| Objectives:The activation of interleukin-1?(IL-1?),Toll-4 receptor(TLR-4)and nuclear transcription factor(NF-?B)signaling pathways is closely related to the occurrence of inflammatory response and cell apoptosis in the body.Our previous cell model confirmed that the down-regulation of hyaluronan synthase 2/3(HAS-2/3)can directly affect the morphology and function of Cytoskeleton(CSK).The expression of RhoA pathway can be up-regulated at the molecular level of nucleic acid and protein,and the expression of CSK related proteins in cartilage can be regulated,leading to functional degeneration and increased apoptosis of chondrocytes.Therefore,it remains to be further studied whether HAS-2/3 can promote cartilage degeneration by regulating the up-regulation of the expression level of inflammation-related factors,and whether the down-regulation of HAS-2/3 can also up-regulate the expression level of RhoA signaling pathway at the animal level,thus inducing chondrocyte functional degeneration.In this study,the changes in the expression of related inflammatory factors and CSK after the down-regulation of HAS-2/3 expression were studied through cell experiments.The expression of RhoA signaling pathway after the down-regulation of HAS-2/3 expression was studied from the animal experiment level,and the role of HAS-2/3 in the regulation of CSK in cartilage was discussed.It provides a new direction for the early prevention and treatment of osteoarthritis and other related cartilage degenerative diseases.Methods:C28/I2 normal human chondrocytes were transfected with SHHAS-2/3 mediated by lentiviral vector to construct HAS-2/3 gene silenced cell model.Laser Scanning Confocal Microscope(LSCM)was used to detect the expression of inflammatory factors IL-?,TLR-4,NF-kB P65 and the damage degree of CSK after the down-regulated expression of HAS-2/3.To investigate the mechanism of these factors in cartilage CSK damage.Meanwhile,an experimental animal model was established by intraarticular injection of SHHAS-2/3 silencing HAS-2/3 gene.RT-PCR,Western Blot and other molecular biological hand sections were used to detect HAS-2/3,RhoA/ROCK1/ROCK2 mRNA and HAS-2/3,RhoA/ROCK1/ROCK2 protein expression levels in experimental animal chondrocytes.To investigate the effect of HS-2/3 down-regulation on RhoA signaling pathway in animals.The effect of HS-2/3 down-regulation on chondrocytes was observed by embedding pathological specimens and HE staining.Immunohistochemistry was used to observe the effect of down-regulation of HAS-2/3 expression on CSK related proteins in cartilage at animal level.Results:1.Cell experiment:After HAS-2/3 silenced,(1)IL-1? expression and cartilage CSK were detected by immunofluorescence and LSCM.The results showed that there was a significant correlation between HAS-2/3 and IL-1?(P<0.05).After HAS-2/3 silenced,IL-1? expression level in chondrocytes of the experimental group was significantly increased.The severity of CSK damage was significantly correlated with the expression level of IL-1?(P<0.05).(2)TLR-4 expression and CSK were detected by immunofluorescence and LSCM in chondrocytes.The results showed that there was a significant correlation between HAS-2/3 and TLR-4(P<0.05).After HAS-2/3 silenced,the expression level of TLR-4 in chondrocytes of the experimental group was significantly increased.There was a significant correlation between CSK damage severity and TLR-4 expression level(P<0.05).(3)Cell immunofluorescence,the expression of NF-KBp65 detected by LSCM and the condition of cartilage CSK,HAS-2/3 was significantly correlated with NF-KBp65(P<0.05).After HAS-2/3 silenced,the expression level of NF-KBp65 in chondrocytes of the experimental group was significantly increased.The severity of CSK damage was significantly correlated with the expression level of NF-KBP65(P<0.05).2.Animal experiment:after silencing HAS-2/3 gene by lentivirus transfection technology,the articular cartilage of the experimental group showed degenerative changes.(1)HE staining:in the experimental group(SH-HAS2 group and SH-HAS-3 group),the cartilage layer of articular cartilage tissue was thinned,the number of cells was reduced,some cells were reduced in volume,irregular arrangement,fibrosis was observed,and cartilage tissue was damaged.(2)Vimentin immunohistochemistry:the number of positive cells in the experimental group(SH-HAS2 group and SH-HAS3 group)was significantly reduced,a few positive cells were seen in the shallow and middle layer of the cartilage tissue of rats,and the number of deep positive cells was very small,and the CSK of the articular cartilage was destroyed.(3)The results of Tublin immunohistochemistry showed that the number of positive cells in the experimental group(SH-HAS2 group and SH-HAS3 group)was significantly reduced,and the number of positive cells in the shallow layer was not significantly different from that in the control group,but the number of positive cells in the middle layer and deep layer of cartilage was very small,and the expression of CSK related proteins in articular cartilage was reduced and CSK was destroyed.(4)The mRNA expression levels of HAS-2,HAS-3 and RhoA/ROCK1/ROCK2 signaling pathway were detected by RT-PCR,and the mRNA expression levels of HAS-2 and HAS-3 in the experimental group were significantly decreased(P<0.05).MRNA expression levels of RhoA/ROCK1/ROCK2 were significantly up-regulated(P<0.05);(5)W-B detected the protein expression levels of HAS-2,HAS-3 and RhoA/ROCK1/ROCK2,and the protein expression levels of HAS-2 and HAS-3 were significantly decreased in the experimental group(P<0.05),while the protein expression levels of RhoA/ROCK1/ROCK2 were significantly increased in the experimental group(P<0.05).Conclusions:1.The down-regulation of HAS-2/3 expression can lead to the up-regulation of IL-1? expression,the activation of NF-kB signaling pathway,and the destruction of cartilage CSK,suggesting that HAS-2/3 HAS a regulatory effect on the inflammatory response of chondrocytes,and HAS a protective effect on the maintenance of cartilage CSK;2.In animal experiments,the expression of HAS-2/3 in articular chondrocytes was inhibited by intraarticular injection of SHHAS-2/3 lentivirus at the animal level,and mRNA and protein levels of RhoA/ROCK1/ROCK2 classical signaling pathway were increased;3.In animal experiments,the down-regulation of any of HAS-2/3 can lead to articular cartilage degeneration and cartilage CSK destruction.The mechanism may be that down-regulation of HAS-2/3 activates RhoA/ROCK1/ROCK2 classical signaling pathway,leading to chondrocyte injury;4.HAS-2/3 plays an important role in maintaining the normal function of cartilage CSK and protecting chondrocytes. |
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