Maslinic acid alleviates intervertebral disc degeneration by inhibiting the PI3K/AKT and NF-κB signaling pathways.

Intervertebral disc degeneration (IDD) is the cause of low back pain (LBP), and recent research has suggested that inflammatory cytokines play a significant role in this process. Maslinic acid (MA), a natural compound found in olive plants ( Olea europaea), has anti-inflammatory properties, but its potential for treating IDD is unclear. The current study aims to investigate the effects of MA on TNFα-induced IDD in vitro and in other in vivo models. Our findings suggest that MA ameliorates the imbalance of the extracellular matrix (ECM) and mitigates senescence by upregulating aggrecan and collagen II levels as well as downregulating MMP and ADAMTS levels in nucleus pulposus cells (NPCs). It can also impede the progression of IDD in rats. We further find that MA significantly affects the PI3K/AKT and NF-κB pathways in TNFα-induced NPCs determined by RNA-seq and experimental verification, while the AKT agonist Sc-79 eliminates these signaling cascades. Furthermore, molecular docking simulation shows that MA directly binds to PI3K. Dysfunction of the PI3K/AKT pathway and ECM metabolism has also been confirmed in clinical specimens of degenerated nucleus pulposus. This study demonstrates that MA may hold promise as a therapeutic agent for alleviating ECM metabolism disorders and senescence to treat IDD.

Rosuvastatin suppresses TNF-α-induced matrix catabolism, pyroptosis and senescence via the HMGB1/NF-κB signaling pathway in nucleus pulposus cells.

Intervertebral disc degeneration is mainly caused by irregular matrix metabolism in nucleus pulposus cells and involves inflammatory factors such as TNF-α. Rosuvastatin, which is widely used in the clinic to reduce cholesterol levels, exerts anti-inflammatory effects, but whether rosuvastatin participates in IDD remains unclear. The current study aims to investigate the regulatory effect of rosuvastatin on IDD and the potential mechanism. In vitro experiments demonstrate that rosuvastatin promotes matrix anabolism and suppresses catabolism in response to TNF-α stimulation. In addition, rosuvastatin inhibits cell pyroptosis and senescence induced by TNF-α. These results demonstrate the therapeutic effect of rosuvastatin on IDD. We further find that HMGB1, a gene closely related to cholesterol metabolism and the inflammatory response, is upregulated in response to TNF-α stimulation. HMGB1 inhibition or knockdown successfully alleviates TNF-α-induced ECM degradation, senescence and pyroptosis. Subsequently, we find that HMGB1 is regulated by rosuvastatin and that its overexpression abrogates the protective effect of rosuvastatin. We then verify that the NF-κB pathway is the underlying pathway regulated by rosuvastatin and HMGB1. In vivo experiments also reveal that rosuvastatin inhibits IDD progression by alleviating pyroptosis and senescence and downregulating HMGB1 and p65. This study might provide new insight into therapeutic strategies for IDD.

Chromobox homolog 4 overexpression inhibits TNF-α-induced matrix catabolism and senescence by suppressing activation of the NF-κB signaling pathway in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is featured as enhanced catabolism of extracellular matrix (ECM) in the nucleus pulposus (NP), in which tumor necrosis factor-alpha (TNF-α)-related cell senescence is involved. Chromobox homolog protein 4 (CBX4) exhibits anti-inflammatory effects and shows promising therapeutic potential. Thus, in the present study, we explore the role of CBX4 in IDD. Immunohistochemistry staining reveals that CBX4 expression is decreased in severe degenerative NP tissues compared to mild degenerative tissues, and real-time PCR and western blot analysis results show that CBX4 expression is downregulated under TNF-α stimulation in NP cells. siRNA and adenoviruses are used to knockdown or overexpress CBX4, respectively. The results demonstrate that CBX4 knockdown augments the catabolism of ECM in human NP cells, while CBX4 overexpression in rat NP cells restores the ECM degradation induced by TNF-α, as illustrated by immunofluorescence and western blot analysis. In addition, transcriptome sequencing results reveal the regulatory effect of CBX4 on the cell cycle, and further western blot analysis and senescence-associated ß-galactosidase staining assay indicate that CBX4 overexpression alleviates cell senescence in the presence of TNF-α. Moreover, the phosphorylation of p65, which indicates the activation of NF-κB signaling, is measured by western blot analysis and immunofluorescence assay, and the results reveal that CBX4 overexpression reduces the TNF-α-induced increase in the p-p65/p65 ratio. In addition, the effect of CBX4 overexpression in NP cells is suppressed by NF-κB agonist. In summary, our results indicate that CBX4 overexpression can suppress TNF-α-induced matrix catabolism and cell senescence in the NP by inhibiting NF-κB activation. This study may provide new approaches for preventing and treating IDD.

Comparison of anterior or posterior approach in surgical treatment of thoracic and lumbar tuberculosis: a retrospective case-control study.

BACKGROUND: With the widespread use of the posterior surgery, more and more surgeons chose posterior surgery to treat thoracic and lumbar tuberculosis. But others still believed that the anterior surgery is more conducive to eradicating the lesions, and easier to place larger bone pieces for bone graft fusion. We compared the clinical and radiological outcomes of anterior and posterior surgical approaches and presented our views. METHODS: This study included 52 thoracic and lumbar tuberculosis patients at Sun Yat-sen Memorial Hospital from January 2010 to June 2018. All cases underwent radical debridement, nerve decompression, intervertebral bone graft fusion and internal fixation. Cases were divided into anterior group (24 cases) and posterior group (28 cases). Statistical analysis was used to compare the clinical effectiveness, radiological outcomes, complications and other related information. RESULTS: Patients in the anterior group and the posterior group were followed up for an average of 27.4 and 22.3 months, respectively. There were no statistically significant differences between groups in the preoperative, postoperative and last follow-up VAS score, ASIA grade and Cobb angle of local kyphosis. Moreover, there were no statistically significant differences in the improvement of neurological function, loss of kyphotic correction, total incidence of complications, operative time, intraoperative blood loss and hospital stay between the two groups (P > 0.05). But there was greater correction of kyphosis, earlier bone fusion, lower incidence of poor wound healing, less interference with the normal spine and less internal fixation consumables and medical cost in the anterior group (P < 0.05). CONCLUSIONS: Both anterior and posterior approaches are feasible for thoracic and lumbar tuberculosis. While for thoracic and lumbar tuberculosis patients with a single lesion limited in the anterior and middle columns of the spine without severe kyphosis, the anterior approach surgery may have greater advantages in kyphosis correction, bone fusion, wound healing, protection of the normal spine, and medical consumables and cost.

Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP.

Intervertebral disc degenerative disease (IDD) is the most common degenerative spine disease, which leads to chronic low back pain and symptoms in the lower extremities. In this study, we found that RORα, a member of the retinoid-related orphan receptor family, is significantly elevated in nucleus pulposus tissue in IDD patients. The elevation of RORα is associated with increased apoptosis of nucleus pulposus (NP) cells. Therefore, we applicated a well-established inverse agonist of RORα, SR3335, to investigate its role in regulating NP cell metabolism and apoptosis. To further investigate the mechanism that SR3335 regulates the pathogenesis of IDD in vitro, tumor necrosis factor alpha (TNF-α) stimulation was used in human NP cells to mimic the hostile environment that leads to degeneration. We found that SR3335 treatment reversed the trend of increased apoptosis in NP cells induced by TNF-α treatment. Next, TNF-α treatment upregulated the expression of type II collagen and aggrecan and downregulated MMP13 (matrix-degrading enzyme matrix metalloproteinase 13) and ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4). However, these effects were reversed after SR3335 treatment. Furthermore, we find that SR3335 mediated the effect in NP cells by regulating the YAP signaling pathway, especially by affecting the phosphorylation state of YAP. In conclusion, the reduction of matrix degradation enzymes and apoptosis upon SR3335 treatment suggests that SR3335 is a promising drug in reversing the deleterious microenvironment in IDD patients.

BRD9 Inhibition Attenuates Matrix Degradation and Pyroptosis in Nucleus Pulposus by Modulating the NOX1/ROS/NF-κB axis.

Intervertebral disc degeneration (IDD) is considered to be the leading cause of low back pain (LBP). The progression of IDD is closely related to the inflammatory microenvironment, which results in extracellular matrix degradation and cell death. One of the proteins, which have been shown to participate in the inflammatory response, is the bromodomain-containing protein 9 (BRD9). This study aimed to investigate the role and mechanism of BRD9 in regulating IDD. The tumor necrosis factor-α (TNF-α) was used to mimic the inflammatory microenvironment in vitro. Western blot, RT-PCR, immunohistochemistry, immunofluorescence, and flow cytometry were used to demonstrate the effect of BRD9 inhibition or knockdown on matrix metabolism and pyroptosis. We found that the expression of BRD9 was upregulated as IDD progressed. BRD9 inhibition or knockdown alleviated TNF-α-induced matrix degradation, reactive oxygen species (ROS) production, and pyroptosis in rat nucleus pulposus cells. Mechanistically, RNA-seq was used to investigate the mechanism of BRD9 in promoting IDD. Further investigation revealed that BRD9 regulated NOX1 expression. Inhibition of NOX1 could abrogate matrix degradation, ROS production, and pyroptosis caused by BRD9 overexpression. In vivo, the radiological and histological evaluation showed that the pharmacological inhibition of BRD9 alleviated IDD development in rat IDD model. Our results indicated that BRD9 could promote IDD via the NOX1/ROS/ NF-κB axis by inducing matrix degradation and pyroptosis. Targeting BRD9 may be a potential therapeutic strategy in treating IDD.

MicroRNA-155 suppressed cholesterol-induced matrix degradation, pyroptosis and apoptosis by targeting RORα in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is associated with low back pain, yet its inherent mechanism remains obscure. Hypercholesteremia was regarded as a risk factor for IDD, and our previous study showed that cholesterol accumulation could elicit matrix degradation in the nucleus pulposus (NP). MicroRNA-155 (miR-155) was substantiated as protective in IDD, but its role in cholesterol-induced IDD was unclear. The present study investigated whether miR-155 could mediate cholesterol-related IDD and its internal mechanisms. In vivo experiments revealed high-fat diet-induced hypercholesteremia in wild-type (WT) mice along with the occurrence of IDD, whereas Rm155LG transgenic mice showed milder NP degeneration, as evidenced by Saffron O-fast green (SF) staining and immunohistochemistry (IHC). Meanwhile, IHC showed that NLRP3 and Bax expression was also suppressed in Rm155LG mice. In vitro studies using Western blotting (WB) and immunofluorescence (IF) confirmed that the miR-155 mimic could alleviate cholesterol-induced matrix degradation, apoptosis and pyroptosis in NP. Moreover, RORα was upregulated in severely degenerated NP compared to mild IDD. It was also noted that RORα was suppressed in Rm155LG mice. In this study, we demonstrated that miR-155 could target RORα and that inhibition of RORα could prevent cholesterol-induced matrix degradation, apoptosis, and pyroptosis in NP, indicating the protective effect of miR-155 in cholesterol-induced IDD by targeting RORα.

Melatonin reverses tumor necrosis factor-alpha-induced metabolic disturbance of human nucleus pulposus cells via MTNR1B/Gαi2/YAP signaling.

Background: Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Methods: Western blotting, RT-qPCR and immunohistochemistry were used to detect the expression of melatonin membrane receptors (MTNR1A/B) and TNF-α in human NP tissues. In vitro, human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model was constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. Results: The upregulation of TNF-α and downregulation of melatonin membrane receptors (MTNR1A/B) were observed in degenerative NP tissues. Then we demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α-impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α-induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Conclusions: Our results revealed that melatonin can reverse TNF-α-impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD.

Irisin Ameliorates Intervertebral Disc Degeneration by Activating LATS/YAP/CTGF Signaling.

Unbalanced metabolism of an extracellular matrix (ECM) in nucleus pulposus cells (NPCs) is widely acknowledged as the primary cause of intervertebral disc degeneration (IDD). Irisin, a novel myokine, is cleaved from fibronectin type III domain-containing 5 (FNDC5) and has recently been proven to regulate the metabolism of ECM. However, little is known about its potential on NPCs and the development of IDD. Therefore, this study sought to examine the protective effects and molecular mechanism of irisin on IDD in vivo and in vitro. Decreased expression levels of FNDC5 and anabolism markers (COL2A1 and ACAN) but increased levels of catabolism markers (ADAMTS4) were found in degenerative nucleus pulposus (NP) tissues. In a punctured-induced rat IDD model, irisin treatment was found to significantly slow the development of IDD, and in TNF-α-stimulated NPCs, irisin treatment partly reversed the disorder of ECM metabolism. In mechanism, RNA-seq results suggested that irisin treatment affected the Hippo signaling pathway. Further studies revealed that with irisin treatment, the phosphorylation levels of key factors (LATS and YAP) were downregulated, while the expression level of CTGF was upregulated. Moreover, CTGF knockdown partially eliminated the protective effects of irisin on the metabolism of ECM in NPCs, including inhibiting the anabolism and promoting the catabolism. Taken together, this study demonstrated that the expression levels of FNDC5 were decreased in degenerative NP tissues, while irisin treatment promoted the anabolism, inhibited the catabolism of the ECM in NPCs, and delayed the progression of IDD via LATS/YAP/CTGF signaling. These results shed light on the protective actions of irisin on NPCs, leading to the development of a novel therapeutic target for treating IDD.

Cholesterol Induces Pyroptosis and Matrix Degradation via mSREBP1-Driven Endoplasmic Reticulum Stress in Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is closely associated with low back pain, but its underlying mechanism remains unclear. Cholesterol is an essential nutrient in mammalian cells. Alterations in cholesterol levels lead to impairments in cell physiology, such as cell proliferation and signal transduction. Previous clinical studies demonstrated that hypercholesterolemia could be a potential risk factor for IDD, but how cholesterol induces IDD remains unknown. The current study aimed to explore the regulatory role of cholesterol in IDD development and the potential underlying mechanisms. It was found that different forms of cholesterol levels were elevated in degenerative nucleus pulposus (NP) tissues in both humans and Sprague-Dawley rats. Rats fed a high cholesterol diet (HCD) exhibited degenerative features in the lumbar intervertebral disc compared with those fed a standard diet. Interestingly, this effect could be abolished by cholesterol-lowering drug atorvastatin. In NP cells treated with TNF-α and IL-1ß, a significantly higher level of cholesterol was observed. These results suggested a pivotal role of cholesterol in the progression of IDD. We also observed accelerated pyroptosis in NP cells and extracellular matrix (ECM) degradation in the rat NP cells treated with exogenous cholesterol. We further demonstrated that endoplasmic reticulum stress was responsible for cholesterol-induced pyroptosis and ECM degradation. Moreover, RNA-seq analysis revealed that the mature form of SREBP1 (mSREBP1), an important regulator of lipid metabolism, is involved in regulating endoplasmic reticulum stress in knockdown experiments. In conclusion, this study demonstrated that cholesterol could induce pyroptosis in NP cells and ECM degradation by activating endoplasmic reticulum stress through stimulating mSREBP1 in IDD.

Inhibition of nuclear receptor RORα attenuates cartilage damage in osteoarthritis by modulating IL-6/STAT3 pathway.

Osteoarthritis (OA) is characterized by cartilage destruction, chronic inflammation, and local pain. Evidence showed that retinoic acid receptor-related orphan receptor-α (RORα) is crucial in cartilage development and OA pathogenesis. Here, we investigated the role and molecular mechanism of RORα, an important member of the nuclear receptor family, in regulating the development of OA pathologic features. Investigation into clinical cartilage specimens showed that RORα expression level is positively correlated with the severity of OA and cartilage damage. In an in vivo OA model induced by anterior crucial ligament transaction, intra-articular injection of si-Rora adenovirus reversed the cartilage damage. The expression of cartilage matrix components type II collagen and aggrecan were elevated upon RORα blockade. RNA-seq data suggested that the IL-6/STAT3 pathway is significantly downregulated, manifesting the reduced expression level of both IL-6 and phosphorylated STAT3. RORα exerted its effect on IL-6/STAT3 signaling in two different ways, including interaction with STAT3 and IL-6 promoter. Taken together, our findings indicated the pivotal role of the RORα/IL-6/STAT3 axis in OA progression and confirmed that RORα blockade improved the matrix catabolism in OA chondrocytes. These results may provide a potential treatment target in OA therapy.

Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p.

BACKGROUND: Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. METHODS: BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. RESULTS: MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. CONCLUSION: MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.