phase. The expression of type Ⅱ collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.
基金Nature Science Foundation of Zhejiang Province(Y20H060063,LY19H060005,LQ18H060003,LZ22H090003)National Natural Science Foundation of China(NO.82072465,NO.81772379,NO.81972096,NO.82172457,NO.82002327)+1 种基金China Postdoctoral Science Foundation(2017M612011)Scientific Research Fund of Zhejiang Provincial Education Department(Y201941476).
文摘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.
基金Supported by the Beijing Municipal Administration of Hospitals Youth program,code:20160807 Capital Medical University basic-clinical research cooperation fund project,code:16JL43
文摘Objective: We have reported the presence of discogenic visceral pain secondary to anterior herniation of the lumbar disc. The aim of this study was to observe the inflammatory response of the sympathetic trunk to an autologous degenerative nucleus pulposus(NP) injection under fluoroscopy. Methods: A total of 72 rats were used. In 24 rats,the autologous NP suspension was injected into the right sympathetic trunk. Next,food intake and body weight of each rat were monitored for 14 days. Fourteen days after the injection,the right lumbar sympathetic trunk was harvested for histological assessment,and protein levels of IL-1 β,IL-6,and TNF-α were quantified via ELISA. Results: In the NP-treated group,endoneural hyperemia and intensive infiltration of inflammatory cells could be identified in sections of the sympathetic trunk by the hematoxylin and eosin(H&E) stain. Meanwhile,elevated concentrations of IL-1 β,IL-6,and TNF-α were determined in the sympathetic trunk of the NP group as compared to that of the na?ve and control groups,which indicated the development of an inflammatory response. Furthermore,food intake and body weight of rats in the NP group decreased significantly. Conclusions: The results indicated that an inflammatory response in the sympathetic trunk can be caused by anterior herniation of the lumbar disc,which can generate a sympathetic inflammatory pain response.
基金Supported by National Natural Science Foundation of China:Differential proteomics-based research on the intervention effects,and mechanism of scraping therapy,treating lumbar disc herniation(No.81473791)Natural Science Foundation of Basic Research Program in Jiangsu Province:Proteomics research on the intervention effects,and mechanism of scraping therapy for lumbar disc herniation based on i TRAQ technology(No.BK20141464)
文摘OBJECTIVE: To investigate the analgesic effect of Gua Sha and its underlying mechanism in rats with noncompressive lumbar disk herniation induced by autologous nucleus pulposus.METHODS: A rat model of noncompressive lumbar disk herniation was established and rats were randomly divided into model group, sham group, and Gua Sha group(24 in each group). Gua Sha was performed from the 5 th day after the surgery, once every other day, 3 times for a course of treatment,and totally 3 courses. The thermal withdrawal latency was evaluated using the intelligent hot plate one day before the surgery, and on days 4(the day before the treatment), 10(the end of the firstcourse), 16(the end of the second course) and 22(the end of the third course). On days 4, 10, 16 and22, six rats in each group were picked randomly and their blood samples were drawn to assess the expression of interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-alpha(TNF-α).RESULTS: Compared to rats in the sham group, the application of nucleus pulposus to right L5 dorsal root ganglion induced prolonged thermal hyperalgesia, and up-regulated the expression of IL-1β,IL-6 and TNF-α in serum(P < 0.01). The therapy of Gua Sha attenuated thermal hyperalgesia potently,inhibited the expression of IL-1β, IL-6 and TNF-α in a time-dependent manner(P < 0.01). There were no significant differences in the thermal withdrawal latency and the expression of inflammatory cytokines between the sham and Gua Sha groups at the end of the treatment(P > 0.01).CONCLUSION: The current study showed that Gua Sha might alleviate thermal hyperalgesia in rats with lumbar disc herniation induced by autologous nucleus pulposus via inhibiting the expression of proinflammatory cytokins.
基金support by the National Nature Science Foundation of China,Nos.81830033,61761166004(both to JGZ)。
文摘There are many documented sex differences in the clinical course,symptom expression profile,and treatment response of Parkinson’s disease,creating additional challenges for patient management.Although subthalamic nucleus deep brain stimulation is an established therapy for Parkinson’s disease,the effects of sex on treatment outcome are still unclear.The aim of this retrospective observational study,was to examine sex differences in motor symptoms,nonmotor symptoms,and quality of life after subthalamic nucleus deep brain stimulation.Outcome measures were evaluated at 1 and 12 months post-operation in 90 patients with Parkinson’s disease undergoing subthalamic nucleus deep brain stimulation aged 63.00±8.01 years(55 men and 35 women).Outcomes of clinical evaluations were compared between sexes via a Student’s t-test and within sex via a paired-sample t-test,and generalized linear models were established to identify factors associated with treatment efficacy and intensity for each sex.We found that subthalamic nucleus deep brain stimulation could improve motor symptoms in men but not women in the on-medication condition at 1 and 12 months post-operation.Restless legs syndrome was alleviated to a greater extent in men than in women.Women demonstrated poorer quality of life at baseline and achieved less improvement of quality of life than men after subthalamic nucleus deep brain stimulation.Furthermore,Hoehn-Yahr stage was positively correlated with the treatment response in men,while levodopa equivalent dose at 12 months post-operation was negatively correlated with motor improvement in women.In conclusion,women received less benefit from subthalamic nucleus deep brain stimulation than men in terms of motor symptoms,non-motor symptoms,and quality of life.We found sex-specific factors,i.e.,Hoehn-Yahr stage and levodopa equivalent dose,that were related to motor improvements.These findings may help to guide subthalamic nucleus deep brain stimulation patient selection,prognosis,and stimulation programming for optimal therapeutic efficacy in Parkinson’s disease.
