High molecular weight hyaluronan reduced the inflammatory response in human chondrocytes exposed to LPS: involvement of jagged/notch pathway

Purpose. The aim of this study is to investigate the effects of high molecular weight hyaluronan (HMW-HA) in human primary chondrocytes stimulated by lipopolysaccharides (LPS). Since studies revealed the involvement of NOTCH signaling in inflammatory processes, JAGGED/NOTCH pathway was investigated. Methods. Chondrocytes were previously stimulated with LPS and subsequently treated with HMW-HA at increasing concentrations. A further set of plates were treated with NOTCH1/2 siRNAs before LPS treatment. mRNA levels of TLR-4, NOTCH1/2, JAGGED1, MAPK, RBP-J, IL-6, IL-1β, and MMP-13 were evaluated by qPCR. NF-kB (p65) activation was determined by a commercial assay. The protein levels of TLR-4, NOTCH1/2 and pro-inflammatory cytokines were measured by ELISA and western blot. Results. LPS induced high expression of TLR-4, MAPK, JAGGED1, NOTCH1/2, RBP-J, IL-6, IL-1β and MMP-13, as well as NF-kB activation. The pre-treatment of chondrocytes with HMW-HA, previous stimulated with LPS, reduces significantly the inflammatory response. Discussion. Stimulation of TLR-4 triggers activation of downstream signaling molecules such as MAPK and finally NF-kB. Furthermore, this receptor may prime NOTCH pathway that through JAGGED1 activate pro-inflammatory cytokine transcription. LPS stimulation was able to produce a strong inflammatory response. On the contrary, HMW-HA reduced the mRNA and protein levels of pro-inflammatory mediators, as well as NF-kB activation. In addition, it decreased MAPK, JAGGED1, NOTCH1/2, and RBP-J mRNA and protein levels. As HA acts by blocking TLR4 activation, specific NOTCH1/2 siRNAs were used to verify if the inflammatory response could be further influenced. Results proved that IL-6, IL-1β and MMP-13 were further reduced, those suggesting that NOTCH-1/2 signaling can be activate independently by TLR-4 activation. Conclusion. These results suggest that HMW-HA exert a protective effects on human primary chondrocytes via regulating JAGGED/NOTCH pathway.