3.6 mir-99a-5p negatively regulates TLR8 activation of PI3K/AKT
pathway to regulate knee cartilage injury in rabbits.
Through the previous sequencing, many pathways were found to be involved
in the immune response after cartilage injury, we can find the
PI3K/AKT-related pathway is highly correlated (Fig. 6A).Based on this,
we chose the PI3K/AKT related pathway as the research object to explore
whether TLR8 achieves immune response through this pathway. Firstly, the
joint fluid in the rabbit trauma model (1week) was co-cultured with
normal chondrocytes, and then different time points from 0 to 48 hours
were selected as the study nodes(Fig. 6B).Then, the fluorescence values
of PI3K, AKT, P65 and TLR8 were detected by immunofluorescence.
According to the fluorescence map, the fluorescence values showed
different changes with the change of time(Fig. 6C-F). These changes
suggest that this pathway is involved in the immune response to
cartilage injury. Secondly, fluorescence values of PI3K,AKT,P65 and TLR8
was further analyzed, we can find that the value of 12h is the highest,
which are statistically different from 0h and 12h(Fig.S7).Both
PI3K,AKT,P65and TLR8 reached the highest value at 12 hours, indicating
that they had synchronous changes. It is speculated that TLR8 may
achieve immune response through the PI3K/AKT pathway. That is, it
reaches its peak at 12 hours, and then become gradual decline with the
time go on (Fig.6G).
Next, the cultured cells were then further measured for PI3K,AKT,P65 and
TLR8 expression levels. First,
PCR detection results can be seen
after 12 h of cell culture, the expression levels of PI3K,AKT,P65 and
TLR8 reached the highest, and then gradually decreased, and their
changing trends were basically the same(Fig.6H-K). Such results were
also further confirmed by WB detection (Fig.6L-P).
In order to further determine whether AKT pathway is involved in immune
response, PI3K
inhibitor(PI3K-IN-1, MCE HY-12068)
and agonist(Recilisib,MCE HY-101625)
were added in cell culture, respectively. After 12 h co-culture,
AKT and P65 showed synchronous
changes with the inhibitor or agonist, it can be reflected by PCR and WB
results.(Fig.6Q-T,S8).Therefore, we boldly hypothesized that TLR8
regulates the related inflammatory response through the PI3K/AKT pathway
in the early immune response after cartilage injury.
Discussion
Cartilage injury is very common in clinic, which mainly includes chronic
injury and acute injury. Acute injury, such as osteoarthritis or
rheumatoid arthritis,is mainly caused by chronic cartilage
lesions.However, acute injuries are mainly caused by trauma, such as
tibial plateau fracture and ankle fracture which involving in the
articular surface.
Most studies focus on chronic cartilage injury, such as OA and
RA.Meanwhile, many animal models of chronic cartilage injury have been
established, such as DMM, ACTL, type II collagenase, and so on. Although
these models were able to approximate the process of chronic cartilage
injury, they were not able to reflect acute cartilage injury. Therefore,
in order to better study the immune response process of acute cartilage
injury, we chose the cartilage impingement model. It can not only
approximate the process of trauma, but also mainly study the short-term
immune response after trauma.
Through the study on the impact injury model of rabbit knee joint, we
found that the cartilage injury was the most severe and the Mankin’s
score was the highest about one week after the injury, and the
expression changes of COL1A1, COL2A1 and MMP13 were also the largest at
this time. This simultaneous change in histology and molecular. This
indicates that the inflammation peaks in the first week after cartilage
injury.
Many studies of articular cartilage injury have shown that there is a
strong inflammatory response to joint injury.This reaction has been
shown to involve synovial cells, chondrocytes and bone cells in and
around injured joints, and if left unchecked, can lead to post-traumatic
osteoarthritis27.
Cartilage cells and extracellular matrix due to trauma, degeneration of
debris all can become DAMP, and then identified by TLRs receptors on the
surface of the cartilage cells, inflammatory signaling pathways and
activate the corresponding release decomposition factor, cause the
damage of cartilage cell apoptosis, but the release of inflammatory
factors can lead to the excessive release of DAMP, thus forming a
vicious cycle, Exacerbates inflammatory response and chondrocyte
apoptosis28.
It is important to understand these inflammatory responses and develop
successful intervention strategies to treat and ultimately prevent
arthritis following joint injury. Oxidative stress is associated with
the occurrence and development of
OA29, and studies have
shown that the severity of OA can be reduced by the use of antioxidants
or drugs that target reactive oxygen species (ROS) mechanisms in joints.
Cartilage injury is accompanied by physiological processes such as
chondrocyte proliferation, subchondral bone remodeling and chronic
synovitis. Production of intra-articular pro-inflammatory cytokines such
as interleukins(ILs) leads to production of ROS, such as peroxide,
hydroxyl radical and nitric oxide (NO), accompanied by down-regulation
of antioxidants such as superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase
(GPX)30,
31. The resulting oxidative stress leads
to upregulation of decomposition enzymes, degradation of the
extracellular matrix (ECM), reduced matrix synthesis, joint
inflammation, and chondrocyte death and senescence, thereby contributing
to the overall progression of the
disease32. In OA, When
articular cartilage is subjected to excessive mechanical load,
mitochondria within chondrocytes release ROS, resulting in chondrocyte
death, joint tissue inflammation and degradation of matrix
components32. Clearly,
oxidative stress is implicated in the progression of various phenotypes
of OA, making it a therapeutic target that could impact a broad spectrum
of patients. In acute injury, a large number of inflammatory mediators
such as IL-1B, IL-6, IL-8, TNF-αand NO were found in the surrounding
tissues and synovial fluid, indicating that many inflammatory factors
were involved in cartilage
injury28,
33.
In the process of cartilage damage, DAMP, as an alarm signal, is
transmitted by necrotic and damaged cells to the surrounding environment
and activates immune cells. These endogenous molecules interact with
cellular receptors (TLRs) to further stimulate innate immune responses
that initiate signaling pathways that can trigger additional joint
inflammation or initiate tissue
repair34. Most domestic
and foreign studies have shown that the abnormal activation of TLR2 and
TLR4 in OA aggravates cartilage injury by affecting the activation of
NF-κB pathway. Studies have also shown that single gene mutations in the
TLR3 promoter region and increased TLR3 gene expression are associated
with the susceptibility to cartilage
injury35.
Our findings also indicated that TLR8 expression was the highest one
week after cartilage injury, which was consistent with cartilage
morphology and expression of related factors such as COL1A1and so on. In
addition, TLR1-9 showed the greatest variation in TLR8 expression,
indicating its highest participation in inflammatory immunity after
cartilage injury. In order to better verify the involvement of TLR8 in
immune response after cartilage injury, we constructed plasmids and
SiRNA to overexpress or inhibit the expression of TLR2, TLR4 and TLR8,
respectively; found they all changed COL1A1,COL2A1 and MMP13 to varying
degrees, among which TLR8 had the greatest change. It was also found
that the expression of TLR8 and CCK8 was inversely proportional,
indicating that when TLR8 increased, the proliferation ability of cells
was inhibited, and further experiments showed that TLR8 could promote
the phagocytosis ability of cells.That results are consistent with
expectations, indicating that TLR8 is involved in the immune process
after cartilage injury and has an important role.
What are some of the molecules involved in the immune processes that
TLR8 is involved in? Firstly, bioinformatics analysis revealed an
interesting mir-RNA, mir-99a-5P, which is changed the most. Secondly,
further research found that TLR8 was mainly expressed in the endosomes
and lysosome of cells, and the expression sites of Mir-99a-5p were found
to be highly overlapped, suggesting that the two may be highly
correlated. Thirdly, the binding between mir-99a-5p and TLR8 was further
confirmed by RNA pull-down experiment. Base on the research,mir-99a-5p
may plays a negative feedback role in the TLR8-mediated immune response.
How to evaluate the severity of the cartilage damage more effectively in
clinical practice is always a problem, including the clinical symptoms
of patients, such as pain and range of motion, as well as radiographic
evaluation.However, combining inflammatory factors to detect and measure
cartilage injury is a meaningful research direction. Through a
comparative study on the content level of mir -99a-5p blood in human
body, we find that when cartilage was damaged, the level of mir -99a-5p
in the blood of a patient with a cartilage injury was lower than normal,
and there was a statistical difference. This is an interesting
phenomenon, so we speculate that when cartilage damage worsens, namely
when osteoarthritis occurs, due to the high expression of TLR8, the
expression of mir-99a-5p is negatively regulated. Here we make a bold
assumption that whether mir-99a-5p can be used as an indicator of
cartilage damage in clinical practice is worth further discussion and
research.
In order to further clarify the relationship between them, we selected
rabbits as research objects and treated them with inhibition and high
expression of TLR8 and mir-99a-5p respectively. We detected the related
factors involved in cartilage injury immunity and found that TLR8 and
mir -99a-5p not only affected the transcription of MyD88, IRF7, Iκ-Bα
and NF-κB, but also participated in the process of cell apoptosis, such
as IL-6, TNF, caspase-9 and BCL2. However, the effect of mir -99a-5p and
TLR8 presents the opposite. By double fluorescence it not only
reconfirmed the expression of TLR8 and its tight binding to mir-99a-5p,
but also revealed that nuclear transfer of NF-κB occurred after
cartilage injury, which confirmed the effect of TLR8 on NF-κB.
Through the above studies, we found the importance of TLR8 in cartilage
injury immunity and clarified its localization in cells. The close
relationship between Mir-99a-5p and TLR8 was found, and TLR8 was
confirmed to be involved by analyzing the changes of mir-99a-5p.
Moreover, the alteration of mir-99a-5p provides a research direction for
cartilage injury in clinical work, and it may become a marker of
cartilage injury.Of course, this study only found the changes of
mir-99a-5p and TLR8 in cartilage injury, and we need to further clarify
and study their mutual relationship and mechanism.
In order to further explore the specific process of TLR8 involvement in
inflammation, we selected the highest correlation relevant PI3K/AKT
pathway as the research object. Through co-culture of rabbit articular
fluid in the trauma model and normal cartilage, it could be found that,
PI3K, Akt, P65 and TLR8 reached the highest value at 12 hours after
culture. Through their synchronous performance, we speculated that
PI3K/Akt pathway was involved in TLR8-mediated inflammatory response
This is the first study to find the change of TLR8 in cartilage injury,
which confirms that tlr8 is involved in cartilage repair and plays an
important role in innate immunity through two experimental animals. At
the same time, an important mRNA mir-99a-5p was found by gene analysis,
and the interaction and close correlation between mir-99a-5p and TLR
were verified by pull-down and other techniques, providing a clinical
research direction and therapeutic choice for the treatment of cartilage
injury. Of course, this study also has its limitations. First of all, we
only conducted preliminary zoological studies, and further human related
histological studies are needed. Secondly, further discussion on
mechanism is needed to provide stronger support for our conclusion.
5. Declarations