3.2 TLR8 molecule changes obviously in rabbit impingement knee cartilage injury.
TLRs receptors are important pattern recognition receptors in innate immune response. To date, a total of 13 TLRs have been detected in humans and mice, among which TLR1-TLR10 is expressed in humans. The function of TLR10 in the human body is unclear, therefore, we detected the expression level of TLR1-TLR9 one week after knee cartilage injury in rabbits(Fig.S3).The results showed TLR8,TLR4,TLR2 expression was significantly changed during cartilage injury, and TLR8 expression changes were significantly stronger than the others(Fig.2A,B,C,E).Western Blot analysis of the injured cartilage samples showed that TLR8 was more obvious than TLR2 and TLR4 at different time points of injury(Fig.2D).Immunohistochemical staining indicated that the number of TLR8 positive cells changed significantly at different time points compared with the number of TLR2 and TLR4 positive cells(Fig.2F).We further developed a model of impact-type chondrocyte injury in vitro to overexpress or inhibit the expression of TLRs to verify its effect on cartilage injury. Normal cartilage cells of rabbit knee joint were extracted and co-cultured with the damaged joint fluid of rabbit knee joint cavity after 1 week of cartilage injury. After 24 hours of co-culture, TLR2, TLR4 and TLR8 genes in chondrocytes were overexpressed and inhibited to observe whether they had repair effects on injured chondrocytes. We build virus plasmid (Pcdna3.1-EGFP-T2A-Puro, Fig.S4) which contain CDS region of the gene transfected into cells to overexpress the gene of TLR2, TLR4 and TLR8, at the same time use si-RNA transfected into cells for down-regulate the expressions of TLR2, TLR4 and TLR8. The overexpression and inhibition of TLR8 had the most significant effect on the expression of MMP13, COL1A1 and COL2A1 mRNA(Fig.2G-I). Meanwhlie, the overexpression and inhibition of TLR8 also affect the expression of MMP13, COL1A1 and COL2A1 at the protein level(Figure 2J-M).From the above, we conclude that TLR8 is the main pattern recognition receptor in the innate immune response to impingement cartilage injury.