Massage ameliorates lumbar disc herniation-related radicular pain in rats by suppressing TLR4/NLRP3 inflammasome signaling transduction.

BACKGROUND: Lumbar disc herniation (LDH) is a common spinal disease that can cause severe radicular pain. Massage, also known as Tuina in Chinese, has been indicated to exert an analgesic effect in patients with LDH. Nonetheless, the mechanism underlying this effect of massage on LDH remains unclarified. METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. A rat LDH model was established by autologous nucleus pulpous (NP) implantation, followed by treatment with or without massage. A toll-like receptor 4 (TLR4) antagonist TAK-242 was administrated to rats for blocking TLR4. Behavioral tests were conducted to examine rat mechanical and thermal sensitivities. Western blotting was employed for determining TLR4 and NLRP3 inflammasome-associated protein levels in the spinal dorsal horn (SDH). Immunofluorescence staining was implemented for estimating the microglial marker Iba-1 expression in rat SDH tissue. RESULTS: NP implantation induced mechanical allodynia and thermal hyperalgesia in rat ipsilateral hindpaws and activated TLR4/NLRP3 inflammasome signaling transduction in the ipsilateral SDH. Massage therapy or TAK-242 administration relieved NP implantation-triggered pain behaviors in rats. Massage or TAK-242 hindered microglia activation and blocked TLR4/NLRP3 inflammasome activation in ipsilateral SDH of LDH rats. CONCLUSION: Massage ameliorates LDH-related radicular pain in rats by suppressing microglia activation and TLR4/NLRP3 inflammasome signaling transduction.

Warm Acupuncture Reduces Pain and Inflammation in Rats with Lumbar Disc Herniation Induced by Autologous Nucleus Pulposus Transplantation via Regulating p38MAPK/NF-κB Pathway.

Background: : Warm acupuncture (WA) has analgesic and anti-inflammatory effects. However, the underlying mechanism of these effects remain unclear. Objectives: : To explore the analgesic and anti-inflammatory effects of WA and the potential underlying mechanism in male Sprague-Dawley rats with non-compressive lumbar disk herniation (LDH) caused by autologous nucleus pulposus (NP) transplantation. Methods: : We used low-frequency (2 Hz) electrical stimulation and WA (40℃) to treat GB30 and BL54 acupoints in rats for 30 mins per day. We monitored the paw withdrawal threshold of rats during the experiment and measured serum cytokine levels using commercial kits. Dorsal root ganglion (DRG) tissue pathology was analyzed via H&E staining. We used qRT-PCR to measure the mRNA expression levels of IL-1ß, IL-6, and TNF-α genes in DRG. Western blot was used to analyze the expression levels of IL-1ß, IL-6, TNFα, P-p38MAPK, p38MAPK, P-IκBα, IκB α, and NF-κB p65 proteins. Results: : WA treatment significantly increased the pain threshold of rats, reduced serum IL-6, PEG2, NO, SP, NP-Y, and MMP-3 levels, and effected histopathological improvements in the DRG in rats. Moreover, WA treatment significantly downregulated the expression levels of inflammation-associated genes (Il-1ß, Il-6, and Tnf-α) and proteins (IL-1ß, IL-6, TNF-α, P-p38MAPK, P-IκBα, and NF-κB p65) in the DRG of non-compressive LDH rats. Conclusion: : WA can alleviate pain and inhibit inflammatory response in rats with non-compressive LDH caused by autologous NP transplantation, and these effects are likely associated with the inhibition of the p38MAPK/NF-κB pathway.

Therapeutic effect and mechanism of Yougui Wan in rats with intervertebral disk degeneration.

OBJECTIVE: To explore the potential mechanism of Yougui Wan on deformed lumbar intervertebral disk structure in rats. METHODS: Thirty male Sprague-Dawley rats were randomly divided into 3 groups, with 10 rats in each group. The animals in the blank control group were healthy rats without specific treatment, and those in the model group and traditional Chinese medicine (TCM) group were used to establish the intervertebral disk degeneration (IDD) model by puncturing the annulus. Four weeks after modeling, rats in the TCM group were administered Yougui Wan by gavage for 2 consecutive weeks. Serum interleukin-6 (IL-10), macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNF-α) levels were measured by ELISA, and the protein expression levels of collagen II and Notch1 in intervertebral disk tissues were examined by Western blotting. Apoptosis was detected by the TUNEL method. RESULTS: Compared with those in the blank group, IL-10, MIF and TNF-α levels in the model group and TCM group were increased (P < 0.05), the protein expression levels of collagen II were decreased, and the protein expression levels of Notch1 were increased. Compared with those in the model group, the levels of IL-10 in the TCM group were increased (P < 0.05), the levels of MIF and TNF-α were decreased (P < 0.05), the protein expression levels of collagen II were increased, and the protein expression levels of Notch1 were decreased. CONCLUSION: Yougui Wan can inhibit the inflammatory response in IDD rats, reduce the degradation of extracellular matrix, reduce apoptosis in nucleus pulposus cells, and alleviate intervertebral disk degeneration. The mechanism may be related to the regulation of the Notch signaling pathway.

[Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway].

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-ß-galactosidase(SA-ß-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1ß(IL-1ß), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1ß and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1ß and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Traditional Chinese Medicine formula Bu-Shen-Huo-Xue-Fang (BSHXF) protects nucleus pulposus cells against the inflammatory and oxidative stress-induced degenerative changes.

Degeneration of the intervertebral disc is primarily caused by the loss of nucleus pulposus cells (NPCs) and extracellular matrix (ECM) (IDD). Bu-Shen-Huo-Xue-Fang (BSHXF), a traditional Chinese medicine decoction, has been used to treat IDD in clinical; nevertheless, the active components and underlying molecular mechanisms remain unknown. BSHXF improved IL-1ß and H2O2 stimulation-induced injuries on NPCs by promoting cell viability, increasing ECM deposition, inhibiting cell senescence, and decreasing the levels of inflammatory factors. The active ingredients in BSHXF were identified by LC-MS/MS analysis; three active ingredients from the principal drugs, Aucubin, Tanshinol, and Tanshinone II A promoted NPC viability; and Aucubin and Tanshinol promoted NPC viability more. Aucubin and Tanshinol, respectively, improved H2O2 stimulation-induced injuries on NPCs by promoting cell viability, increasing ECM deposition, inhibiting cell senescence, and decreasing the levels of inflammatory factors. The activator of NF-κB and Wnt signaling pathways attenuated Aucubin and Tanshinol's protective effects by promoting ECM degradation and NPC senescence. Aucubin, Tanshinol, and Tanshinone II A were identified as the most potent compounds in BSHXF protection against degenerative changes in NPCs. The NF-κB and Wnt signaling pathways might be involved in the protective effects of Aucubin and Tanshinol against H2O2-induced degenerative changes.

BSHXF-medicated serum combined with ADSCs regulates the TGF-ß1/Smad pathway to repair oxidatively damaged NPCs and its component analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lower back pain (LBP) is a common and frequent clinical condition, and intervertebral disc degeneration (IDD) is recognized as the leading cause of LBP, typically manifested by increased nucleus pulposus cell (NPC) senescence and death. In recent years, the treatment of IDD with stem cell injections has had great potential compared to surgical treatment. Combining the two may achieve better results, as BuShenHuoXueFang (BSHXF) is an herbal formula that improves the survival rate of transplanted stem cells and enhances their efficacy. AIM OF THE STUDY: We aimed to qualitatively and quantitatively analyze BSHXF-medicated serum and investigate the molecular mechanism of BSHXF-mediated serum in promoting the differentiation of adipose mesenchymal stem cells (ADSCs) into NPCs and delaying the senescence of NPCs by regulating the TGF-ß1/Smad pathway. MATERIALS AND METHODS: In this study, an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometer (UPLC-Q-TOF-MS) was used to establish a method for the analysis of rat serum samples to track the active components in vivo; the oxidative damage model of NPCs was induced by T-BHP, and a Transwell chamber was used to construct a coculture system of ADSCs and NPCs. Flow cytometry was used to determine the cell cycle; SA-ß-Gal staining was used to assess cell senescence; ELISA was used to detect IL-1ß, IL-6 inflammatory factors, CXCL-1, CXCL-3, CXCL-10 chemokines, and TGF-ß1 in the supernatants of ADSCs and NPCs. WB was used to detect COL2A1, COL1A1, and Aggrecan in ADSCs to assess the manifestation of NP differentiation in ADSCs, and the WB method was used to detect COL2A1, COL1A1, Aggrecan, p16, p21, p53, and p-p53 protein expression in NPCs to reflect the cellular senescence status and to detect TGF-ß1, Smad2, Smad3, p- Smad2, and p- Smad3 protein expression in NPCs to reflect the pathway condition. RESULTS: We finally identified 70 blood components and their metabolites, including 38 prototypes, from the BSHXF-medicated serum. Compared with that in the nonmedicated serum group, the TGF-ß1/Smad pathway was activated in the medicated serum group, ADSCs moved toward NPC characteristics, the number of NPCs in the S/G2M phase increased, the number of senescent NPCs decreased, IL-1ß and IL-6 inflammatory factors in the Transwell decreased, CXCL-1, CXCL-3, and CXCL-10 chemokines decreased, and the expression of p16, p21, p53 and p-p53 proteins in NPCs was inhibited. CONCLUSION: By regulating the TGF-ß1/Smad pathway, BSHXF-medicated serum promoted ADSCs to NPCs, effectively alleviated the cycle blockage of NPCs after oxidative damage, encouraged the growth and proliferation of NPCs, delayed the aging of NPCs, improved the deteriorating microenvironment around NPCs, and repaired oxidatively damaged NPCs. The combination of BSHXF or its compounds with ADSCs has great potential for the treatment of IDD in the future.

Pharmacological network analysis of the functions and mechanism of kaempferol from Du Zhong in intervertebral disc degeneration (IDD).

Background: Senescence and apoptosis of the nucleus pulposus cells (NPCs) are essential components of the intervertebral disc degeneration (IDD) process. Senescence and anti-apoptosis treatments could be effective ways to delay or even stop disc degeneration. IDD has been treated with Eucommia ulmoides Oliver (Du Zhong, DZ) and its active ingredients. However, the roles and mechanisms of DZ in NPC apoptosis and senescence remain unclear. Methods: Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to select the main active ingredients of DZ with the threshold of oral bioavailability (OB) â€‹≥ â€‹30% and drug-likeness (DL) â€‹≥ â€‹0.2. GSE34095 contained expression profile of degenerative intervertebral disc tissues and non-degenerative intervertebral disc tissues were downloaded for different expression genes analysis. The disease targets genes of IDD were retrieved from GeneCards. The online tool Metascape was used for functional enrichment annotation analysis. The specific effects of the ingredient on IL-1ß treated NPC cell proliferation, cell senescence, reactive oxygen species (ROS) accumulation and cell apoptosis were determined by CCK-8, SA-ß-gal staining, flowcytometry and western blot assays. Results: A total of 8 active compounds of DZ were found to meet the threshold of OB â€‹≥ â€‹30% and DL â€‹≥ â€‹0.2 with 4151 drug targets. After the intersection of 879 IDD disease targets obtained from GeneCards and 230 DEGs obtained from the IDD-related GSE dataset, a total of 13 hub genes overlapped. According to functional enrichment annotation analysis by Metascape, these genes showed to be dramatically enriched in AGE-RAGE signaling, proteoglycans in cancer, wound healing, transmembrane receptor protein tyrosine kinase signaling, MAPK cascades, ERK1/2 cascades, PI3K/Akt signaling pathway, skeletal system, etc. Disease association analysis by DisGeNET indicated that these genes were significantly associated with IDD, intervertebral disc disease, skeletal dysplasia, and other diseases. Active ingredients-targets-signaling pathway networks were constructed by Cytoscape, and kaempferol was identified as the hub active compound of DZ. In the IL-1ß-induced IDD in vitro model, kaempferol treatment significantly improved IL-1ß-induced NPC cell viability suppression and senescence. In addition, kaempferol treatment significantly attenuated IL-1ß-induced ROS accumulation and apoptosis. Furthermore, kaempferol treatment partially eliminated IL-1ß-induced decreases in aggrecan, collagen II, SOX9, and FN1 levels and increases in MMP3, MMP13, ADAMTS-4, and ADAMTS-5. Moreover, kaempferol treatment significantly relieved the promotive effects of IL-1ß stimulation upon p38, JNK, and ERK1/2 phosphorylation. ERK1/2 inhibitor PD0325901 further enhanced the effect of kaempferol on the inhibition of ERK1/2 phosphorylation, downregulation of MMP3 and ADAMTS-4 expression, and upregulation of aggrecan and collagen II expressions. Conclusion: Kaempferol has been regarded as the major active compound of DZ, protecting NPCs from IL-1ß-induced damages through promoting cell viability, inhibiting cell senescence and apoptosis, increasing ECM production, and decreasing ECM degradation. MAPK signaling pathway may be involved. The translational poteintial of this article: This study provides in vitro experimental data support for the pharmacological effects of kaempferol in treating IDD, and lays a solid experimental foundation for its future clinical application in IDD treatment.

Transforaminal Full-endoscopic Discectomy for Gas-containing Herniated Nucleus Pulposus at L5-S1 Under Local Anesthesia†:†A Case Report.

The vacuum phenomenon is often observed in degenerative disc disease, whereas gas-containing disc herniation is relatively rare. Full-endoscopic discectomy at the lumbar spine level via a transforaminal approach, which was established and subsequently refined over the last two decades, requires only an 8-mm skin incision and causes minimal damage to the paravertebral muscles. Foraminoplasty, performed with a high-speed drill, is a useful technique to enlarge the foramen, especially when applied at the L5-S1 level, where the trajectory is limited because of anatomical structures such as the iliac crest. Here, we report a case of gas-containing lumbar disc herniation at L5-S1 that was successfully treated by transforaminal full-endoscopic discectomy. The patient was a 62-year-old man with low back pain and pain in the plantar aspect of the right great toe. Magnetic resonance and computed tomography scans demonstrated gas-containing lumbar disc herniation at L5-S1 on the right. Following foraminoplasty, transforaminal full-endoscopic lumbar discectomy was successfully performed under local anesthesia. The patient's symptoms improved immediately after the surgery. Transforaminal full-endoscopic surgery can be effective and minimally invasive even when performed for gas-containing disc herniation. J. Med. Invest. 69 : 328-331, August, 2022.

Study on swelling, compression property and degradation stability of PVA composite hydrogels for artificial nucleus pulposus.

Artificial nucleus pulposus (ANP) replacement as an alternative to the treatment of cervical spondylosis aims to relieve pain and restore the normal cervical motion. In this study, the PVA/PVP and PVA/Pectin composite hydrogels (CH)s with different concentrations were prepared by the freezing-thawing process, and their performances were tested. The effect of different concentrations on both kinds of PVA CHs were evaluated and analysed. The results demonstrated that both kinds of CHs had good swelling property (¿190%), compressive stress-strain characteristic response and stable performance, and they were not easy to degrade (¡9%). The elastic modulus of the PVA/PVP CH was close to that of nucleus pulposus prosthesis, and the weight loss ratio of the PVA/PVP CH was lower than that of PVA/Pectin CH under load condition. Further, the experimental results showed that the PVA/PVP CH with 15 wt% solute and 1 wt% PVP content had the best comprehensive performance, which may provide significant advantages for use in future clinical application in replacing nucleus pulposus.

Panax notoginseng saponin reduces IL-1ß-stimulated apoptosis and endoplasmic reticulum stress of nucleus pulposus cells by suppressing miR-222-3p.

Background: It is well documented that the malignant biological behaviors of nucleus pulposus cells (NPCs) could trigger intervertebral disc degeneration (IDD). Panax notoginseng saponin (PNS) is a traditional Chinese medicine that inhibits osteoclastogenesis. However, its effects on the phenotypes of NPCs in IDD remains largely unknown. This study sought to examine the role of PNS in IDD and its regulatory mechanism. Methods: First, human NPCs (hNPCs) were treated with interleukin-1 beta (IL-1ß) to induce an IDD cell model. Cell proliferation and apoptosis were estimated by Cell Counting Kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Western blot was employed to examine the levels of proteins related to apoptosis and endoplasmic reticulum (ER) stress. Enzyme-linked immunosorbent assays (ELISAs) were used to test inflammatory factors levels. Immunofluorescence (IF) assays were used to determine the nuclear translocation of nuclear factor-kappa beta (NF-κB) p65. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)was used to detect miR-222-3p expression. Results: We discovered that PNS enhanced the viability but reduced the apoptosis, inflammation, and ER stress response of IL-1ß-induced hNPCs in a concentration-dependent manner. Additionally, PNS significantly reduced miR-222-3p expression in the IL-1ß-induced hNPCs. Notably, these PNS effects were reversed by the upregulation of miR-222-3p. Conclusions: In summary, PNS appears to facilitate the proliferation and attenuate the apoptosis, inflammatory response, and ER stress response of IL-1ß-induced hNPCs by inhibiting miR-222-3p expression. Our findings provide a theoretical basis for a novel drug application in IDD research.

Gamma-oryzanol alleviates intervertebral disc degeneration development by intercepting the IL-1ß/NLRP3 inflammasome positive cycle.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a highly prevalent musculoskeletal disorder characterized by a local inflammatory response associated with the IL-1ß/NLRP3 inflammasome positive feedback loop. Rice bran-derived gamma-oryzanol (Ory) as a sterol ferulate has attracted much attention due to its powerful anti-inflammatory, hypoglycemic and hypolipidemic health effects. As a clinical pharmaceutical for autonomic disorders, Ory's role in musculoskeletal degenerative disease remains unknown. PURPOSE: This study aims to validate the role of Ory in IVDD and explore the potential mechanism. STUDY DESIGN: Establishing the in vitro and in vivo IVDD models to detect the protective effect and molecular mechanism of Ory. METHOD: The anti-ECM degradation, antioxidant and anti-NLRP3 inflammasome activation effects of Ory on IL-1ß-stimulated nucleus pulposus (NP) cells were assessed by immunoblotting and immunofluorescence, etc. MRI, S-O staining and immunohistochemistry were performed to estimate the effects of Ory administration on acupuncture-mediated IVDD in rats at imaging and histological levels. RESULTS: Ory treatment inhibited IL-1ß-mediated ECM degradation, oxidative stress and NLRP3 inflammasome activation in NP cells. By interfering with NF-κB signaling and ROS overproduction, Ory interrupted IL-1ß/NLRP3-inflammasome positive cycle. In vivo experiments showed that Ory delayed acupuncture-mediated IVDD development. CONCLUSION: Our results support the potential application of Ory as a therapeutic compound for IVDD.

Arctigenin inhibits apoptosis, extracellular matrix degradation, and inflammation in human nucleus pulposus cells by up-regulating miR-483-3p.

BACKGROUND: Arctigenin (ATG) is the active ingredient of the Chinese herbal medicine Arctium lappa, with anti-inflammatory and antioxidant effects. Excessive inflammation and cell apoptosis are important causes of intervertebral disc degeneration (IDD). Hence, this study probed into the possible role of ATG in IDD. METHODS: Interleukin (IL)-1ß (10 ng/ml) was adopted to induce human nucleus pulposus cells (HNPCs) as a cell model for IDD. The effects of different concentrations of ATG (0, 2, 5, 10, 20, 50 µmol/L) on the viability of HNPCs and effects of ATG (10, 50 µmol/L) on the viability of IL-1ß-induced HNPCs were detected by cell counting kit-8 (CCK-8). After IL-1ß-induced HNPCs were transfected with miR-483-3p inhibitor and/or treated with ATG, cell viability and apoptosis were determined by CCK-8 and flow cytometry; the expressions of miR-483-3p, extracellular matrix (ECM)-related genes, and inflammation-related genes were measured by quantitative real time polymerase chain reaction (qRT-PCR), and expressions of ECM/apoptosis/NF-κB pathway-related proteins were quantified by Western blot. RESULTS: ATG had no significant effect on the viability of HNPCs but could promote the viability of IL-1ß-induced HNPCs. ATG inhibited apoptosis, ECM degradation, inflammation, and activation of NF-κB pathway in HNPCs induced by IL-1ß, but promoted the expression of miR-483-3p. MiR-483-3p inhibitor reversed the above-mentioned regulatory effects of ATG. CONCLUSION: Arctigenin suppresses apoptosis, ECM degradation, inflammation, and NF-κB pathway activation in HNPCs by up-regulating miR-483-3p.

[Efficacy of Yishen Huoxue Tongluo() medicated serum on apoptosis of nucleus pulposus cells of human intervertebral disc under overload static pressure].

OBJECTIVE: To explore the efficacy of Yishen Huoxue Tongluo() decoction containing serum on the apoptosis of human disc nucleus pulposus cells under the overload static pressure stress and its related mechanism. METHODS: Human nucleus pulposus cells were divided into three groups. The blank group had no intervention. The model group and traditional Chinese medicine serum intervention group were treated with 3 MPa compressive stress in vitro for 2, 4 and 6 hours. The changes and differences of morphology, growth status and ultrastructure of intervertebral disc nucleus pulposus cells were observed. The apoptosis rate of nucleus pulposus cells and nuclear factor kappa-B p65 (NF-κB p65), SRY-related high mobility group box 9 (SOX9), C/EBP-homologous protein (CHOP), matrix metalloprotein-13 (MMP-13) and corresponding gene expression were detected. RESULTS: At the same time, compared with the blank group, the nucleus pulposus cells in the model group were smaller in volume, less in cytoplasm and worse in growth; the nucleus pulposus cells in the traditional Chinese medicine serum intervention group were slightly larger in volume, more complete in morphology, richer in cytoplasm and better in growth. Under the same action time, the ultrastructure of nucleus pulposus cells in blank group was complete, and the structures of primary and secondary processes were not broken;and the ultrastructure of model group and traditional Chinese medicine serum intervention group were damaged, the main and secondary processes were broken to varying degrees, and there was no significant difference between the two groups. At the same time, the apoptosis rate of nucleus pulposus cells in model group was higher than that in blank group, while the apoptosis rate of nucleus pulposus cells in the traditional Chinese medicine serum intervention group was lower than that inmodel group, the difference was statistically significant (P<0.05); with the increase of action time, there was no significant difference in the apoptosis rate of nucleus pulposus cells between blank group and traditional Chinese medicine serum intervention group, and the apoptosis rate of nucleus pulposus cells in model group was increased. Compared with model group, the expression of NF-κB p65, CHOP, MMP-13 were decreased and SOX9 was increased in traditional Chinese medicine serum intervention group at the same time (P<0.05);with the increase of action time, the expression of NF-κB p65, CHOP and MMP-13 were increased, and the expression of SOX9 was decreased in blank group and model group(P<0.05), and the expression level of model group was higher than that of blank group(P<0.05). Overall observation by gene expression, under the same action time, the relative quantifications of NF-κB p65, CHOP and MMP-13 in traditional Chinese medicine serum intervention group were lower than that in model group, while SOX9 was increased (P<0.05);compared with model group, the relative quantifications of NF-κB p65, CHOP and MMP-13 in blank group were decreased(P<0.05), and the relative quantification of SOX9 was increased(P<0.05);with the increase of action time, the relative quantifications of NF-κB p65, CHOP and MMP-13 of nucleus pulposus cells in blank group and model group were increased and SOX9 was decreased(P<0.05). CONCLUSION: Yishen Huoxue Tongluo() decoction can reduce the apoptosis of nucleus pulposus cells under overload and static pressure, and has the effect of delaying the degeneration of nucleus pulposus cells. Its mechanism may be related to the decrease of CHOP, MMP-13 expression and the increase of SOX9 expression by inhibiting NF-κB p65 signal pathway of nucleus pulposus cells.

Ozone as Modulator of Resorption and Inflammatory Response in Extruded Nucleus Pulposus Herniation. Revising Concepts.

Ozone therapy has been used to treat disc herniation for more than four decades. There are several papers describing results and mechanism of action. However, it is very important to define the characteristics of extruded disc herniation. Although ozone therapy showed excellent results in the majority of spinal diseases, it is not yet fully accepted within the medical community. Perhaps it is partly due to the fact that, sometimes, indications are not appropriately made. The objective of our work is to explain the mechanisms of action of ozone therapy on the extruded disc herniation. Indeed, these mechanisms are quite different from those exerted by ozone on the protruded disc herniation and on the degenerative disc disease because the inflammatory response is very different between the various cases. Extruded disc herniation occurs when the nucleus squeezes through a weakness or tear in the annulus. Host immune system considers the nucleus material to be a foreign invader, which triggers an immune response and inflammation. We think ozone therapy modulates this immune response, activating macrophages, which produce phagocytosis of extruded nucleus pulposus. Ozone would also facilitate the passage from the M1 to M2 phase of macrophages, going from an inflammatory phase to a reparative phase. Further studies are needed to verify the switch of macrophages.

Curcumol Alleviates the Inflammation of Nucleus Pulposus Cells via the PI3K/Akt/NF-κB Signaling Pathway and Delays Intervertebral Disk Degeneration.

BACKGROUND: Intervertebral disk degeneration (IVDD) is closely associated with inflammatory environments. Curcumol has been shown to alleviate inflammation in various disease models, but its effects on IVDD remain unclear. In this study, we sought to determine the mechanism of curcumol in tumor necrosis factor (TNF)-α-induced nucleus pulposus cells and a mouse IVDD model. METHODS: Nucleus pulposus cells were pretreated with curcumol and then exposed to TNF-α. Cell viability was analyzed using CCK-8, and the messenger ribonucleic acid and protein levels of inflammatory cytokines and PI3K/Akt/NF-κB-related signaling molecules were detected using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. The mouse IVDD model was established by puncturing the C6/7 level of the caudal spine, and then it was treated with curcumol after surgery. Alcian blue/orange G staining was performed to evaluate the severity of intervertebral disk damage, and immunohistochemistry was performed to detect the expression of TNF-α. Toxicologic effects of curcumol were measured by performing hematoxylin-eosin staining and enzyme-linked immunosorbent assay. RESULTS: Curcumol reduced IL-1ß, IL-6, and TNF-α production in NPCs, and the phosphorylation of proteins in the PI3K/Akt/NF-κB signaling pathway was also decreased. The PI3K/Akt/NF-κB-related signaling molecules decreased when TNF-α-induced NPCs were treated with a PI3K inhibitor; however, curcumol did not reverse these effects. In vivo, curcumol ameliorated the progression of IVDD at the early stage and did not exert toxicologic effects. CONCLUSIONS: These results suggest a potential therapeutic use of curcumol to alleviate inflammation via the PI3K/Akt/NF-κB signaling pathway and delay the progression of IVDD.

The potential role of melatonin in retarding intervertebral disc ageing and degeneration: A systematic review.

Intervertebral disc degeneration (IDD) is a common degenerative disease of the musculoskeletal system that develops with age. It is regarded as the main cause of chronic low back pain in the elderly. IDD has various causes, including ageing, mechanical overloading, and nutritional deficiency. Melatonin is a pleiotropic indole hormone secreted by the pineal gland and plays an important role in resisting various degenerative diseases. The serum levels of melatonin decline with age and are reported to be negatively correlated with the symptomatic and histopathological scores of IDD. In vivo studies have shown that exogenous administration of melatonin could maintain the structural integrity of the intervertebral disc and inhibit the development of IDD. Mechanistically, by interacting with its membrane or intracellular receptors, melatonin can promote autophagic flux, scavenge free radicals, inhibit the release of pro-inflammatory factors, and block apoptotic pathways, thereby enhancing anti-stress abilities and matrix anabolism in different types of disc cells. Therefore, melatonin supplementation may be a promising therapeutic strategy for IDD. This review aimed to summarize the latest findings regarding the therapeutic potential of melatonin in IDD.

Dehydrocostus Lactone Attenuates the Senescence of Nucleus Pulposus Cells and Ameliorates Intervertebral Disc Degeneration via Inhibition of STING-TBK1/NF-κB and MAPK Signaling.

The progression of intervertebral disc degeneration (IDD) is multifactorial with the senescence of nucleus pulposus (NP) cells and closely related to inflammation in NP cells. Dehydrocostus lactone (DHE) is a natural sesquiterpene lactone isolated from medicinal plants that has anti-inflammatory properties. Thus, DHE may have a therapeutic effect on the progression of IDD. In this study, NP cells were used to determine the appropriate concentration of DHE in vitro. The role of DHE in tumor necrosis factor-α (TNF-α)-induced activation of inflammatory signaling pathways and cellular senescence, together with anabolism and catabolism of extracellular matrix (ECM) in NP cells, was examined in vitro. The therapeutic effect of DHE in vivo was determined using a spinal instability model of IDD in mice. The TNF-α-induced ECM degradation and the senescence of NP cells were partially attenuated by DHE. Mechanistically, DHE inhibited the activation of NF-κB and MAPK inflammatory signaling pathways and ameliorated the senescence of NP cells caused by the activation of STING-TBK1/NF-κB signaling induced by TNF-α. Furthermore, a spinal instability model in mice demonstrated that DHE treatment could ameliorate progression of IDD. Together, our findings indicate that DHE can alleviate IDD changes and has a potential therapeutic function for the treatment of IDD.

In the presence of TGF-ß1, Asperosaponin VI promotes human mesenchymal stem cell differentiation into nucleus pulposus like- cells.

BACKGROUND: The regeneration of nucleus pulposus (NP) cells is an effective method to prevent intervertebral disc degeneration (IVDD). In this study, we investigated the role of Asperosaponin VI (ASA VI), isolated from a traditional Chinese medicine (TCM), the root of Dipsacus asper Wall, in promoting human mesenchymal stem cell (HMSC) proliferation and differentiation into NP-like cells and explored the possible mechanism of action. METHODS: The effects of ASA VI on HMSC viability and proliferation were determined by the XTT method and EDU staining. Then, Real-time qPCR, immunocytochemistry and immunofluorescence assays were used to measure the effect of ASA VI on the expression of extracellular matrix (ECM) components, such as COL2A1, aggrecan, SOX9, KRT19, PAX1, and glycosaminoglycans (GAGs), in NP cells. In addition, Western blot assay was used to measure the expression of p-ERK1/2 and p-smad2/3. RESULTS: ASA VI was able to promote the proliferation and differentiation of HMSCs into NP-like cells, and the optimum concentration was 1 mg/L. Western blot assay indicated that the possible mechanism might be related to the activation of p-ERK1 / 2 and p-Smad2 / 3. CONCLUSIONS: ASA VI can promote the proliferation and differentiation of HMSCs into NP-like cells, which can potentially be used as a treatment for IVDD.

Duhuo jisheng decoction suppresses matrix degradation and apoptosis in human nucleus pulposus cells and ameliorates disc degeneration in a rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: The lower back pain (LBP) caused by intervertebral disc (IVD) degeneration brings a heavy burden to society. A classic treatment method of Chinese medicine, fangji-duhuo jisheng decoction (DHJSD), has been effective in the clinical treatment of LBP, although the underlying mechanism remains unknown. AIM OF THE STUDY: In this work, the main objective was to study the effects of DHJSD on in vitro IVD degeneration of human nucleus pulposus (NP) cells after pressure treatment and on an in vivo interrupted IVD degeneration rat model. MATERIALS AND METHODS: The effects of DHJSD on the viability of NP cells were detected using Cell Counting Kit-8. RT-qPCR, western blotting, TUNEL assay, transmission electron microscopy, and immunofluorescence staining were performed to explore the molecular mechanism underlying protection against compression-induced matrix degradation and apoptosis in NP cells by DHJSD. Furthermore, the effects of DHJSD on IVD degeneration in a rat IDD model were also determined. RESULTS: We found that DHJSD increased the viability of NP cells in a concentration- and time-dependent manner. Furthermore, DHJSD significantly reduced compression-induced NP matrix degeneration and apoptosis, activated autophagy, and inhibited the p38/MAPK signaling pathway in NP cells subjected to compression. Autophagy inhibitor 3-MA and p38/MAPK signaling pathway activator anisomycin reversed the beneficial effects of DHJSD in NP cells, indicating that DHJSD protects against IVD degeneration by autophagy activation and P38/MAPK signaling pathway inhibition. Furthermore, DHJSD treatment effectively delayed IVD degeneration in a puncture-induced IDD rat model. CONCLUSIONS: DHJSD prevents compression-induced matrix degradation and cell apoptosis through regulating autophagy and the P38/MAPK signaling pathway. The mechanism underlying the effects of DHSJD elucidated in this study provides a new direction for LBP treatment.