| Background and purpose:Chronic low back pain(CLBP)is one of the clinically important disabling factors,and appropriately 60%-80% people worldwide will experience the symptoms of CLBP in their lifetime.Previous studies have found that intervertebral disc degeneration is the most important pathogenic cause for CLBP.From an anatomical point of view,the intervertebral disc is mainly composed of the upper and lower cartilage-like endplate structures,the jellylike nucleus pulposus in the middle part,and the surrounding annulus fibrosus structure.Among those structures,the degeneration of nucleus pulposus tissue is widely considered to be the initiating factor for intervertebral disc degeneration.Intervertebral disc degeneration is a multifactorial disease,often closely related to genetic and environmental factors.The occurrence of intervertebral disc degeneration is often characteristized by a decrease in the extracellular matrix synthesis capacity of nucleus pulposus cells,an increase in extracellular matrix degradation,and an excessive secretion of inflammatory factors and infiltration of inflammation-related cells.These pathophysiological changes will further lead to extracellular matrix remodeling,including increased expression of matrix metalloproteinases(MMPs)and a disintegrin and metalloproteinase with thrombospondin motifs proteins(ADAMTS),and degradation of type Ⅱ collagen and aggrecan.At the same time,the degenerated intervertebral disc is accompanied by neurovascular ingrowth and infiltration,which eventually leads to the loss of the biomechanical function of the intervertebral disc,and causes the symptoms of CLBP.As a result,delaying the degenerative changes of the extracellular matrix in the nucleus pulposus tissue of the intervertebral disc and maintaining the number of nucleus pulposus cells are of great significance for the prevention and treatment of intervertebral disc degeneration.In this study,high-throughput proteomic sequencing was used to analyze the differences in protein expression between normal human intervertebral disc nucleus pulposus tissue and degenerated nucleus pulposus tissue,and to explore the change characteristics of differentially expressed protein or pathway in degenerated intervertebral disc nucleus pulposus tissue.Further,the researchers intend to systematically elucidate the intrinsic links between differentially expressed protein or pathway and intervertebral disc degeneration and the key molecules that promote intervertebral disc degeneration by combining human nucleus pulposus cell transcriptomic sequencing method with in vivo animal model and in vitro cell model experiments.The results of this study will lay a theoretical foundation for the future development of new treatments for intervertebral disc degeneration.Part Ⅰ:Proteome analysis revealed the correlation between tissue renin-angiotensin system and intervertebral disc degenerationMethods:(1)The normal and degenerated nucleus pulposus tissues of intervertebral disc were collected during operation according to the Pfirrmann scores of the intervertebral disc under T2-weighted magnetic resonance images(MRI);(2)A part of the normal or mildly degenerated nucleus pulposus tissue and the degenerated nucleus pulposus tissue were subjected to proteome analysis,and another part of the normal or mildly degenerated nucleus pulposus tissue was used to extract primary nucleus pulposus cells in vitro;(3)Toluidine blue staining and immunofluorescence for type II collagen were used to identify the obtained cells;(4)Bioinformatics was performed to analyze the proteomic sequencing results to detect differential expressed proteins and related pathways;(5)Histological and molecular biological analysis methods(immunohistochemistry,tissue TUNEL assay,realtime quantitative fluorescent PCR,and Western blot)were used to explore the expression changes of the components of the renin-angiotensin system in intervertebral disc nucleus pulposus tissue with different degenerative grades;(6)Enzyme-linked immunosorbent assay was used to explore the differences of angiotensin II levels in peripheral blood between normal and patients with intervertebral disc degeneration;(7)Mice model of intervertebral discs degeneration was established and lumbar intervertebral disc degeneration in mouse model was evaluated by MRI and histological analysis;(8)The expression changes of RAS components in the nucleus pulposus tissue of model mice were evaluated by molecular biology technique;(9)Enzyme-linked immunosorbent assay to explore the level of angiotensin Ⅱ in peripheral blood in normal mice and mice with intervertebral disc degeneration.Results:(1)The researcher successfully obtained primary nucleus pulposus cells in vitro;(2)Proteomic sequencing found that nucleus pulposus tissue of the degenerated intervertebral disc showed higher expression of renin-angiotensin system components;(3)In vitro histological and molecular biological analysis methods confirmed that inflammatory stimulation can lead to the activation of RAS,which was positively related to the intensity of inflammation;(4)Low concentration of AngⅡ facilitated to the activation of local RAS,while high concentration of AngⅡ inhibited local RAS activation.(5)Compared with normal individuals,the level of angiotensin II in peripheral blood of patients with intervertebral disc degeneration was significantly higher;(6)The RAS component in the intervertebral disc tissue of mice with lumbar intervertebral disc degeneration was activated,and the peripheral blood level AngⅡ of mice was higher than that of the sham-operated group,which was consistent with the characteristics of peripheral blood AngⅡ level in patients with intervertebral disc degeneration.Conclusions:(1)The researchers successfully isolated and cultured primary human nucleus pulposus cells,and verified the obtained nucleus pulposus cells.(2)The researchers used TMT high-throughput semi-quantitative proteomic analysis to reveal the activation of RAS in degenerated human intervertebral disc tissue,and the results of western blot and immunohistochemistry of human nucleus pulposus tissue samples confirmed the correlation of activated RAS with the severity of intervertebral disc degeneration.(3)The level of angiotensin Ⅱ in peripheral blood of patients had a good correlation and predictive ability for the grade of intervertebral disc degeneration.(4)In vitro cell experiments and animal model experiments further verified the correlation between the activated RAS and intervertebral disc degeneration.The above research results provide a sufficient foundation for the subsequent exploration of the internal mechanism of angiotensin II in regulating the degeneration of intervertebral disc.Part Ⅱ : The effects of angiotensin Ⅱ on the function of nucleus pulposus cells and potential mechanismsMethods:(1)Transcriptome sequencing analysis of human nucleus pulposus cells treated with angiotensin II in vitro;(2)Cell counting kit-8(CCK8)method to detect the effects of angiotensin II on the biological function of nucleus pulposus cells;(3)Senescenceβ-galactosidase staining kit was used to detect the effect of angiotensin Ⅱ on the senescence of nucleus pulposus cells;(4)Mitochondrial membrane potential kit was used to detect the effect of angiotensin Ⅱ on the mitochondrial membrane potential of nucleus pulposus cells;(5)TUNEL method was used to detect the effect of angiotensin Ⅱ on apoptosis of nucleus pulposus cells;(6)The effect of angiotensin Ⅱ on the level of intracellular reactive oxygen was evaluated;(7)Enzyme-linked immunosorbent assay was used to detect the effect of AngⅡ on the secretion of inflammatory factors in nucleus pulposus cells;(8)The effect of angiotensin Ⅱ on the degeneration and fibrosis phenotype of nucleus pulposus cells was detected by immunofluorescence;(9)The effect of high-level angiotensin Ⅱ on the natural degeneration of intervertebral disc in aging spontaneously hypertensive rats.Results:(1)Transcriptomic results showed that the expression of degeneration-related genes,inflammatory response-related genes,fibrosis-related genes,and nerve angiogenesisrelated genes in nucleus pulposus cell was significantly increased after angiotensin II treatment;(2)KEGG analysis showed angiotensin Ⅱ treatment activated cell cytokine and its receptor interaction pathway(Cytokine-cytokine receptor interaction),tumor necrosis factor pathway(TNF signaling pathway),interleukin 17 pathway(IL-17 signaling pathway),and rheumatoid arthritis-related pathways(Rheumatoid arthritis);(3)Angiotensin II inhibited the activity of nucleus pulposus cells and promoted the senescence of nucleus pulposus cells;(4)Angiotensin II promoted apoptosis of human nucleus pulposus cells;(5)Angiotensin II increased the level of oxidative stress in nucleus pulposus cells;(6)Angiotensin II promoted the degeneration and fibrosis phenotype of human nucleus pulposus cells;(7)Angiotensin Ⅱ promoted the activation of inflammasome and the secretion of inflammatory factors in human nucleus pulposus cells;(9)Aging spontaneously hypertensive rats showed more severe intervertebral disc degeneration than control rats.Conclusions:(1)Angiotensin Ⅱ significantly promoted the expression of genes related to nucleus pulposus cell degeneration,fibrosis and neovascularization;(2)Angiotensin Ⅱ can reduce the cell activity of nucleus pulposus cells,and increase apoptosis,senescence,oxidation stress,inflammation and activation of nucleus pulposus cell,and enhance the polarization of M1 macrophages;(3)Angiotensin Ⅱ can promote the degeneration and fibrosis phenotype of human nucleus pulposus cells;(4)High-level Angiotensin Ⅱ in peripheral blood and local activated RAS in nucleus pulposus tissue can accelerate the occurrence of intervertebral disc degeneration in aging SHR;(5)Nrf2/NF-κB signaling pathway may exert a critical role in the regulation of RAS in promoting intervertebral disc degeneration.In conclusion,angiotensin II is involved in the onset and development of intervertebral disc degeneration by activating oxidative stress and inflammatory responses in nucleus pulposus cells,leading to cell apoptosis,senescence,and degeneration-related phenotypes.Part III: The therapeutic effects of angiotensin Ⅱ receptor antagonist(candesartan)on intervertebral disc degeneration induced by needle injuryMethods:(1)Cell counting kit-8(CCK8)method was utilized to assess the effects of candesartan on the cell viability of nucleus pulposus cells after angiotensin II treatment;(2)TUNEL assay was performed to detect the effects of candesartan on nucleus pulposus cells after angiotensin II treatment.(3)Real-time quantitative fluorescence PCR was carried out to evaluate the effects of candesartan on the expression of degeneration-related genes in nucleus pulposus cells after angiotensin Ⅱ treatment;(4)Cell immunofluorescence was used to detect candesartan effects of candesartan on the inflammatory response,oxidative stress,and degenerative and fibrotic phenotypes of nucleus pulposus cells after angiotensin II treatment;(5)Mice model of intervertebral disc degeneration was established by acupuncture at the caudal vertebrae and the therapeutic effect of candesartan on intervertebral disc degeneration was evaluated by imaging and histological methods.Results:(1)In vitro cell experiments showed that candesartan alleviated angiotensinⅡ-mediated apoptosis,inflammatory response,and oxidative stress in nucleus pulposus;(2)In vitro cell experiments showed that candesartan alleviated angiotensin Ⅱ-mediated degeneration phenotype in nucleus pulposus cells;(3)Imaging and histomorphological analysis showed that candesartan could alleviate acupuncture-mediated coccygeal disc degeneration in mice;(4)Tissue immunofluorescence analysis showed that candesartan could alleviate acupuncture-mediated expression of inflammation-related proteins and factors in the nucleus pulposus cells;(5)Tissue immunofluorescence analysis showed that candesartan could alleviate the acupuncture-mediated decrease of cell viability in nucleus pulposus cells;(6)Tissue immunofluorescence analysis showed that candesartan could alleviate acupuncture-mediated degeneration and fibrosis phenotypes of nucleus pulposus cells in mice.Conclusions: Within a certain concentration range(10n M-100 n M),the AGTR1 antagonist,candesartan,can alleviate the degeneration of nucleus pulposus cells by reducing apoptosis,inflammatory response,and oxidative stress in the nucleus pulposus in vitro.In experiments in vivo,candesartan treatment can alleviate acupuncture-mediated local cell apoptosis and inflammatory response in the nucleus pulposus tissue of the intervertebral disc,and exert an effective therapeutic effect in delaying the degeneration of the intervertebral disc.In conclusion,inhibition of RAS components is expected to become a new clinical target for the treatment of intervertebral disc degeneration in the future.Summary:In this study,we first used high-throughput proteomics to analyze human normal and degenerative disc nucleus pulposus tissues and found that the renin-angiotensin system was significantly activated in degenerative disc nucleus pulposus tissues.At the same time,histological studies revealed that in the degenerated intervertebral disc nucleus pulposus,the expression of renin-angiotensin system components was significantly increased.In vitro cell experiments and animal model experiments also further verified the correlation between the activation of RAS and intervertebral disc degeneration,and there was an AngⅡ-RAS feedback mechanism at the level of nucleus pulposus cells..Furthermore,by analyzing the levels of peripheral blood angiotensin Ⅱ in normal people and patients with intervertebral disc degeneration,it is clear that angiotensin Ⅱ showed a good correlation and predictive ability with the grade of intervertebral disc degeneration.Subsequently,in cell experiments,the researcher combined transcriptomic analysis and cell experimental studies to reveal that angiotensin Ⅱ could lead to intervertebral disc degeneration by promoting the dysfunction of human nucleus pulposus cells and related molecular mechanisms.Animal model experiments also confirmed that RAS overactivation can accelerate intervertebral disc degeneration.Finally,the researcher discovered the therapeutic effect of candesartan on antagonizing angiotensin II-induced nucleus pulposus cell dysfunction and alleviating acupuncture-mediated intervertebral disc degeneration through in vivo and in vitro experiments.The results of this study will provide a new theoretical basis for the formulation of new treatment plans for intervertebral disc degeneration in the future. |
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