| Background and PurposeOsteoarthritis(OA)is a kind of degenerative disease involving all joints in the whole body,and has a high prevalence rate in all kinds of people in the world.End-stage osteoarthritis patients often need joint replacement surgery to relieve pain and restore physiological function,but joint replacement surgery is often accompanied by many complications and failure rates,and patient satisfaction is not high.Cartilage injury and degeneration are important pathological manifestations of osteoarthritis.Due to the particularity of cartilage tissue,chondrocytes are the only cells that can synthesize and maintain the shape of cartilage tissue.Any damage to chondrocytes will eventually damage the function and integrity of cartilage tissue.In recent years,mechanical stress load has been widely studied as an important role in the occurrence and development of osteoarthritis.Some scholars have found that high mechanical stress load is closely related to various programmed cell death events.Ferroptosis is a newly developed programmed cell death mode,which is different from apoptosis,necrosis and pyrosis.Ferroptosis is characterized by lipid peroxidation damage to cell intima structure,which is mainly manifested by increased ROS content in cells,decreased activity and expression of key regulatory protein GPX4,mitochondrial shrinkage and mitochondrial membrane thickening.Piezo1 is an ion-channel protein proven to exist on human chondrocytes.By selectively permeating various ions,mainly Ca2+,in response to different forms of mechanical stress stimulation,Piezo1 protein works to convert pressure signals into electrochemical signals.Based on the important role of cartilage cells in the development of osteoarthritis,and the potential link between ferroptosis and chondrocyte damage by the mechanical stress,Piezo1 protein,this study explores several questions:(1)Whether ferroptosis and Piezol protein are involved in osteoarthritis induced by high mechanical stress.(2)What role does chondrocyte ferroptosis play in the occurrence and development of osteoarthritis,and what effect does regulate chondrocyte ferroptosis have on osteoarthritis?(3)How Piezol,is activated by the high mechanical stress,regulates the activity of chondrocytes.And whether there is a link between the mechanical stress,Piezo1,and ferroptosis in chondrocytes,and by what molecular pathway.Methods(1)The cartilage tissues of human knee osteoarthritis patients in weight-bearing and nonweight-bearing areas were collected for mRNA transcriptome sequencing to explore the difference in protein expression caused by high mechanical stress.At the same time,part of the cartilage tissues was observed by transmission electron microscopy to determine whether chondrocytes showed characteristic manifestations of ferroptosis under high mechanical stress.In vitro cell mechanical stress stimulation culture model was designed and prepared.Primary mouse chondrocytes were extracted and subjected to high mechanical stress stimulation to detect the expression level of GPX4,a key regulatory protein of iron death.Mitochondria of chondrocytes were observed by transmission electron microscopy.(2)Construct chondrocyte specific knockout mice(Col2al-CreERT GPX4flox/flox)of ferroptosis regulatory protein GPX4.Specific knockout the GPX4 expression in chondrocytes and induced osteoarthritis model,then detected by Micro-CT,crosin solid green staining,immunohistochemical staining,to examine the effect of chondrocyte ferroptosis on the progression of osteoarthritis.On the other hand,overexpression of the regulatory protein FSP1(Ferroptosis Suppressant protein 1)of the Ferroptosis bypass regulatory pathway and supplementation of regulatory factor coenzyme Q10 were used to inhibit the ferroptosis of chondrocytes induced by GPX4 knockout,so as to explore the influence of the regulation of chondrocyte ferroptosis on the occurrence and development of osteoarthritis.(3)Wild-type mouse primary chondrocytes were extracted and stimulated by Piezol protein-specific inhibitor(GsMTx4)in vitro then detect the changes in calcium content,cell activity,intracellular glutathione GSH level,the expression of mitochondrial morphology,activity,membrane potential,as well as GPX4 and metabolism related parameters,to clarify whether Piezo1 protein is involved in the high mechanical stress-induced chondrocyte ferroptosis and its role.Secondly,the role of Piezo1 protein in the development of osteoarthritis was determined by injection of GsMTx4 into the joint of wild-type mice and then detected by Micro-CT,saffron solid green staining,and immunohistochemical staining.Primary chondrocytes of GPX4 chondrocyte specific knockout mouse(Col2al-CreERT GPX4flox/flox)were extracted,GPX4 knockdown of chondrocytes in vitro was performed with 4-OH-TAM,and then cultured with mechanical stress stimulation in vitro to observe the changes of intracellular calcium content,cell activity,the intracellular levels of reduced glutathione GSH,mitochondrial morphology,activity and membrane potential,the expressions of GPX4 and metabolic related parameters to explain the GPX4-mediated ferroptosis in chondrocytes may be the key downstream pathway after Piezo1 protein is activated by high mechanical stress.Finally,extracellular calcium ions were removed,and mechanical stress stimulation culture was performed in vitro again to observe the changes of intracellular calcium ion content,cell activity,intracellular reduced glutathione GSH level,mitochondrial morphology,activity and membrane potential,as well as the expression of GPX4 and metabolic related indexes.To explore whether Piezo1-mediated calcium influx is the regulator between Piezo1,a protein activated by high mechanical stress,and iron death in chondrocytes mediated by GPX4.Results(1)High mechanical stress decreased GPX4 protein expression level,and induced chondrocyte mitochondrial membrane thickening and mitochondrial shrinkage.MRNA sequencing results of loading and non-loading cartilage in patients with osteoarthritis showed that the level of GPX4,was significantly lower in loading cartilage than in non-loading cartilage(P<0.01).Meanwhile,the results of transmission electron microscopy showed that the chondrocytes in loading zone showed the characteristics of ferroptosis,including mitochondrial membrane thickening,mitochondrial shrinkage and mitochondrial crest disappearance.Furthermore,chondrocytes stimulated by high mechanical stress(1MPa,1HZ)in vitro significantly decreased GPX4 expression and transcription levels(P<0.01).Transmission electron microscopy analysis of chondrocytes showed that the mitochondria of chondrocytes a characteristic phenotype of ferroptosis after being stimulated by high mechanical stress.MRNA transcriptome analysis of normal and high-stress chondrocytes showed significantly lower GPX4 levels(P<0.01).In the mouse knee joint,GPX4 protein was mainly located in the superficial layer of cartilage.At the same time,the expression level of GPX4 in the surface layer of the knee cartilage was significantly decreased after the establishment of the mouse knee DMM osteoarthritis model.(2)Knockout GPX4 protein in mouse osteo-articular chondrocytes aggravated the degree of osteoarthritis,and targeted inhibition of chondrocyte ferroptosis could alleviate the damage of bone and joint.In mouse arthritis,GPX4 protein is mainly localized in the surface chondrocytes.By building GPX4 chondrocyte specific knockout mice(Col2al-CreERT GPX4flox/flox),it was found that the loss of GPX4 could aggravate the degree of osteoarthritis and reduce the anabolic products of chondrocytes,increased the expression of matrix protease.Further,the articular cavity of GPX4 chondrocyte specific knockout mice was transfected with FSP1 overexpression adeno-associated virus to enhance the expression level of FSP1.The results showed that the overexpression of FSP1 alleviated the degree of osteoarthritis aggravated by GPX4 deletion.FSP1 uses coenzyme Q10 as a key substrate in ferroptosis metabolism,so we studied its therapeutic value for osteoarthritis aggravated by GPX4 deficiency by oral administration of coenzyme Q10.The results showed that oral coenzyme Q10 could effectively relieve the severity of osteoarthritis aggravated by GPX4 deficiency.(3)The high mechanical stress induces chondrocytes ferroptosis by activating the calcium influx mediated by the Piezo1 protein.In previous experiments,it was found that in addition to the difference in the expression of GPX4,the mechanical stresses are also responsible for the different expression of Piezo1,in particular,the Piezo1 protein whose transcriptional levels are elevated in the highly stressed group.The mouse chondrocytes were stimulated with high mechanical stress with or without Piezo1 protein inhibitor(GsMTx4).The results show that:compared with the high mechanical stress group,the inhibition of Piezo1 protein resulted in decreased intracellular calcium content,increased intracellular GSH level,increased GPX4 protein expression,decreased intracellular ROS content,restoration of intracellular mitochondrial activity and membrane potential,and decreased cell damage.In addition,intraarticular injection of GsMTx4 injected increased the GPX4 protein levels in bone and articular chondrocytes,alleviated the destruction of articular cartilage in mouse DMM model.Further,primary chondrocytes of GPX4 chondrocyte specific knockout mouse(Col2a1CreERT GPX4flox/flox)were extracted and the expression of GPX4 was knocked down in vitro,followed by high mechanical stress stimulation and GsMTx4.The results show that:after GPX4 knockdown,high mechanical stress stimulation could still induce the increase of intracellular calcium ion and the decrease of GSH level,and the addition of GsMTx4 could inhibited the level of calcium ion and restored the content of GSH.However,after GPX4 knockdown,a large number of chondrocytes were also inactivated then whatever stimulated by high mechanical stress,or added GsMTx4,chondrocyte activity did not show significant changes.Similarly,both mitochondrial membrane potential and mitochondrial activity showed low expression after GPX4 knockdown,and mechanical stress stimulation did not induce corresponding changes.Finally,to demonstrate the critical role of calcium influx in mechanical stress-induced chondrocyte ferroptosis,we prepared calcium-free culture-medium and cultured chondrocytes with calcium-free culture-medium both at the time of mechanical stress stimulation and within 24 hours after stimulation.The results showed that after extracellular calcium ions were removed,the GSH level could not be reduced by high mechanical stress stimulation,the intracellular ROS content was significantly decreased,mitochondrial activity was enhanced,membrane potential was increased,GPX4 expression level was increased,Col2 level was increased,matrix protease ADAMTS5 and MMP-13 expression levels were significantly decreased,and chondrocyte activity was significantly increased.Conclusion1.High mechanical stress load can induce chondrocyte ferroptosis.2.Conditional knockout of GPX4 expression in chondrocytes can aggravate the degree of osteoarthritis in mice,aggravate the catabolism of chondrocytes and inhibit anabolism.3.After conditional knockout of GPX4 expression in chondrocytes,overexpression of FSP1 or supplement of coenzyme Q10 can strengthen the ferroptosis bypass pathway,which can reduce the degree of cartilage injury in osteoarthritis,indicating that chondrocyte ferroptosis can aggravate the progression of OA.4.The ion channel protein Piezo1 is involved in the high-mechanical stress-induced chondrocyte ferroptosis.5.Inhibiting Piezo1 activity could reduce the damage to cartilage in osteoarthritis.6.The high-mechanical stress-induced chondrocyte ferroptosis is achieved by Piezo1,mediated calcium ion influx. |
