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.