Effect And Mechanisms Of HAS2 Gene Up-regulation On Articular Cytoskeleton

Objectives: Osteoarthritis(OA)is currently the most common articular lesion in worldwide.With the increase of human life expectancy,both the incidence and prevalence of OA will also rise further.Hyaluronan synthase 2(HAS2)is of great significance in the maintenance of normal cartilage function,but its uncontrolled expression may result in adverse effects.Our previous in vivo and in vitro experiments confirmed that down-regulation of HAS2 can disrupt cytoskeleton(CSK)in cartilage,leading to the increased apoptosis rate of chondrocytes and degeneration of cartilage.However,the effect of HAS2 up-regulation on chondrocytes remains to be further explored.This study aimed to find out the influence of HAS2 gene up-regulation on Rho A/ROCK signaling pathway,inflammatory mediators as well as the morphological and functional changes of chondrocytes and CSK in vitro,and to explore the mechanism and pathway through which HAS2 regulates articular cytoskeleton,so as to find new targets for the early prevention and clinical treatment of OA.Methods: The subjects in this study were C28/I2 human chondrocyte cell lines,4-Methylumbelliferone(4-MU)was used to establish in vitro OA chondrocyte model,and in vitro HAS2 gene overexpression chondrocyte model was constructed by transfection of OE-HAS2 lentivirus.After up-regulating HAS2 gene expression in healthy chondrocytes and OA chondrocytes,flow cytometry was used to detect the changes in the apoptosis rate of chondrocytes,Quantitative real-time RT-PCR(RTq PCR)and Western-Blot(W-B)were used to detect the expression levels of HAS2,Rho A,ROCK1 and ROCK2 so as to verify the relationship between HAS2 and Rho A/ROCK pathway,and Laser scanning confocal microscope(LSCM)was used to detect the changes of immunofluorescence intensity of inflammatory mediators – IL-1β,TLR-4 and NF-κB – and CSK.Results: 1.For healthy chondrocytes,compared with the control group,in the HAS2-overexpressed experimental group:(1)flow cytometry results showed that the apoptosis rate of chondrocytes did not change significantly(P > 0.05).(2)RT-q PCR results showed that the expression level of HAS2 m RNA was significantly increased(P < 0.05),while the expression levels of Rho A,ROCK1 and ROCK2 m RNA were not significantly changed(P > 0.05).(3)W-B results showed that the expression level of HAS2 protein was significantly increased(P < 0.05),while the expression levels of Rho A,ROCK1 and ROCK2 proteins were not significantly changed(P > 0.05).(4)LSCM observation results showed that the immunofluorescence intensity of IL-1β,TLR-4,NF-κB and CSK had no significant changes(P > 0.05).2.For OA chondrocytes,compared with the control group,in the HAS2-overexpressed experimental group:(1)flow cytometry results showed that the apoptosis rate of chondrocytes was significantly decreased(P < 0.05).(2)RT-q PCR results showed that the expression level of HAS2 m RNA was significantly increased(P < 0.05),while the expression levels of Rho A,ROCK1 and ROCK2 m RNA were significantly decreased(P < 0.05).(3)W-B results showed that the expression level of HAS2 protein was significantly increased(P < 0.05),while the expression levels of Rho A,ROCK1 and ROCK2 proteins were significantly decreased(P < 0.05).(4)LSCM observation results showed that the immunofluorescence intensity of IL-1β,TLR-4,NF-κB was significant decreased(P < 0.05),while the immunofluorescence intensity of CSK was significantly increased(P < 0.05).Conclusions: 1.In vitro experiments,HAS2 gene overexpression mediated by OE-HAS2 lentivirus transfection can up-regulate HAS2 m RNA and protein expression levels in both healthy and OA chondrocytes.2.In healthy chondrocytes,overexpression of HAS2 gene has no significant effect on the normal morphology or function of chondrocytes and CSK in cartilage.3.In OA chondrocytes,the up-regulation of HAS2 gene can inhibit the activation of Rho A/ROCK signaling pathway,reduce the expression of IL-1β,TLR-4,NF-κB,reduce the destruction of CSK as well as the apoptosis of chondrocytes,and slow down the degeneration of chondrocytes.