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Objective: To explore the effect of baicalin on human nucleus pulposus cells (NPCs) in response to interleukin (IL)-1β stimulation., Methods: Viability of NPCs was measured by cell counting kit-8 (CCK-8) assay. TUNEL staining assay and flow cytometry were performed to detect apoptotic cell death of NPCs. Western blot analysis was conducted to detect the expression levels of proteins. Enzyme-linked immunosorbent assay (ELISA) was applied for the determination of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and IL-6. Oxidative stress indicators including reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity were measured., Results: Baicalin attenuated IL-1β-caused cell viability reduction and apoptosis in NPCs. IL-1β-induced increase in Bax expression and decrease in Bcl-2 expression were attenuated by baicalin treatment. IL-1β-induced production of iNOS, COX-2, IL-6, and TNF-α in NPCs was inhibited by baicalin treatment. Baicalin treatment reversed IL-1β-induced increase in ROS production and MDA level, as well as decrease in SOD activity. Furthermore, baicalin treatment elevated the expression levels of Col II and Aggrecan and downregulated the expression levels of MMP3, MMP13, and ADAMTS5 in IL-1β-induced NPCs. A total of 402 related targets of baicalin and 134 related targets of intervertebral disk degeneration were found, and nine intersection targets were screened out. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mitogen-activated protein kinase (MAPK) pathway was found to be involved in the effects of baicalin., Conclusions: Baicalin exhibited protective effects on IL-1β-caused cell viability reduction, apoptosis, oxidative stress, inflammation, and extracellular matrix degradation in NPCs. In addition, we found c-Jun N-terminal kinase (JNK) and p38 MAPK pathways as targets of baicalin through bioinformatic analysis.

Context: Cyanidin has been shown to have therapeutic potential in osteoarthritis. However, it is unclear whether cyanidin prevents the progression of intervertebral disc degeneration (IVDD)., Objective: This study evaluates the effects of cyanidin on IVDD in vitro and in vivo ., Materials and Methods: Nucleus pulposus cells (NPCs) isolated from lumbar IVD of 4-week-old male Sprague-Dawley (SD) rats were exposed to 20 ng/mL IL-1β, and then treated with different doses (0-120 µM) of cyanidin for 24 h. SD rats were classified into three groups ( n  = 8) and treated as follows: control (normal saline), IVDD (vehicle), IVDD + cyanidin (50 mg/kg). Cyanidin was administered intraperitoneally for 8 weeks., Results: The IC 50 of cyanidin for NPCs was 94.78 µM, and cyanidin had no toxicity at concentrations up to 500 mg/kg in SD rats. Cyanidin inhibited the apoptosis of NPCs induced by IL-1β (12.73 ± 0.61% vs. 18.54 ± 0.60%), promoted collagen II (0.82-fold) and aggrecan (0.81-fold) expression, while reducing MMP-13 (1.02-fold) and ADAMTS-5 (1.40-fold) expression. Cyanidin increased the formation of autophagosomes in IL-1β-induced NPCs, and promoted LC3II/LC3I (0.83-fold) and beclin-1 (0.85-fold) expression, which could be reversed by chloroquine. Cyanidin inhibited the phosphorylation of JAK2 (0.47-fold) and STAT3 (0.53-fold) in IL-1β-induced NPCs. The effects of cyanidin could be enhanced by AG490. Furthermore, cyanidin mitigated disc degeneration in IVDD rats in vivo ., Discussion and Conclusions: Cyanidin improved the function of NPCs in IVDD by regulating the JAK2/STAT3 pathway, which may provide a novel alternative strategy for IVDD. The mechanism of cyanidin improving IVDD still needs further work for in-depth investigation.

Intervertebral disc degeneration (IDD) is a complex degradative disorder associated with inflammation. Emodin, an anthraquinone derivative, possesses strong anti-inflammatory activity. This study focused on the in vitro therapeutic action of emodin in a cellular model of IDD. Human nucleus pulposus cells (NPCs) were stimulated with interleukin‐1 β (IL-1 β) to induce inflammation. Cell Counting Kit-8 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays were performed to evaluate the viability and apoptosis of NPCs, respectively. Caspase-3 activity was measured to indirectly assess cell apoptosis. Western blot analysis was performed to detect protein expression levels. Reverse transcription‐polymerase chain reaction was performed for the detection of relative mRNA levels of tumor necrosis factor- α (TNF- α) and IL-6. Enzyme-linked immunosorbent assay was performed to analyze TNF- α and IL-6 secretion. Our results showed that emodin treatment mitigated IL-1β-induced reduction of cell viability in NPCs. Moreover, the increase in reactive oxygen species (ROS) production, apoptotic rate, and caspase-3 activity in IL-1 β -stimulated NPCs was reduced by emodin treatment. Treatment with emodin also abolished IL-1 β -induced inflammation in NPCs, as indicated by reduced secretion of IL-6 and TNF- α. Besides, the increase in expression levels of phosphorylated p65 and nuclear p65 in IL-1 β -stimulated NPCs was suppressed by emodin treatment. Furthermore, inhibition of nuclear factor kappa B (NF- κ B) activation with pyrrolidine dithiocarbamate aggravated the protective effects of emodin. These results suggested that emodin protected NPCs against IL-1 β -induced apoptosis and inflammation via inhibiting ROS-mediated activation of NF- κ B. [ABSTRACT FROM AUTHOR]

Nucleus pulposus (NP) apoptosis is mainly charged for the pathological process of Intervertebral disc degeneration (IVDD). Our previous study revealed that Resveratrol (RSV) combined with 17β-estradiol (E2) was more effective in cutting down IL-1β induced NP cell apoptosis via PI3K/AKT pathway. The present study further evaluated the effect of RSV and E2 in the anti-apoptosis process of IVDD. Human nucleus pulposus (NP) cells culture system and IL-1β inducing apoptosis model were constructed in this research. RSV and E2 were used to inhibit apoptosis. FACS (Fluorescence-activated cell sorting) and CCK-8 (Cell Counting Kit-8) assays were respectively used to determine apoptotic incidence and cell viability of NP cells. Quantitative RT-PCR was used to determine expression of target genes in mRNA level, and western blot analysis was performed to detect the changes of related protein expression. RSV combined with E2 attenuated IL-1β-induced cell apoptosis and recovered cell viability. Blockers for mTOR and GSK-3β abated the effect of RSV and E2. RSV combined with E2 obviously increased activated P-mTOR and P-GSK-3β, which contributes to the downregulation of caspase-3. Activated P-NF-kappa B was not involved in the anti-apoptosis process of RSV and E2. Combination of Resveratrol and 17β-estradiol efficiently resisted IL-1β induced apoptosis of NP cell, mainly through PI3K/AKT/mTOR/caspase-3 and PI3K/AKT/GSK-3β pathway. [ABSTRACT FROM AUTHOR]

Embelin is a natural benzoquinone compound that displays a beneficial effect in various inflammatory-related diseases. However, the effect of embelin on degeneration of intervertebral disc (IDD), a chronic inflammatory disorder, has not been reported. This study was attempted to explore the therapeutic action of embelin on IDD in vitro. Network pharmacology analysis was performed for evaluating the link between embelin and IDD. The human nucleus pulposus cells (NPCs) were stimulated with IL-1β to induce inflammation. Cell viability of NPCs was assessed by CCK-8 assay. Western blotting was conducted to detect the expression levels of PI3K, p-PI3K, Akt, p-Akt, cleaved caspase-3, caspase-3, Bax, Bcl-2, p65 and p-p65. Apoptotic deaths of NPCs were examined by TUNEL assay. The production of COX-2, IL-6, IL-8, and TNF-α was examined by ELISA. It can be seen that 16 overlapping genes were selected from 109 possible targets of embelin and 342 possible targets of IDD. KEGG pathway enrichment analysis showed that the PI3K/Akt signaling pathway was a close link between embelin and IDD. We found that embelin dose-dependently improved the cell viability in IL-1β-stimulated NPCs. Embelin elevated the relative levels of p-PI3K/PI3K and p-Akt/Akt in IL-1β-stimulated NPCs. IL-1β induced a significant increase in apoptotic deaths of NPCs, which was attenuated by embelin treatment. IL-1β-induced alternations in expression levels of apoptotic-related proteins including cleaved caspase-3, Bax and Bcl-2 were prevented by embelin treatment. Pretreatment with LY294002 (an inhibitor of PI3K) reversed the inhibitory effect of embelin on IL-1β-induced apoptosis in NPCs. Embelin treatment caused inhibitory effects on the IL-1β-stimulated production of COX-2, IL-6, IL-8, and TNF-α, which were abolished by LY294002 treatment. Furthermore, embelin treatment prevented IL-1β-induced phosphorylation of p65 in NPCs, while LY294002 elevated the embelin-caused decrease in p-p65/p65 level. Overall, embelin protected human NPCs against IL-1β-stimulated apoptosis and inflammation by regulating the PI3K/Akt signaling pathway. These findings provided new ideas for the clinical usage of embelin in the prevention and treatment of IDD. • KEGG pathway analysis suggests that PI3K-Akt signaling is a possible regulator for the role of embelin against IDD. • IL-1β-induced cell viability reduction and apoptosis are suppressed after treatment with embelin. • Embelin has an anti-inflammatory effect in IL-1β-stimulated NPCs. • The mechanism of action of embelin involves the PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

• Tyrosol reversed IL-1β-induced inactivation of the PI3K/Akt pathway in HNPCs. • Tyrosol resisted IL-1β-induced viability decrease and apoptosis in HNPCs by PI3K/Akt pathway. • Tyrosol suppressed inflammation and ECM degradation in IL-1β-stimulated HNPCs by PI3K/Akt pathway. • Sirt1 was upregulated by tyrosol and modulated PI3K/Akt pathway in IL-1β-stimulated HNPCs. Intervertebral disc degeneration (IDD) is the major pathogenesis of lower back pain. Tyrosol is a polyphenolic compound that exhibits anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Herein, we explored the effects and mechanisms of tyrosol on IDD progression in interleukin (IL)-1β-stimulated human nucleus pulposus cells (HNPCs). Cell viability and apoptosis were detected by CCK-8 and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was examined to evaluate inflammation. The mRNA expression of matrix metalloproteinases (MMPs) (MMP-3/9/13), collagen type II, SRY-related high mobility group box 9 (SOX-9), and aggrecan was measured by qRT-PCR. Protein levels of silent information regulator 2 homolog 1 (Sirt1), phosphorylated protein kinase B (p-Akt), Akt, collagen type II, SOX-9, and aggrecan were determined by western blot. Results showed that tyrosol attenuated IL-1β-induced viability reduction, apoptosis, and caspase-3/7 activity in HNPCs. The increase in the production of TNF-α, IL-6, NO, and PGE2 in IL-1β-treated HNPCs was abolished by tyrosol treatment. Tyrosol treatment reversed IL-1β-induced upregulation of MMP-3, MMP-9, and MMP-13, and downregulation of collagen II, SOX-9, and aggrecan in HNPCs. Additionally, tyrosol treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in IL-1β-stimulated HNPCs. Sirt1 was upregulated by tyrosol, and Sirt1 silencing inhibited Akt phosphorylation in HNPCs. Sirt1 knockdown attenuated the effects of tyrosol on IL-1β-induced apoptosis, inflammation, and ECM remodeling in HNPCs. In summary, upregulation of Sirt1 by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1β-stimulated HNPCs through activation of PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]