Analyses of Transcriptomics upon IL-1β-Stimulated Mouse Chondrocytes and the Protective Effect of Catalpol through the NOD2/NF-κB/MAPK Signaling Pathway

Abstract

The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1β (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 μM catalpol 2 h prior to treatment with IL-1β (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 μM and 100 μM) could significantly reduce NO, IL-6, IL-1β, and TNF-α in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1β. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-α mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKBα protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-κB and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1β. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of IκBα, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.

Integrated Network Pharmacology and Experimental Validation Approach to Investigate the Mechanisms of Radix Rehmanniae Praeparata - Angelica Sinensis - Radix Achyranthis Bidentatae in Treating Knee Osteoarthritis

“…41 Catalpol is not only the main active component of Rehmannia glutinosa, but also its primary active component has been reported that can prevent IL-1β-induced chondrocyte injury through inhibiting the NF-κB pathway. 25 Glabralactone, a Coumarin Compound from Angelica Sinensis exhibited an anti-inflammatory activity in in vitro and in vivo through TRIF-Dependent IRF-3 signaling and NF- κB pathways. 26 Another study has shown the Angelica Sinensis polysaccharide has the ability to protect chondrocytes from H 2 O 2 -induced apoptosis via its antioxidant effects.…”

“…According to earlier research, Radix Rehmanniae Praeparata has many biological effects, such as anticancer and anti-inflammatory effects, that can help treat bone illnesses such as OA. 24 , 25 Angelica Sinensis possesses anti-inflammatory and antiapoptotic properties and has been widely used in TCM for treating various diseases, such as inflammation, infections, and OA. 26 , 27 The Chinese herb Radix Achyranthis Bidentatae was reported to have antioxidative and anti-inflammatory effects.…”

Anti‐inflammatory effect and mechanism of catalpol in various inflammatory diseases

“…Catalpol significantly downregulated PGE2, COX‐2, IL‐8, and monocyte chemoattractant protein‐1, and downregulated phosphorylation of nuclear factor κB‐α inhibitors (Cai et al, 2022). Catalpol alleviates inflammatory damage to chondrocytes by inhibiting NOD2 (nucleotide‐binding oligomerization domain 2) signaling activation, reducing phosphorylation of ERK, p38, and JNK, inhibiting degradation of kappa B alpha, inhibiting p65 translocation to the nucleus, and reducing the release of inflammatory cytokines (Pang et al, 2023).…”

“…Catalpol significantly downregulated PGE2, COX-2, IL-8, and monocyte chemoattractant protein-1, and downregulated phosphorylation of nuclear factor κB-α inhibitors(Cai et al, 2022). Catalpol alleviates inflammatory damage to chondrocytes by inhibiting NOD2 (nucleotide-binding oligomerization domain 2) signaling activation, reducing phosphorylation of ERK, p38, and JNK, inhibiting degradation of kappa B alpha, inhibiting p65 translocation to the nucleus, and reducing the release of inflammatory cytokines(Pang et al, 2023).3.7 | Eye diseases3.7.1 | Corneal neovascularization (CNV)CNV is the pathological basis of many ocular diseases, often caused by ocular infections, inflammation, degenerative changes, and trauma(Nicholas & Mysore, 2021). The mechanism of development is not fully understood, but it is now recognized that dynamic imbalance between proangiogenic and antiangiogenic factors(Sharif & Sharif, 2019).…”

Suppression of CGRP and TRPV1 Expression in Dorsal Root Ganglia of Knee Osteoarthritis Rats by Huojing Decoction via TrkA/MKK3/6/p38 Pathway

“…Inflammation plays a crucial role in preserving the equilibrium of the knee joint's aging and internal environment. IL-1β, a versatile cytokine, not only provokes inflammation but also collaborates with other inflammatory factors to intensify the inflammatory response, ultimately fostering cartilage degradation [7]. As the disease advances, sustained inflammatory stimulation and progressive neuron damage may lead to pain sensitization and the loss of peripheral nerve fibers.…”