Research progress of necroptosis and ferroptosis in knee osteoarthritis

ZONG Yi-zhou, DAI Yi, LI Zhe, YAN Wei-wei, LEI Mao-kun, YUAN Chang-shen,2,DUAN Kan,2✉

1.Guangxi University of Traditional Chinese Medicine, Nanning 530000, China

2.Department of Extremities and Orthopedics, Dongge District, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530000, China

Keywords:

ABSTRACT

Knee Osteoarthritis (KOA) is a chronic degenerative disease characterized by articular cartilage wear, ligament injury, synovial inflammation and articular osteophytes.Its clinical manifestations mainly include joint swelling and pain, limited movement and other discomfort, and even knee deformation or muscle weakness in severe cases[1].According to epidemiological statistics[2], KOA affects about 650 million people in the world, of which 22.9% are over 40 years old, and its incidence is negatively correlated with the level of education, bringing a heavy burden to the medical security system.At present, the etiology of the disease is not clear,and it is mostly related to age, weight, trauma and environmental changes.The commonly used treatment methods include proper exercise, weight control, heat therapy, anti-inflammatory and analgesic treatment, joint injection and traditional Chinese medicine manipulation[3, 4].Although they all have certain effects, they cannot fundamentally solve the problem.The disease is prone to continue to deteriorate, and joint replacement is often used at the end of the stage, which is often accompanied by high surgical costs and long postoperative recovery period[5].Therefore, it is particularly important to explore new specific treatment options for KOA.Recent studies have shown[6] that necroptosis and ferroptosis are programmed cell death modes regulated by genes, which play an important role in the occurrence and development of KOA and may be the potential mechanism for the prevention and treatment of KOA.This article reviews necroptosis and ferroptosis and its latest research on in KOA.

1.Necroptosis and KOA

1.1An overview of necroptosis

Necroptosis is a novel way of regulating cell death, which is mediated by Receptor-interacting protein kinase 1/ Receptorinteracting protein kinase 3 (RIPK1 /RIPK3) and uses Mixed lineage kinase domain-like proteins(MLKL) as executing proteins[7].It combines the characteristics of apoptosis and necrosis,and can be activated when apoptosis is blocked, but it is different from apoptosis[8].Apoptosis is the first discovered programmed cell death, characterized by cell concentration, nuclear breakdown,chromatin condensation and so on, which is often triggered by death receptors and mediated by caspase, and it is an active death process in which specific cells are sacrificed for greater benefits of the organism[9].Necroptosis is a non-caspase dependent cell death process that shares upstream signaling with apoptosis, and it often show the similar cell necrosis cytological changes, such as cytoplasmic hemihyalinization, organelle swelling, cell membrane rupture and cell content release, and the contents of the ruptured necrotic cells can trigger a strong inflammatory response, adversely affecting the surrounding tissues[10].At present, Necrostatin-1(Nec-1) is a relatively effective inhibitor of necroptosis, which was first discovered in the chemical screening of its antagonist, and can protect cells from necrosis and apoptosis by inhibiting RIPK1 kinase activity[11].

1.2 Mechanism of necroptosis

Necroptosis is a regulatory response produced by cells under environmental stress, which is usually triggered by a variety of different factors, such as Tumor necrosis factor-α (TNF-α),Reactive oxygen species (ROS) and inflammatory factors.Among them, TNF-α mediated signaling pathway is the most in-depth study.When cells are stimulated by the external environment,TNF-α, which induces necroptosis, is released and Tumor necrosis factor receptor1 (TNFR1) is activated,TNF-α binds to TNFR1 and recruits RIPK1 and other proteins to form complex I on the cell membrane[12].Under normal circumstances, RIPK1 in complex I can activate Nuclear factor-kB (NF-kB) signaling pathway after undergoing ubiquitination modification, and then promote inflammation and maintain cell survival rate by inhibiting caspase-8 activity[7].However, when RIPK1 is deubiquitinated, the activity of proteins acting on NF-kB signaling pathway decreases,RIPK1 is separated from complex I and forms complex II with recruited Caspase-8 and other proteins, which jointly mediate the cell death pathway.As a molecular switch regulating apoptosis and necroptosis, Caspase-8 plays an important role in maintaining body homeostasis[13].When the activity of Caspase-8 is stimulated, it can form complex Ⅱa by inhibiting the activity of RIPK1 and RIPK3,which promotes the occurrence of cell apoptosis; when the activity of Caspase-8 is inhibited, the activities of RIPK1 and RIPK3 are higher than before, thus inducing the formation of complex Ⅱb and causing necroptosis of cells[14].

RIPK1 and RIPK3 are characteristic proteins of necroptosis, and they have similar RIP homotypic interaction motif (RHIM) that can be used as binding sites.In the necroptosis pathway, RIPK1 is first activated by phosphorylation, and then the activated RIPK1 binds to RIPK3 through the RHIM domain, forming the RIPK1/RIPK3 complex, namely the necrosome[15].Necrosome is an important platform for RIPK3-mediated MLKL[16].With the formation of necrosomes, MLKL, the phosphorylated substrate of RIPK3,is aggregated.RIPK3 in necrosome acts on MLKL that form phosphorylated polymers and translocate them to the cell membrane to perform cell necrosis.

1.2 Application of necroptosis in KOA

The research of necroptosis involves the mechanism of its generation and action, as well as its application in KOA.In order to study the role of RIPK3 axis in the pathogenesis of KOA, Jeon J[17] et al.induced a KOA mouse model by surgery and compared RIPK3 expression.The results showed that the damage degree of KOA was positively correlated with RIPK3 expression within a certain range, and when RIPK3 kinase inhibitor was used to intervene it, the progress of KOA was halted.RIPK3 kinase inhibitors may be a potential clinical intervention for the treatment of OA.Green-lipped mussel (GLM) is a natural dietary supplement that has the function of alleviating pain and protecting joints[18].In order to explore the mechanism of GLM in KOA, the researchers made GLM act on KOA mice and observed the corresponding indicators, and found that GLM played a protective role in KOA by reducing the expression levels of necroptosis mediators RIPK1,RIPK3 and MLKL in joints[19].Similarly, researchers have found a breakthrough in the treatment of KOA in the way of necrotizing pathways and genes.Zhong W[20] and Gong Y[21] et al.found that small molecule inhibitors VX-11e and AZ-628 could reduce the necroptosis of chondrocytes in KOA by reducing the expression of necrotic key proteins RIPK3 and MLKL, and then inhibit the loss of articular cartilage and subchondral bone in KOA.Yuan Changshen et al.found that the genes IL1B, MYC, CYBB and EGFR play a key role in the regulation of necroptosis and can be significantly differentially expressed in rat KOA cartilage, which is a breakthrough in the treatment of KOA at the gene level and provides a reliable idea for further research and targeted therapy of KOA[22].In addition, the oxidation and mechanical trauma was also proved to be associated with necroptosis in osteoarthritis.According to the oxidative stress and the release of cytokines in the experiment, they found a possible link between cartilage damage and necroptosis, so blocking the pathogenic factors and molecular mechanisms become a means of prevention and treatment of osteoarthritis, also provide a kind of important method for treatment of KOA[23].

2.Ferroptosis and KOA

2.1 An overview of ferroptosis

Ferroptosis is a novel mode of regulated cell death caused by iron accumulation, lipid peroxidation and plasma membrane damage.Its occurrence is driven by iron-dependent phospholipid peroxidation and involves a variety of biological processes, such as redox reaction, iron metabolism, lipid and glucose metabolism, and a variety of signal transduction related to diseases[24].Ferroptosis has morphological and pathological features different from necroptosis.In terms of morphology, the main manifestations of ferroptosis are mitochondrial outer membrane rupture, mitochondrial shrinkage,and reduction or even disappearance of mitochondrial ridges.In terms of pathology, the increase of Lipid peroxidation (LPO) and iron in cells is the main characteristic of ferroptosis.As the inducing factor of ferroptosis, LPO is usually formed under the action of specific enzymes such as ester oxygenase, cyclooxygenase and cytochrome P450.When the content of LPO increases, it will promote ferroptosis by regulating the integrity of lipid film, and the excess iron in cells will often induce ferroptosis through the massive production of ROS mediated by Fenton reaction.At present, the regulatory pathways of ferroptosis can be roughly divided into three categories.The first type is regulated by Glutathione (GSH) and Glutathione peroxidase4 (GPX4) pathway, which can reduce the synthesis of GSH by inhibiting the system Xc-, thus inhibiting the activity of GPX4,so it reduces the antioxidant capacity of cells and leads to ferroptosis[25].The second type is regulated by the regulatory mechanism of iron metabolism.Protease OTUD1 can increase the absorption of iron by cells by regulating the OTUD1-IREB2-TFRC pathway related to ferritin activity and promote the production of iron accumulation by cells[26].Finally, It regulated by pathways related to lipid metabolism, Acyl-CoA synthetase long-chain family member4/Lysophosphatidylcholine acyltransferase 3(ACSL4/LPCAT3 ) can accelerate the accumulation of lipid peroxides in cells and induce the occurrence of ferroptosis by affecting the activity and transmembrane properties of polyunsaturated fatty acids[27].

2.2 The mechanism of ferroptosis

At present, the research on the mechanism of ferroptosis is mainly mediated by GSH/GPX4 pathway.The occurrence of ferroptosis is induced by the small molecule Erastin and is potentially regulated by lipid repair enzymes including GSH and GPX4.GSH is a tripeptide composed of cysteine, glutamic acid and glycine, which has become an important antioxidant in human body because of its sulfhydryl structure[28].Gpx4 is a classical inhibitor of ferroptosis with GSH as the substrate, it can reduce GSH and convert phospholipid hydroperoxides into lipid alcohols to inhibit ferroptosis.When its activity decreases, it will promote ferroptosis.When cells are exposed to inflammatory stimuli, hyperlipidemia,or iron overload, the cystine/glutamate antitransporter system XClocated on the cell membrane decreases its activity in response to the small molecule Erastin.Under normal physiology, system Xc- will ingest cystine through the cell membrane in a 1:1 ratio and expel glutamate to maintain the stability of oxidative stress.When the activity of system Xc- is inhibited, the rate of cystine transfer into the cell decreased, and the production of cysteine using cystine as raw material is reduced, and then the synthesis of GSH is decreased, and the expression of Gpx4 is decreased.ROS and lipid-ROS in chondrocytes cannot be metabolized by the GSH reductase reaction pathway catalyzed by GPX4 and accumulate, resulting in ferroptosis[12, 29].

At the same time, external environmental stimulation can also promote the increase of intracellular free iron and induce ferroptosis.When cells are stimulated externally, extracellular Fe3+is transferred from transferrin receptor 1 on the cell membrane to the cell body and reduced to Fe2+under the action of transmembrane epithelial antigen of metal reductase prostate 3[30].Subsequently, the reduced Fe2+is transported by divalent metal transporter 1 to an unstable iron pool in the cytoplasm.Under normal physiology, excess iron is transported out of the cell by iron transporters, and when the iron export mechanism is impaired, intracellular iron accumulation occurs.

2.3 Application of ferroptosis in KOA

With the deepening of research on KOA, ferroptosis has become a new breakthrough in the treatment of KOA.In order to study the effect of chondrocyte ferroptosis on KOA, Yao X et al.[31] used interleukin-1β and ferric ammonium citrate to induce chondrocytes of mouse knee joint and treated the induced cells with Ferrostatin-1(Fer-1).The results showed that chondrocyte ferroptosis promoted the progression of KOA and Fer-1 could inhibit the progression of KOA by inhibiting ferroptosis.Zhou X[32] et al., in order to investigate whether D-mannose, which has anti-inflammatory effect, interferes with chondrocyte ferroptosis in KOA, administered it to mice with knee cartilage degeneration, and observed that D-mannose could alleviate the progression of osteoarthritis by inhibiting the sensitivity of chondrocytes to ferroptosis.As the current first-line drug for the treatment of type 2 diabetes mellitus,metformin has been proved by many studies to have moderate antisystemic inflammation effect[33].Yan J[34] et al.in order to explore the effect of metformin on ferroptosis in KOA chondrocytes and make it act on chondrocytes induced by ferroptosis, found that the reduction of articular cartilage damage was related to the inhibition of ferroptosis related proteins by metformin.In terms of traditional Chinese medicine, Wenjing Tongluo decoction, as a derivative of Guizhi and Fuzi decoction, can regulate angiogenesis and inhibit the growth of sensory nerves through a variety of ways, which plays an important role in relieving KOA pain[35].In order to explore the mechanism of its inhibition of angiogenesis, the researchers used drug-containing serum to act on umbilical vein endothelial cells, and found that Wenjing Tongluo decoction could reduce the proliferation of vascular endothelial cells by activating the ferroptosis pathway,which also indicated that it could achieve the purpose of treating KOA by regulating the ferroptosis pathway[36].In terms of gene research, some researchers have found that the ferroptosis related gene HMOX1 is highly expressed in the cartilage tissue of KOA and is positively correlated with the severity of KOA, and finding a pathway to inhibit the expression of HMOX1 has become a potential idea for the treatment of KOA[37].In addition, the iron chelating agents desferriamine and the carotenoid astaxanthin have also been shown to affect the progression of chondrocyte ferroptosis and have a protective effect on KOA chondrocytes, and their application in the articular cavity has become a better solution for the treatment of KOA[38, 39].

3.The association between necroptosis and ferroptosis

Necroptosis and ferroptosis are programmed cell death with high research value, both belong to necrotizing cell death.Although they are two different ways of cell death, they are closely related in some ways.For one thing, they are all affected by mitochondrial function.Mitochondria play an important role in regulating cell survival and death, and their functions are usually related to ROS production and ATP consumption,when cells are under stress or under pathological invasion, the channels responsible for regulating the exchange of cytoplasmic matrix with mitochondrial substances and ions are inhibited, intracellular ATP is consumed, and ROS production increases, and the generated ROS can not only promote the autophosphorylation of RIPK1 and the recruitment of RIPK3 to induce necroptosis, but also promote the accumulation of lipid peroxides to cause ferroptosis[40, 41].On the other hand, Ca2+is involved in both necroptosis and ferroptosis.As an important mineral element for bone development, Ca2+plays an important role in regulating body growth and energy metabolism.When cells are subjected to external abnormal stimulation, a large amount of extracellular Ca2+influx due to excessive activation of calcium ion channels in the cell membrane, coupled with the release of Ca2+in intracellular organelles, the concentration of Ca2+increases rapidly,and the excess Ca2+in the cytoplasm transfers to mitochondria,resulting in energy supply disorders and oxidative stress, resulting in cell death[42, 43].Finally, they both promote the body to mount an immune response.After the activation of necroptosis pathway,the necrotic cell membrane is broken, and the released cell contents act as DAMPS to stimulate the surrounding cells to produce an inflammatory response , and the metabolism of arachidonic acid in iron-dead cells mediated by ester oxygenase and cyclooxygenase can also lead to the release of inflammatory mediators to generate immune response , accompanied by a large number of natural immune factor release, Such as DAMPS et al.[44].

4.Application of necroptosis and ferroptosis in KOA

Articular chondrocytes are the only cellular component of knee joint cartilage, and their survival is of great significance for the protection of knee joint; Osteoblasts are the functional cells of joint bone formation, and their dysregulation often leads to osteoplasia or degeneration of joint; Both of them are important components of the knee joint, and only when they work normally can they prevent knee joint lesions[45, 46].Previous researchers have found that articular chondrocytes can induce ferroptosis by producing excessive ROS in the environment of iron accumulation, and the latest studies have proved that excessive iron in osteoblasts can also lead to necroptosis after ROS increase, the main mechanism is that excessive ROS in cells can activate the RIPK1/RIPK3 pathway and increase the expression of RIPK1 and RIPK3 in cells[31, 47].Therefore, both necroptosis and ferroptosis can mediate knee joint lesions when iron accumulation and ROS increase, and the inhibition of these two common inducing factors may be an important method to prevent and treat KOA.In addition, some researchers from Chinese Academy of Sciences studied the co-regulatory mechanism between necroptosis and ferroptosis by chemical compounds and gene screening, and found that the small molecule 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO) added into necroptosis cells can inhibit RIPK1 kinase activity by inhibiting the activity of Heat shock protein 90 (HSP90),so the phosphorylation of RIPK1, RIPK3 and MLKL is reduced, and the occurrence of necroptosis is reduced; In the ferroptosis pathway, CDDO inhibits the occurrence of autophagy by inhibiting the activity of HSP90, thereby reducing the degradation of GPX4 to inhibit ferroptosis[48].Therefore, small molecule CDDO can effectively control these two types of death with protein HSP90 as the medium, and it becomes a more suitable choice for its related drugs and other HSP90 inhibitors when necroptosis and ferroptosis exist in KOA.

5.Summary and Prospect

KOA is a common musculoskeletal disease in clinical practice,and its pathogenic factors are complex and diverse, which troubles many middle-aged and elderly people.Although there are many treatment methods, it is difficult to have a radical cure.With the emergence of necroptosis and ferroptosis, some new discoveries have opened a new field for the treatment of KOA, such as RIPK3 kinase inhibitors, GLM, Fer-1, D-mannose, etc.which have become a new breakthrough point for the treatment of KOA.In particular, the joint study of necroptosis and ferroptosis has proved that the same factors and targets exist between them, which provides a direction for single drug treatment of KOA caused by two or even multiple factors.Necroptosis and ferroptosis are two new cell death modes with their own characteristics and are related to each other.From the study of external small molecules to the study of signaling pathways and to the study of gene targets, all these reflect the further in-depth research on the treatment of KOA, and provide some scientific theoretical support for the prevention and treatment of KOA.Even so, we should still recognize that the research on necroptosis and ferroptosis in the treatment of KOA is still incomplete, some correlation studies are still in the stage of animal experiments, lack of clinical human trial confirmation, and there is no report on how to prevent KOA through the two programmed death methods.In addition, there is a certain relationship between different cell death patterns, whether different inhibitors can be used in combination and the effect of use remains to be proven by scientific experiments.Therefore, we need continuous research to provide feasible methods and more evidence-based evidence for the treatment of KOA.

Authors’ contributions

The literature search and article writing were completed by graduate student Zong Yizhou, the article content revision was supervised by graduate students Dai Yi, Li Zhe, Yan Weiwei and Lei Maokun, and the article conception was provided by deputy chief physician Yuan Changshen and chief physician Duan Kan

Conflicts of Interest: All the authors of this article declare that they have no conflicts of interest in the writing of this article.