Corresponding author:
Cong Sui, Department of Orthopaedics, The first Afffliated Hospital of Anhui medical University, Hefei, Anhui Province, P.R. China.
Email:suicong@ahmu.edu.cn.
Abstract
Background: Traumatic cartilage injury is an important cause of osteoarthritis (OA) and limb disability, and Toll-like receptors (TLRs) mediated innate immune response has been confirmed to play a crucial role in cartilage injury.In the previous study, we found that the activation of TLR8 molecules in injured articular cartilage was more obvious than other TLRs by establishing an animal model of knee impact injury in rabbits, and the changes of TLR8 molecules could significantly affect the process of articular cartilage injury and repair.
Objective: To verify how mir-99a-5p regulates TLR8 receptor mediated innate immune response to treat traumatic cartilage injury.
Methods: The impact of a heavy object on the medial condyle of the rabbit’s knee joint caused damage to the medial condylar cartilage. Through pathological and imaging analysis, it was demonstrated whether the establishment of an animal model of traumatic cartilage injury was successful. Establishing a cell model by virus transfection of chondrocytes to demonstrate the role of TLR8 in the innate immune response to impact cartilage injury. Through transcriptome sequencing, potential targets of TLR8, mir-99a-5p, were predicted, and basic experiments were conducted to demonstrate how they interact with innate immune responses to impact cartilage damage.
Results: TLR8 is a receptor protein of the immune system, which is widely expressed in immune cells. In our study, we found that TLR8 expression is localized in lysosomes and endosomes.Mir-99a-5p can negatively regulate TLR8 to activate PI3K-AKT molecular pathway and aggravate cartilage damage.Inhibiting TLR8 expresson can effectively reduce the incidence of articular cartilage damage.
Conclusion: Based on the results from this study, mir-99a-5p may be an effective molecular marker for predicting traumatic cartilage injury and targeting TLR8 is a novel and promising approach for the prevention or early treatment of cartilage damage.
Keyword: Articular cartilage injury, Mir-99a-5p, TLR8, Innate immune response, PI3K-AKT
Introduction:
Articular cartilage injury is a disease that widely involves people of all ages and has serious consequences. Its main causes include trauma, infection, inflammation, degeneration, congenital and metabolic diseases. In recent years, with the expansion of injury-causing energy spectrum and the enhancement of population aging trend, the incidence of traumatic articular cartilage injury has been increasing year by year. According to statistics, the annual number of cartilage injury cases in China is 65 million cases, and the incidence of injury in specific populations such as athletes can reach 22% - 50%1.The early symptoms of traumatic cartilage injury are hidden. Only 30% - 40% of the patients have clinical symptoms and seek medical advice. Pathological specimens are not easy to obtain. This leads to insufficient clinical diagnosis of traumatic cartilage injury, which is an important reason why the incidence of OA increases year by year due to cartilage degeneration in the later period2.
There are many methods for experimental animal models of articular cartilage injury,such as mechanical immobilization,adding local immobilization with papuloprotease, using collagenase, cutting off the medial and lateral collateral ligaments, cutting off the anterior and posterior cruciate ligaments.The medial semilunar plate was excised, and the patellar bone was excised to produce segmental instability3.The human acute stress cartilage injury is often caused by collision, such as the collision between bones caused by falling from a high place, the contact collision between joints and external hard objects.The more similar the injury mechanism of animal model of cartilage injury to that of human, the more valuable the treatment and related research will be.Some scholars used the impact method to establish an animal model of knee cartilage injury in rabbits, which is simple to operate, basically similar to the pathological process of human articular cartilage injury, and has good comparability4. It can simultaneously obtain normal knee joint synovial fluid samples of the same experimental animal, and is a good animal experimental model.New berry used vertical impact frames with different weights (1.33kg, 0.43kg) to fall from different heights (46cm, 20cm) to close the medial femoral condyle of rabbits, causing different degrees of damage to femoral articular cartilage5. They found that cartilage damage was very similar to clinical pathology, which could be used as an animal model of articular cartilage damage.Mastbergen established the dog cartilage injury model by performing scratches on the cartilage surface without damaging the subchondral bone6. According to the research methods of relevant literature, we improved and made a rabbit model of articular cartilage injury caused by impact of knee joint, and carried out relevant pathological staining and MRI examination to confirm that the model was successful.
TLRs family is an important sensor for detecting heterogeneous pathogens and endogenous danger signals in innate immune response, and is involved in a variety of physiological processes7. So far, 13 kinds of TLRs have been detected in humans and mice, of which TLR1-TLR9 is expressed in both humans and mice. TLR10 in mouse genome has no clear function, while TLR11-TLR13 is only found in mouse genome8. These TLRs can recognize various microbial pathogens and synthetic ligands, including lipids, lipoproteins, polysaccharides, proteins or nucleic acids with different molecular weights.Numerous studies have shown that the abnormal activation of TLR2 and TLR4 mediates the process of cartilage injury by affecting the activation of NF-κB pathway9, 10. Some studies have also shown that single gene mutation in the promoter region of TLR3 and elevated expression of TLR3 gene are associated with susceptibility to cartilage injury11. Hoshikawa N found that TLR7 is the target of miR-21, and miR-21 in cartilage tissue of cartilage injury rat model can lead to cartilage damage aggravation and the occurrence of OA in the later stage by activating TLR712. The polymorphism of TLR9-T-1486C gene is related to the progressive aggravation of cartilage damage. The change of TLR9 expression caused by different alleles may lead to the aggravation of cartilage damage13. We found that the expression of TLR1-TLR9 was increased in varying degrees in the animal model of knee cartilage injury induced by impact in rabbits, but the activation of TLR8 was more obvious than that of other TLRs.
TLR8 is expressed in a variety of physiological cells, such as monocytes, macrophages, DC cells, mast cells and microglia cells. It is often considered as the natural receptor of single stranded RNA and a powerful activator of natural immune response after virus infection7. After TLR8 is recognized and bound to the ligand, the TIR domain containing the adaptor myeloid differentiation factor MyD88 begins to change. The binding of TLR8 and MyD88 stimulates the recruitment of IL-1 receptor-associated protein kinases (IRAK) and leads to the activation of downstream mitogen-activating proteins (MAPKs) and IκB kinase (IKK) complexes. Thus, the expression of MAPK family members phosphorylated activated transcription factor activator protein 1 (AP-1) is increased, and the nuclear translocation of IKK complex and transcription factor (NF-κB) is further promoted to control the expression of proinflammatory cytokines.In addition, members of the interferon regulatory factor family of transcription factors can be activated in MyD88 dependent pathway and induce the production of type I interferon (IFN)8. In addition to its role in antiviral immunity, TLR8 also plays an important role in a variety of physiological diseases. For example,the activation of TLR8 in pancreatic cancer can promote the proliferation of tumor cells and enhance their drug resistance14. In systemic lupus erythematosus (SLE), miR-21 acts as a ligand to mediate the activation of STAT1 regulated by estrogen and promote the activation of TLR8, thereby promoting the expression of related inflammatory factors15. Activation of TLR8 in ischemic stroke also promotes neuronal apoptosis and T cell-mediated inflammation16.
Recent studies have found that miRNA molecules have a negative regulatory effect on TLRs signaling pathway.For example,let-7i inhibits the translation process by interacting with the 3 ’untranslated region (3’ UTR region) of TLR4 mRNA at the post-transcriptional level. TLR4 is negatively regulated17.Bazzoni found that miR-9 activates the NF-κB pathway by regulating the TLR2, TLR4 and TLR7/8 receptors of polymorphonuclear (PMN) and monocytes18. These studies provide a theoretical basis for the understanding and intervention of innate immune-related diseases.By sequencing and proteomic analysis, we found that miR-99a-5p could negatively regulate TLR8 receptor-mediated innate immune responses in animal models of impingent cartilage injury.mir-99a-5p belongs to the miR-99 family and is expressed at low levels in a variety of human malignant tumors and inflammatory diseases. Zhu et al.19 confirmed that miR-99a is a suppressor gene,which can inhibit cell proliferation and induce cell apoptosis by regulating the activation of T cells. Jaiswal et al.20 found that miR-99a may be involved in the regulation of TNF-α and other inflammatory factors, leading to the activation of related inflammatory pathways. Other studies have shown that miR-99a inhibits the invasion and migration of tumor cells by targeting NADPH oxidase 4 (NOX4) -mediated ROS generation pathway21. Mir-99a regulates the formation and differentiation of rat mesenchymal stem cells by targeting bone morphogenetic protein receptor (BMPR2) gene22.
Phosphatidylinositol 3-kinase (PI3Ks) protein family is involved in the regulation of cell proliferation, differentiation, apoptosis, glucose transport and other cellular functions. Increased PI3K activity is often associated with a variety of cancers. PI3K phosphorylates the third carbon atom of the inositol ring of a membrane phospholipid (PI). The proportion of PI in cell membrane is smaller than that of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine, but the content of PI in brain cell membrane is more abundant, up to 10% of the total phosphatidyl. The result of PI3K activation is the generation of a second messenger, PIP3, at the plasma membrane, which binds to the PH domain-containing signaling protein AKT and PDK1(phosphoinositide dependent kinase-1) in cells. Phosphorylation of AKT by PDK1 at Ser308 leads to AKT activation. AKT, also known as protein kinase B(PKB), is a major downstream effector of PI3K. PI3k/Akt signaling pathway is a signaling pathway mediated by enzyme-linked receptors that can regulate life activities. It not only participates in the signal transduction of various production factors, cytokines and extracellular matrix, but also participates in promoting cell proliferation, inhibiting apoptosis, regulating tissue inflammation and tumor growth and invasion.Recent studies have shown that PI3k/Akt signaling pathway is involved in the occurrence of osteoporosis,OA,osteosarcoma and other orthopedic diseases23-25, as well as in the regulation of the proliferation, differentiation and apoptosis of osteoclasts and osteoblasts[28-31].
In this study, we found that the expression of TLR8 was significantly increased in the impact injury model of rabbit cartilage. miR-99a-5p can negatively regulate the innate immune response mediated by TLR8/PI3K/Akt. Targeting the abnormal activation of TLR8 molecules can effectively block the progress of cartilage injury. These results confirm that TLR8 has an important effect on early traumatic cartilage injury, and miR-99a-5p may become an important molecular marker to predict early cartilage injury.