The Sanbi Decoction alleviates intervertebral disc degeneration in rats through intestinal flora and serum metabolic homeostasis modulation.

BACKGROUND: Intervertebral disc degeneration (IVDD) is an essential cause of low back pain (LBP), the incidence of which has risen in recent years and is progressively younger, but treatment options are limited, placing a serious economic burden on society. Sanbi decoction (SBD) is an important classical formula for the treatment of IVDD, which can significantly improve patients' symptoms and is a promising alternative therapy. PURPOSE: The aim of this study is to investigate the safety and efficacy of SBD in the treatment of IVDD and to explore the underlying mechanisms by using an integrated analytical approach of microbiomics and serum metabolomics, as well as by using molecular biology. METHODS: A rat IVDD puncture model was established and treated by gavage with different concentrations of SBD, and clean faeces, serum, liver, kidney, and intervertebral disc (IVD) were collected after 4 weeks. We assessed the safety by liver and kidney weighing, functional tests and tissue staining, the expression of tumor necrosis factor-alpha (TNF-ɑ), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) inflammatory factors in serum was detected by ELISA kits, and X-ray test, magnetic resonance imaging (MRI) examination, immunohistochemistry (IHC), western blotting (WB), hematoxylin-eosin (HE) staining and safranin O-fast green (SO/FG) staining were used to assess the efficacy. Finally, we performed 16S rRNA sequencing analysis on the faeces of different groups and untargeted metabolomics on serum and analyzed the association between them. RESULTS: SBD can effectively reduce the inflammatory response, regulate the metabolic balance of extracellular matrix (ECM), improve symptoms, and restore IVD function. In addition, SBD can significantly improve the diversity of intestinal flora and maintain the balance. At the phylum level, SBD greatly increased the relative abundance of Patescibacteria and Actinobacteriota and decreased the relative abundance of Bacteroidota. At the genus level, SBD significantly increased the relative abundance of Clostridia_UCG-014, Enterorhabdus, and Adlercreutzia, and decreased the relative abundance of Ruminococcaceae_UCG-005 (p < 0.05). Untargeted metabolomics indicated that SBD significantly improved serum metabolites and altered serum expression of 4alpha-phorbol 12,13-didecanoate (4alphaPDD), euscaphic acid (EA), alpha-muricholic acid (α-MCA), 5-hydroxyindoleacetic acid (5-HIAA), and kynurenine (Kyn) (p < 0.05), and the metabolic pathways were mainly lipid metabolism and amino acid metabolism. CONCLUSIONS: This study demonstrated that SBD can extensively regulate intestinal flora and serum metabolic homeostasis to reduce inflammatory response, inhibit the degradation of ECM, restore IVD height and water content to achieve apparent therapeutic effect for IVDD.

A review of Duhuo Jisheng decoction mechanisms in intervertebral disc degeneration in vitro and animal studies.

Intervertebral disc degeneration (IVDD) has become a serious public health problem, placing a heavy burden on society and the healthcare system. Its pathogenesis is not completely clear and may be closely related to mechanical damage, inflammatory factors, oxidative stress and death of nucleus pulposus cells (NPCs). The treatment of IVDD mainly includes conservative treatment and surgery. Conservative treatment is based on hormonal and anti-inflammatory drugs and massage techniques, which can relieve the pain symptoms to a certain extent, but cannot solve the problem from the root cause. Surgical treatment is mainly by removing the herniated nucleus pulposus, but it is more traumatic for IVDD patients, expensive and not suitable for all patients. Therefore, it is extremely important to clarify the pathogenesis of IVDD, to find an effective and convenient treatment and to further elaborate its mechanism of action. The effectiveness of traditional Chinese medicine in the treatment of IVDD has been well demonstrated in clinical medical research. We have been working on the Chinese herbal formula Duhuo Jisheng Decoction, which is a common formula for the treatment of degenerative disc disease. Not only does it have significant clinical effects, but it also has few adverse effects. At present, we found that its mechanism of action mainly involves regulation of inflammatory factors, reduction of apoptosis and pyroptosis of NPCs, inhibition of extracellular matrix degradation, improvement of intestinal flora, etc. However, a few relevant articles have yet comprehensively and systematically summarized the mechanisms by which they exert their effect. Therefore, this paper will comprehensively and systematically explain on it. This is of great clinical significance and social value for elucidating the pathogenesis of IVDD and improving the symptoms of patients, and will provide a theoretical basis and scientific basis for the treatment of IVDD with traditional Chinese medicine.

Derivation and comprehensive analysis of ageing-related genes in intervertebral disc degeneration for prediction and immunology.

Intervertebral disc degeneration (IDD) is triggered primarily by ageing, a process characterized by intrinsic, multifaceted and progressive characteristics. Regarding the crucial senescence genes and underlying regulatory mechanisms leading to the etiology of IDD, there is still some uncertainty. In this study, we used gene expression patterns from the GEO database to create a diagnostic model of IDD using differential ageing-related genes (DARG). We examine the relative dynamics of immune cells by single-sample gene set. On the basis of transcription factor (TF) miRNA and miRNA-mRNA pairs, the regulatory network for transcription and post-transcriptional processes was built. The active therapeutic components and Chinese herbal remedies of the main ageing genes were investigated using a network pharmacology approach. 20 DARGs were combined to create a diagnostic model, and both the training and validation sets had an area under the ROC curve of 1. We found alterations in many cell types in IDD tissue, but mainly in activated dendritic cells, type 17 T helper cells, and mast cells. We identified a regulatory axis for STAT1/miR-4306/PPARA based on the correlations between gene expression and targeting. Active substances (Naringenin and Quercetin) and herbs (Aurantii fructus and Eucommiae cortex) targeting PPARA for the treatment of IDD were discovered through network pharmacology. These results provide a theoretical framework for identifying and treating IDD. For the first time, we were able to diagnose IDD patients using 20 ageing-related indicators. At the same time, TF-miRNA-mRNA in conjunction with network pharmacology enabled the identification of prospective therapeutic targets and pharmacological processes.

Astragaloside IV attenuates IL-1ß-induced intervertebral disc degeneration through inhibition of the NF-κB pathway.

BACKGROUND: Intervertebral disc degeneration (IDD) is the main cause of low back pain. Patients with low back pain may experience significant socio-economic burdens and decreased productivity. Previous studies have shown that inflammation is one of the main causes of IDD. Astragaloside IV (AS IV), a traditional Chinese medicine, has been reported to have therapeutic effects on many inflammation-related diseases; however, the effectiveness of AS IV as the treatment for IDD has not been studied. METHODS: Nucleus pulposus (NP) cells from patients with IDD were used for the experiments. Cell counting kit 8 (CCK8) was used to evaluate the effect of AS IV on the viability of NP cells (NPCs). To mimic IDD in vitro, NPCs were divided into the following groups: control group, interleukin 1ß (IL-1ß) group, and AS IV + IL-1ß group. To analyse the effect of AS IV on IL-1ß-induced IDD, Western blotting, RT-qPCR, flow cytometry, and immunofluorescence assays were performed. To evaluate the effect of AS IV in vivo, a rat model of puncture-induced IDD was established. RESULTS: AS IV effectively alleviated IL-1ß-induced inflammation, apoptosis, and extracellular matrix degeneration in NPCs. We also observed that AS IV decreased the IL-1ß-induced phosphorylation of inhibitor of kappa B-alpha (p-IκBα) in the cytosol, and reduced nuclear translocation of NF-κB p65, indicating that AS IV inhibited the NF-κB pathway. Using the puncture-induced rat IDD model, our results showed that AS IV had a protective effect against the progression of IDD, suggesting that AS IV could alleviate IDD in vivo. CONCLUSIONS: Our results demonstrated that AS IV effectively alleviated IDD in vivo and in vitro, indicating that it could be used as a therapeutic to treat IDD.

Manual therapy regulates oxidative stress in aging rat lumbar intervertebral discs through the SIRT1/FOXO1 pathway.

With the increasing burden of a globally aging population, low back pain has become one of the most common musculoskeletal disorders, caused mainly by intervertebral disc (IVD) degeneration. There are currently several clinical methods to alleviate back pain, but there is scarce attention paid as to whether they can improve age-related IVD degeneration. It is therefore difficult to conduct an in-depth evaluation of these methods. A large number of clinical studies have shown that manual therapy (MT), a widely used comprehensive alternative method, has effects on pain, the mechanisms of which require further study. In this study, MT was performed on aging rats for 6 months, and their behaviors were compared with those of a non-intervention group of aging and young rats. After the intervention, all rats were examined by X-ray to observe lumbar spine degeneration, and the IVD tissues were dissected for detection, including pathological staining, immunofluorescence, Western bolt, etc. This study demonstrated the possibility that MT intervention delay the lumbar IVD degeneration in aging rats, specifically improving the motor function and regulating senescence-associated ß-galactosidase, p53, p21, p16, and telomerase activity to retard the senescence of cells in IVDs. Moreover, MT intervention can modify oxidative stress, increase the expression of SIRT1 and FOXO1 in IVDs and decrease ac-FOXO1 expression, suggesting that MT can reduce oxidative stress through the SIRT1/FOXO1 pathway, thereby playing a role in delaying the aging of IVDs. This study shows that drug-free, non-invasive mechanical interventions could be of major significance in improving the physical function of the elderly.

Vitamin D/VDR in the pathogenesis of intervertebral disc degeneration: Does autophagy play a role?

To date, the underlying mechanisms involved intervertebral disc degeneration (IDD) remain unclear, which has hindered the development of molecular biological therapy for IDD. Autophagy is vital for intracellular quality control and metabolic balance in intervertebral disc cells. Hence, autophagy homeostasis is important. Emerging evidence has implicated vitamin D (VD) and the vitamin D receptor (VDR) in IDD progression because of their effects on different autophagy steps. However, the results of clinical trials in which VD supplementation was assessed as a treatment for IDD are controversial. Furthermore, experimental studies on the interplay between VD/VDR and autophagy are still in their infancy. In view of the significance of the crosstalk between VD/VDR and autophagy components, this review focuses on the latest research on VD/VDR modulation in autophagy and investigates the possible regulatory mechanisms. This article will deepen our understanding of the relationship between VD/VDR and autophagy and suggests novel strategies for IDD prevention and treatment.

High-Throughput Gene and Protein Analysis Revealed the Response of Disc Cells to Vitamin D, Depending on the VDR FokI Variants.

Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor (VDR), observed as associated with spine pathologies. The present study was aimed at investigating-through high-throughput gene and protein analysis-the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)2D3 was administered to the cells with or without interleukin 1 beta (IL-1ß). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.

Systematically characterized mechanism of treatment for lumbar disc herniation based on Yaobitong capsule ingredient analysis in rat plasma and its network pharmacology strategy by UPLC-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Yaobitong capsule (YBTC) was a traditional Chinese medicine (TCM) and it had clinically used to treat lumbar disc degeneration (LDH) for a long time. However, the active ingredients of YBTC absorption into the plasma and its pharmacological mechanism of treatment for LDH still remained unclear. AIM OF THE STUDY: In this study, our research committed to identify the absorbed active ingredients of YBTC in rat plasma, and it may be a potential mechanism of action on LDH by the biological targets regulating related pathways. MATERIALS AND METHODS: An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to identify the absorption components and metabolites of YBTC in rat plasma, and the network pharmacology was further investigated to illuminate its potential mechanism of treatment for LDH by the biological targets regulating related pathways. RESULTS: The network analysis found that 56 components were identified as its main active ingredients including ginsenoside Rg1, ginsenoside Rb1, senkyunolide H, and tetrahydropalmatine, etc. Combining with biological process, cellular component and molecular functions of GO, and kyotoencyclopedia of genes and genomes pathway enrichment analysis to perform network topology analysis on core targets. These active ingredients regulated 29 mainly pathways by 87 direct target genes including MAPK, Ras, PI3K-Akt, and NF-kappa B signaling pathway, etc. CONCLUSION: In this study, the absorption active ingredients of YBTC in rat plasma were firstly combined with the network pharmacology investigation to elucidate its biological mechanism of treatment for LDH in vivo. It inhibited excessive inflammatory reactions, thereby reducing the sensitivity of the nerves to reduce pain and relieve LDH, and potential medicine targets could be identified to clarify the molecular mechanism of YBTCs' regulation of LDH.

Effect of sIL-13Rα2-Fc on the progression of rat tail intervertebral disc degeneration.

BACKGROUND: The incidence of degenerative disc disease caused by intervertebral disc injury is increasing annually, seriously affecting the quality of life of patients and increasing the disease burden on society. The mechanisms of intervertebral disc degeneration include changes in extracellular matrix (ECM) deposition and tissue fibrosis. sIL-13Rα2-Fc potently inhibits interleukin (IL)-13, as well as blocks related cell signaling pathways and inhibits fibrosis in certain tissues. However, it is unknown whether sIL-13Rα2-Fc inhibits fibrosis in injured intervertebral discs and slows the process of degeneration. We hypothesized that sIL-13Rα2-Fc delays the progression of intervertebral disc degeneration by inhibiting intervertebral disc fibrosis and improving ECM deposition. METHODS: A rat tail intervertebral disc degeneration model was established. Pathological changes in rat intervertebral disc tissue were observed by hematoxylin and eosin staining and Masson staining. Glycosaminoglycan (GAG), chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA) contents were quantitatively analyzed by enzyme-linked immunosorbent assay. Type I and type II collagen expression levels were analyzed by reverse transcription-PCR and western blotting. RESULTS: Hematoxylin and eosin staining and Masson staining revealed annulus fibrosus rupture, disordered arrangement, decreased nucleus pulposus tissue, and decreased collagen fiber in the rat intervertebral disc tissue. Following treatment with sIL-13Rα2-Fc, pathological changes in the rat intervertebral disc were reduced. Rat intervertebral disc tissue showed decreased GAG, CS-KS, and (HA) contents, increased type I collagen levels, and decreased type II collagen levels in degenerated intervertebral discs. sIL-13Rα2-Fc intervention increased the contents of GAG, CS, KS, and HA; inhibited the expression of type I collagen; and promoted the expression of type II collagen. CONCLUSION: These results demonstrate that intervertebral disc degeneration is associated with tissue fibrosis. sIL-13Rα2-Fc can regulate type I and type II collagen expression levels by increasing GAG, CS, KS, and HA contents, thereby slowing the progression of intervertebral disc degeneration.

Oxidative stress-induced senescence markedly increases disc cell bioenergetics.

Cellular senescence is a phenotype characterized by irreversible growth arrest, chronic elevated secretion of proinflammatory cytokines and matrix proteases, a phenomenon known as senescence-associated secretory phenotype (SASP). Biomarkers of cellular senescence have been shown to increase with age and degeneration of human disc tissue. Senescent disc cells in culture recapitulate features associated with age-related disc degeneration, including increased secretion of proinflammatory cytokines, matrix proteases, and fragmentation of matrix proteins. However, little is known of the metabolic changes that underlie the senescent phenotype of disc cells. To assess the metabolic changes, we performed a bioenergetic analysis of in vitro oxidative stress-induced senescent (SIS) human disc cells. SIS disc cells acquire SASP and exhibit significantly elevated mitochondrial content and mitochondrial ATP-linked respiration. The metabolic changes appear to be driven by the upregulated protein secretion in SIS cells as abrogation of protein synthesis using cycloheximide decreased mitochondrial ATP-linked respiration. Taken together, the results of the study suggest that the increased energy generation state supports the secretion of senescent associated proteins in SIS disc cells.

The Effect of Adenosine on Extracellular Matrix Production in Porcine Intervertebral Disc Cells.

Compressive loading promotes adenosine triphosphate (ATP) production and release by intervertebral disc (IVD) cells. Extracellular ATP can be rapidly hydrolyzed by ectonucleotidases. Adenosine, one of the adenine derivatives of ATP hydrolysis, can modulate diverse cellular actions via adenosine receptors. The objectives of this study were to investigate the effects of exogenous adenosine on the production of extracellular matrix (ECM; i.e., collagen type II and aggrecan) and ATP of IVD cells and explore the underlying mechanism of action. It was found that adenosine treatment significantly upregulated aggrecan and type II collagen gene expression and the ATP level in IVD cells. Dipyridamole, an adenosine transport blocker, completely suppressed the effects of adenosine on the ATP production and ECM gene expression of the IVD cells, whereas antagonists of adenosine receptors did not significantly affect adenosine-treated IVD cells. The findings suggested that elevated intracellular ATP and upregulation of ECM gene expression by adenosine treatment are mainly due to adenosine uptake rather than receptor activation. Since ECM biosynthesis is a high ATP demanding process, supplementing adenosine could be beneficial as IVD cells are able to utilize it to replenish intracellular ATP and sequentially promote ECM production, which is constantly suppressed by limited nutrition supply due to the avascular nature of the IVD.

[Biological treatment for intervertebral disc degeneration].

Neck shoulder pain or lumbocrural pain caused by intervertebral disc degeneration (IDD) could seriously affect the qualities life of patients. Current treatments mainly focus on alleviating pain and the symptoms of nerve compression, which could not radically stop the process of intervertebral disc degeneration, but conversely lead to high recurrence rate. In recent years, scholars have turned to study the biological treatment for repair and rebuild the intervertebral disc by biological molecular therapy, gene therapy, cell therapy and tissue engineering to solve the problem of intervertebral disc degeneration, while most of the above methods are still in animal experiments or in vitro experiments and the clinical application is still a long way to go.

Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc.

The cartilaginous endplates (CEPs) are thin layers of hyaline cartilage found adjacent to intervertebral discs (IVDs). In addition to providing structural support, CEPs regulate nutrient and metabolic exchange in the disc. In IVD pathogenesis, CEP undergoes degeneration and calcification, compromising nutrient availability and disc cell metabolism. The mechanism(s) underlying the biochemical changes of CEP in disc degeneration are currently unknown. Since calcification is often observed in later stages of IVD degeneration, we hypothesised that elevations in free calcium (Ca2+) impair CEP homeostasis. Indeed, our results demonstrated that the Ca2+ content was consistently higher in human CEP tissue with grade of disc degeneration. Increasing the levels of Ca2+ resulted in decreases in the secretion and accumulation of collagens type I, II and proteoglycan in cultured human CEP cells. Ca2+ exerted its effects on CEP matrix protein synthesis through activation of the extracellular calcium-sensing receptor (CaSR); however, aggrecan content was also affected independent of CaSR activation as increases in Ca2+ directly enhanced the activity of aggrecanases. Finally, supplementing Ca2+ in our IVD organ cultures was sufficient to induce degeneration and increase the mineralisation of CEP, and decrease the diffusion of glucose into the disc. Thus, any attempt to induce anabolic repair of the disc without addressing Ca2+ may be impaired, as the increased metabolic demand of IVD cells would be compromised by decreases in the permeability of the CEP.

Effects of age, replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases.

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.

Hepatocyte growth factor/c-met promotes proliferation, suppresses apoptosis, and improves matrix metabolism in rabbit nucleus pulposus cells in vitro.

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti-apoptotic and anti-inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H2 O2 or TNF-α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase-2 and matrix metalloproteinase-3 and -9 by TNF-α was significantly suppressed by HGF treatment. Expression of c-Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF-α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c-Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities.

In vitro Penetration and in vivo Distribution of Honokiol into the Intervertebral Disc in Rat.

Honokiol is a potential candidate for the treatment of intervertebral disc (IVD) degeneration. In this study, we develop in vitro and in vivo methods to detect the distribution of honokiol in intervertebral discs using high-performance liquid chromatography. A rat tail disc was used for both experimental models. For the in vivo animal experiment, blood samples and tail discs were collected at 15, 30, 60, 120 and 240 min after honokiol administration (30 mg/kg, i.v.). The analyte was separated by a mobile phase of methanol and 10 mM NaH2PO4 buffer at pH 2.8 (78:22, v/v) and pumped through a reversed-phase analytical column (250 × 4.6 mm, particle size 5 µm) at room temperature. The in vitro experimental results demonstrated that honokiol diffused into the intervertebral disc and was concentration-dependent. The active concentration is obtained for the therapeutic level at 15 and 30 min after honokiol administration in the in vivo model.

Organotypic Cultures of Intervertebral Disc Cells: Responses to Growth Factors and Signaling Pathways Involved.

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD's extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration.

[Effect of Acupotome Relaxing on Expression of Type I and II Collagens of Degenerative Cervical Inter- vertebral Discs in Rats].

OBJECTIVE: To observe the effect of acupotome relaxing at cervical acupoints on type I and II collagens of degenerated cervical intervertebral discs in rats, so as to explore its potential mechanism underlying anti-degeneration of intervertebral discs. METHODS: Rats were randomly divided into control, model, Jiaji acupoints, cervical acupoints and medication groups (n = 15 in each group). The rat model of cervical intervertebral disc degeneration due to static-dynamic imbalance was made as previously specified. The Jiaji acupoints were those located along the cervical vertebra 2-7. The cervical acupoints included bilateral "Naokong"(GB 19) , "Naohu" (GV 17) , "Dazhui"(GV 14) , bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11). Acupoints were treated according to the procedures of acupotome for 3 times in ten days with five days' break between every two treatment sessions. Rats of the medication group were intragastrically administered with Jing Fu Kang Granules and ibuprofen daily for ten days. Twenty days after the end of treatment, all rats were sacrificed for further examination of morphological changes of the intervertebral disc tissue. Immunoactivity of protein and mRNA expression levels of collagen type I and II of the intervertebral discs were measured by means of immunohistochemistry and RT-PCR, respectively. RESULTS: In comparison with the control group, the immunoactivity and mRNA expression levels of collagen type I and II of the intervertebral discs were significantly elevated or reduced in rats of the model group, respectively (P < 0.05). After acupotome intervention and medication, the increased and decreased expression levels of type I and II collagen proteins and genes were markedly reversed (P < 0.05). The effects of acupotome relaxing of both cervical and Jiaji acupoints were significantly superior to those of medication in down-regulating expression of type I collagen protein and mRNA, and in up-regulating that of type II collagen protein and mRNA (P < 0.05). No significant differences were found between the cervical acupoints and Jiaji acupoints groups in the above- mentioned outcomes (P > 0.05) . The degree of severity of the degenerated intervertebral discs was the worst in the model group, followed by the medication group, then the Jiaji acupoints group and cervical acupoints group, and the control group the least. CONCLUSION: Acupotome at neck acupoints can regulate the extracellular matrix of the intervertebral disc via inhibiting the transformation between type I and type II collagens, which may contribute to its effect in delaying the degenerative process of the cervical intervertebral discs.

Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells.

INTRODUCTION: Nutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells. METHODS: Thirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting. RESULTS: Cells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II. CONCLUSIONS: SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.

Low level light therapy modulates inflammatory mediators secreted by human annulus fibrosus cells during intervertebral disc degeneration in vitro.

Intervertebral disc degeneration (IVD) is one of the important causes of low back pain and is associated with inflammation induced by interaction between macrophages and the human annulus fibrosus (AF) cells. Low-level light therapy (LLLT) has been widely known to regulate inflammatory reaction. However, the effect of LLLT on macrophage-mediated inflammation in the AF cells has not been studied till date. The aim of this study is to mimic the inflammatory microenvironment and to investigate the anti-inflammatory effect of LLLT at a range of wavelengths (405, 532 and 650 nm) on the AF treated with macrophage-like THP-1 cells conditioned medium (MCM) containing proinflammatory cytokines and chemokines (interleukin-1beta, tumor necrosis factor-alpha, interleukin-6 and 8). We observed that AF cells exposed to MCM secrete significantly higher concentrations of IL-6, IL-8, IL-1ß and TNF-α. LLLT markedly inhibited secretion of IL-6 at 405 nm in a time-dependent manner. Level of IL-8 was significantly decreased at all wavelengths in a time-dependent manner. We showed that MCM can induce the inflammatory microenvironment in AF cells and LLLT selectively suppressed IL-6 and 8 levels. The results indicate that LLLT is a potential method of IVD treatment and provide insights into further investigation of its anti-inflammation effect on IVD.