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.