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