The Mechanism Of Mechanical Stress Regulating Cartilage Development Through The Interaction Between Primary Cilia And Autophagy

Objective 1.To detect the effects of cyclic mechanical stress of different intensities on rat chondrocytes proliferation,viability and phenotype maintenance.2.To evaluate the role of primary cilia and autophagy in the process of cartilage development and their potential interaction relationship.3.To investigate the effect of mechanical stress on the interaction of primary cilia and autophagy and evaluate the role of ERK-m TOR signal axis in this regulation.4.To study the role of inositol polyphosphate 5-phosphatase E(INPP5E)in stress-mediated regulation of primary cilia and autophagy interaction.Method 1.The chondrocytes were isolated from neonatal rats and cultured in vitro.Cells were subjected to different intensities of cyclic mechanical stress by the Four Point Bending System.Western blot assay was used to detect the expressions of proliferation related proteins Cyclin D1,PCNA and cartilage phenotype related protein COL II.RT-PCR was used to detect phenotype related genes COL II and SOX9 expression.Live/dead assay was use to detect the viability changes of chondrocytes.2.The location and expression of primary cilia and autophagy during the development of chondrocytes were observed by transmission electron microscopy and immunofluorescence double-labeled staining.Then,serum free medium was used to induce primary cilia,chloral hydrate to destroy cilia,rapamycin to activate autophagy and 3-methyladenine(3MA)to inhibit autophagy.Immunofluorescence double-labeled staining and western blot assays were used to detect primary cilia and autophagy expression as well as their functional proteins expression.3.Chondrocytes were subjected to different intensities of mechanical stress by Four PointBending System.Immunofluorescence staining and Western blot were used to detect primary cilia expression,length changes and IFT88 expression,and detect the expression of autophagy and related proteins LC3 and Beclin1.After destroying primary cilia by chloral hydrate,Western blot test was used to detect primary cilia and autophagy interaction related proteins expression as well as ERK-m TOR signal axis related P-ERK,P-m TOR expression when under mechanical stress(2500?strain).4.Chondrocytes were subjected to different intensities of mechanical stress in vitro and INPP5 E was detected by Western blot test.si RNA was transfected and RT-PCR,Western blot assays were used to detect the transfection efficiency,and immunofluorescence staining was used to detect primary cilia and autophagy expression.After transfected with INPP5 E si RNA for 2 days,cells were subjected to 2500?strain stress and IFT88,LC3,Cyclin D1 and COL II proteins were detected by Western blot.Animal experiments were also performed to verify the exercise training on cartilage matrix secretion and the expression of IFT88,LC3 B and INPP5 E proteins in post-traumatic osteoarthritis models.Results 1.Low to moderate-intensity stress(1000 and 2500?strain)could increase the expression of Cyclin D1 and PCNA,while the effect of high-intensity stress(4000?strain)was not obvious.Low to moderate-intensity stress had no significant effect on chondrocytes viability,while the number of apoptotic chondrocytes increased after high-intensity stress stimulation.Similarly,Low to moderate-intensity stress could up-regulate the expression of phenotype-related genes COL II,SOX9 and protein COL II,while high-intensity showed an inhibition effect.2.There was co-expression of primary cilia and autophagy in normal chondrocytes,and existed a certain location and expression relationship between the two structures during chondrocyte cycle.Serum-free medium starvation could induce the expression of primary cilia,autophagy and their proteins simultaneously,while chloral hydrate inhibited cilia,autophagy and their proteins.Rapamycin could up-regulate the expression of IFT88 when activating autophagy,and 3-methyladenine could inhibit autophagy and reduce the incidence and length of primary cilia.3.High-intensity stress(4000?strain)down-regulated the incidence and length of primary cilia,while low to moderate-intensity stress(1000?strain and 2500?strain)could up-regulate the expression of IFT88 protein but had no significant effects on cilia incidence and length.Mechanical stress could promote chondrocytes autophagy in a dose-dependent manner,with a peak at 2500 ?strain and showed an inhibitory effect by high-intensity.2500?strain stress could promote IFT88,LC3,Beclin1 expression,increase P-ERK expression and reduce P-m TOR expression at the same time.Chloral hydrate stimulation could inhibit IFT88,LC3,Beclin1 and P-ERK expression but increase P-m TOR expression.Meanwhile,chloral hydrate could also inhibit the related proteins induced by 2500?strain and promote P-m TOR expression.4.Different intensity of mechanical stress could promote chondrocytes INPP5 E protein expression and most obviously at low to moderate intensity(1000?strain and 2500?strain).Primary cilia and autophagy expression were inhibited when knocking down INPP5 E with si RNA.Compared with the control group,2500?strain stress could promote the expression of IFT88,LC3-II,Cyclin D1 and COL II,but decreased when transferring with INPP5 E si RNA at the same time.Appropriate exercise training is beneficial to the matrix secretion of osteoarthritic cartilage with up-regulating the expression of IFT88,LC3 B and INPP5 E in cartilage tissue.Conclusion 1.Appropriate intensity of mechanical stress can promote chondrocytes proliferation and phenotype maintenance,but high-intensity stress has a negative effect on chondrocytes viability.2.There is a certain relationship in location and expression between primary cilia andautophagy in chondrocytes development through interactive regulation.3.Mechanical stress can affect chondrocytes primary cilia and autophagy expression,and this mechanical signals transduction can regulate chondrocyte autophagy expression through primary cilia-ERK-m TOR signaling axis.4.Mechanical stress can promote the expression of INPP5 E,which can participate in the regulation of primary cilia and autophagy interaction under mechanical stress.INPP5 E may be the key point for stress mediated interaction regulation between primary cilia and autophagy.