The Role Of NALP3 Inflammasome In Inflammation Of Differentiated THP-1 Macrophages And Chondrocyte Degradation Induced By Hydroxyapatite

Osteoarthritis(OA)is the most common joint disease characterized by articular cartilage degradation and subchondral bone remodeling,which can seriously affect health and ability.The exact pathogenesis of osteoarthritis remains unclear.Previous studies have shown that immune inflammation caused by cartilage and matrix damage is the key role of a series of pathological injuries.The NALP3(NACHT-LRR-PYD-containing protein 3)inflammasome is an important NOD-like receptor composed of NALP3,ASC(apoptosis associated speck-like protein containing a CARD domain)and caspase-1(cysteine-containing aspartate-specific protease),and its activation plays an important role in the innate immune response.Based on previous studies,we hypothesized that when external mechanical forces cause cartilage destruction,the crystalline hydroxyapatite(HA)is exposed and recognized by pattern recognition receptors of joint synovium macrophages membrane.The immune response is initiated through the NALP3 signaling pathway,and a large number of inflammatory cytokines are secreted.Leading to inflammation and subsequent destruction of cartilage matrix.The study was divided into two parts:(1)the role of NALP3 inflammasome in hydroxyapatite-induced inflammation on differentiated THP-1 macrophages;(2)degradation effects of conditioned medium from hydroxyapatite-stimulated macrophages on chondrocyte.Part 1 The role of NALP3 inflammasome in hydroxyapatite-induced inflammation on differentiated THP-1 macrophagesObjective: To investigate the modulating role of NALP3 inflammasome in hydroxyapatite-induced inflammatory response on human macrophages.Methods: Human macrophages differentiated from THP-1 were experimental target cells.Macrophages were exposed to 25,50,100,150,200 μg/m L hydroxyapatite for 6 h in vitro and then stimulating macrophages at different time points(1.5,3,6,12,24,36 h)with a fixed exposure concentration(100 μg/m L)to obtain the dose-response relationship and time-response relationship.We selected two kinds of NALP3 inflammasome antagonists(Ac-YVAD-cmk,MCC 950)based on previous literature.Macrophages were pretreated with NALP3 inflammasome antagonists as blocking groups.Additionally,stimulating macrophages with silica and blank culture medium as positive control and blank control respectively.Cell viability was determined by CCK-8 kit.Intracellular reactive oxygen species(ROS)level and the activity of lactate dehydrogenase(LDH)were detected by spectrophotometer,which reflect the intracellular oxidative stress level and cell membrane toxicity respectively.Expression levels of inflammatory cytokine(interleukin-1β,IL-1β)in culture medium was measured by enzyme-linked immunosorbent assay(ELISA).The relative expression levels of NALP3,caspase-1,IL-1β,IL-6 and TGF-β m RNA were evaluated by real-time quantitative polymerase chain reaction(RT-q PCR).The protein expression levels of NALP3 and caspase-1 were detected by immunofluorescence.Results:(1)Our results showed that both hydroxyapatite and silica at the same concentration(100 μg/m L)could cause the decline of cell viability,increase of ROS level and inflammatory cytokine IL-1β secretion(P(27)0.05)compared with blank control.Dosedependent decrease of cell viability,and increases of the activity of LDH,secretion of IL-1β,relative expression levels of NALP3,caspase-1,IL-1β and IL-6 m RNA were observed when hydroxyapatite concentrations increased.The expression levels of NALP3 and caspase-1 protein increased when hydroxyapatite concentrations elevated.Suggesting that hydroxyapatite could cause macrophages oxidative stress,NALP3 inflammasome activation and inflammatory response.With the increase of hydroxyapatite exposure time,cell viability of macrophages decreased,while the LDH activity gradually increased,and the IL-1β secretion level increased during 0-6 h,but there was no significant change during 6-24 h.(2)NALP3 inflammasome inhibitors Ac-YVAD-cmk and MCC 950 have no significant effects on macrophage cell viability and LDH activity.Compared with the hydroxyapatite-exposure group alone,Ac-YVAD-cmk and MCC 950 reduced the relative expression levels of NALP3,caspase-1,IL-1β,and IL-6 m RNA,and significantly reduced the levels of IL-1β secretion level(P(27)0.05).It indicates that antagonizing NALP3 inflammasome reduces the level of inflammatory cytokines.Conclusions: Hydroxyapatite could induce suppression of cell viability,enhancing of cytotoxicity and oxidative stress,NALP3 inflammasome activation and inflammatory response on THP-1 differentiated macrophages.Hydroxyapatite-induced inflammatory response was significantly decreased when the NALP3 inflammasome pathway was specifically blocked.Part 2 Degradation effects of conditioned medium from hydroxyapatite-stimulated macrophages on chondrocyteObjective: To investigate the degradation effects of conditioned medium(CM)induced by hydroxyapatite-stimulated macrophages on chondrocytes.Methods: Conditioned medium with different concentrations(represented by the concentration of hydroxyapatite-stimulated macrophages)and hydroxyapatite were used to stimulate SW 1353 chondrosarcoma cells(referred to as chondrocytes)for 6 h in vitro.Additionally,we set NALP3 inflammasome antagonist groups(CM + Ac-YVAD-cmk,CM + MCC 950)and IL-1β block group(CM + anti-IL-1β)for the conditioned medium.Chondrocytes treated with blank culture medium was set as blank control group.Realtime quantitative PCR was performed to detect the m RNA expression levels of collagen type Ⅱ,aggrecan,matrix metalloproteinase(MMP)-3,MMP-13 and apoptosis related gene,such as caspase-3,Bax and Bcl-2.The protein expression level of collagen type Ⅱ was detected by immunofluorescence.Results:(1)The relative expression levels of aggrecan m RNA decreased and caspase-3 m RNA increased when hydroxyapatite directly stimulated chondrocytes,and we did not observe significantly changes in the relative expression levels of collagen type II,MMP-3,MMP-13,Bax and Bcl-2 m RNA(P>0.05).The expression levels of collagen type II,aggrecan,and Bcl-2 m RNA and collagen type II protein decreased,and the expression levels of MMP-3,MMP-13,and caspase-3 m RNA increased when conditioned medium concentrations increased,whereas the expression level of Bax m RNA did not change significantly.(2)Compared with the conditioned medium group,the expression levels of collagen type II,aggrecan,Bcl-2 m RNA and collagen type II protein increased,the expression levels of MMP-3,MMP-13 and caspase-3 m RNA decreased when the NALP3 inflammasome or IL-1β were specifically blocked.Conclusions: The supernatant of hydroxyapatite-stimulated macrophages can cause chondrocytes degradation by inhibiting the expression of collagen type II and aggrecan,increasing the expression of matrix metalloproteinases,and enhancing apoptosis.Specifically block NALP3 inflammasome or IL-1β of macrophages can alleviate these effects.