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Activation of transient receptor potential vanilloid 4 protects articular cartilage against inflammatory responses via CaMKK/AMPK/NF-κB signaling pathway.

ABSTRACT: Transient receptor potential vanilloid 4 (TRPV4) plays an important role in chondrocytes via Ca²⁺ signaling. However, its role in the progression of osteoarthritis is unclear. This study aimed to evaluate the effects of TRPV4 activation on articular cartilage and chondrocytes stimulated with interleukin (IL)-1β. Bovine and human articular chondrocytes were stimulated with various agents, including IL-1β, GSK1016790A (GSK101; a TRPV4 agonist), Compound C (an AMP-activated protein kinase (AMPK) inhibitor), and STO-609 (a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor), and were processed for Western blot analysis and real-time PCR. The dimethylmethylene blue (DMMB) assay and Safranin O staining were also performed. GSK101 reversed the IL-1β-induced increase in expression of matrix metalloproteinase (MMP)-13 and decrease in expression of aggrecan. GSK101 also decreased proteoglycan release in the DMMB assay and retained Safranin O staining of articular cartilage tissue. Furthermore, GSK101 increased AMPK phosphorylation and decreased IL-1β-induced nuclear factor kappa B (NF-κB) phosphorylation. Compound C and STO-609 reversed the suppressive effects of GSK101 on NF-κB activation and MMP-13 expression. In conclusion, TRPV4 activation had chondroprotective effects on articular cartilage stimulated with IL-1β by activating CaMKK/AMPK and suppressing the NF-κB pathway. TRPV4 activators may offer a promising therapeutic option for preventing the progression of osteoarthritis.

SUBMITTER: Hattori K

PROVIDER: S-EPMC8324869 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicroglia-mediated neuroinflammation is important in Alzheimer's disease (AD) pathogenesis. Extracellular deposition of β-amyloid (Aβ), a major pathological hallmark of AD, can induce microglia activation. Adiponectin (APN), an adipocyte-derived adipokine, exerts anti-inflammatory effects in the periphery and brain. Chronic APN deficiency leads to cognitive impairment and AD-like pathologies in aged mice. Here, we aim to study the role of APN in regulating microglia-mediated neuroinflammation in AD.MethodsInflammatory response of cultured microglia (BV2 cells) to AβO and effects of APN were studied by measuring levels of proinflammatory cytokines (tumor necrosis factor α [TNFα] and interleukin-1β [IL-1β]) in cultured medium before and after exposure to AβO, with and without APN pretreatment. Adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) were targeted by small interference RNA. To study the neuroprotective effect of APN, cultured HT-22 hippocampal cells were treated with conditioned medium of AβO-exposed BV2 cells or were co-cultured with BV2 cells in transwells. The cytotoxicity of HT-22 hippocampal cells was assessed by MTT reduction. We generated APN-deficient AD mice (APN-/-5xFAD) by crossing APN-knockout mice with 5xFAD mice to determine the effects of APN deficiency on microglia-mediated neuroinflammation in AD.ResultsAdipoR1 and AdipoR2 were expressed in BV2 cells and microglia of mice. Pretreatment with APN for 2 h suppressed TNFα and IL-1β release induced by AβO in BV2 cells. Additionally, APN rescued the decrease of AMPK phosphorylation and suppressed nuclear translocation of nuclear factor kappa B (NF-κB) induced by AβO. Compound C, an inhibitor of AMPK, abolished these effects of APN. Knockdown of AdipoR1, but not AdipoR2 in BV2 cells, inhibited the ability of APN to suppress proinflammatory cytokine release induced by AβO. Moreover, pretreatment with APN inhibited the cytotoxicity of HT-22 cells co-cultured with AβO-exposed BV2 cells. Lastly, APN deficiency exacerbated microglia activation in 9-month-old APN-/-5xFAD mice associated with upregulation of TNFα and IL-1β in the cortex and hippocampus.ConclusionsOur findings demonstrate that APN inhibits inflammatory response of microglia to AβO via AdipoR1-AMPK-NF-κB signaling, and APN deficiency aggravates microglia activation and neuroinflammation in AD mice. APN may be a novel therapeutic agent for inhibiting neuroinflammation in AD.

Project description:BackgroundGlaucocalyxin B (Gla B) is a type of sesquiterpenoids. At present, there are rare studies on the pharmacological effects and targets of sesquiterpenoids, while multiple sesquiterpenoids have good anti-inflammatory properties. Therefore, in this study, we aimed to investigate the mechanism of Gla B on macrophages and rheumatoid arthritis.MethodsLPS/IFN-γ was used to induce M1 polarization of synovial macrophage (SMG) in vitro, followed by Gla B pretreatment (5 μM and 15 μM). Afterwards, flow cytometry was performed to detect the proportion of M1 cells (F4/80+CD86+), enzyme-linked immunosorbent assay (ELISA) was used to determine the expression levels of M1 cell markers (TNF-α, IL-1β, IL-6, iNOS and IL-12) as well as M2 cell markers (IL-10 and TGF- β1), immunofluorescence (IF) staining was utilized to measure the expression of CD86, the level of ROS was assessed by probe and Western blot was conducted to detect the expression of P65 and p-P65. M1 polarization was detected in SMG cells with P65 silencing after 15 μM Gla B intervention. The culture medium from M1 cell was used to culture cartilage cells in vitro, followed by detection of cartilage cell injury. In animal models, collagen antibodies and LPS were combined to induce RA mouse model. Afterwards, H and E staining was performed to detect pathological changes in mouse joint synovium, safranin O-fast green staining was used to determine cartilage injury, and immunohistochemistry was utilized to detect CD86 and P65 expression. Small molecule-protein docking and co-immunoprecipitation (Co-IP) were used to verify the targeted binding relationship between Gal B and P65.ResultsLPS and IFN-γ could induce M1 polarization in SMG. Gal B could inhibit M1 polarization, decrease the levels of TNF-α, IL-1β, IL-6, iNOS and IL-12, inhibit the expression of P65 and p-P65 while did not affect the expression of IL-10 or TGF-β1. Gal B had no significant effect in SMG cells with P65 silencing. The small molecule-protein docking and Co-IP both showed that Gal B had a targeted binding relationship with P65, and Gal B could inhibit joint injury and inflammation in mice.ConclusionGal B could target the P65 protein. Moreover, Gal B could inhibit the inflammatory injury of articular cartilage in RA by regulating M1 polarization of SMG through inhibiting the NF-κB signaling.

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Activation of transient receptor potential vanilloid 4 protects articular cartilage against inflammatory responses via CaMKK/AMPK/NF-κB signaling pathway.

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