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\begin{document}

\title{The experimental study of mir-99a-5p negative regulation of TLR8
receptor mediated-mediated innate immune response in rabbit knee
cartilage injury}



\author[1]{Jiebin Zhang}%
\author[1]{Ke Zheng}%
\author[2]{Yichao Wu}%
\author[2]{Shengting Zhang}%
\author[2]{Ao Guo}%
\author[2]{Cong Sui}%
\affil[1]{Provincial Second Clinical College of Anhui medical University}%
\affil[2]{First Affiliated Hospital of Anhui Medical University}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{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.%
\end{abstract}%



\sloppy


\textbf{The experimental study of mir-99a-5p negative regulation of TLR8
receptor mediated-mediated innate immune response in rabbit knee
cartilage injury}

Jiebin Zhang\textsuperscript{1,2} ,Ke Zheng\textsuperscript{1,2},Yichao
Wu\textsuperscript{3},Shengting Zhang\textsuperscript{3},Ao
Guo\textsuperscript{3},Cong Sui\textsuperscript{3}

1 Provincial Second Clinical College of Anhui medical University, Hefei,
Anhui Province, P.R. China.

2 Department of Orthopaedics, Anhui Second People's Hosipital, Hefei,
Anhui Province, P.R. China.

3 Department of Orthopaedics, The First Afffliated Hospital of Anhui
medical University, Hefei, Anhui Province, P.R. China.

\textbf{Jiebin Zhang and Ke Zheng contributed equally to this work.}

\textbf{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.

\textbf{Abstract}

\textbf{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.

\textbf{Objective:} To verify how mir-99a-5p regulates TLR8 receptor
mediated innate immune response to treat traumatic cartilage injury.

\textbf{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.

\textbf{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.

\textbf{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.

\textbf{Keyword:} Articular cartilage injury, Mir-99a-5p, TLR8, Innate
immune response, PI3K-AKT

\textbf{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\%\textsuperscript{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
period\textsuperscript{2}.

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
instability\textsuperscript{3}.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
comparability\textsuperscript{4}. 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
cartilage\textsuperscript{5}. 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 bone\textsuperscript{6}. 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 processes\textsuperscript{7}. 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
genome\textsuperscript{8}. 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-\selectlanguage{greek}κ\selectlanguage{english}B pathway\textsuperscript{9, 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 injury\textsuperscript{11}. 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 TLR7\textsuperscript{12}. 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
damage\textsuperscript{13}. 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
infection\textsuperscript{7}. 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 I\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}B) 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)\textsuperscript{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 resistance\textsuperscript{14}. 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
factors\textsuperscript{15}. Activation of TLR8 in ischemic stroke also
promotes neuronal apoptosis and T cell-mediated
inflammation\textsuperscript{16}.

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 regulated\textsuperscript{17}.Bazzoni found that miR-9
activates the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway by regulating the TLR2, TLR4 and TLR7/8
receptors of polymorphonuclear (PMN) and monocytes\textsuperscript{18}.
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.\textsuperscript{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.\textsuperscript{20} found that miR-99a may be involved in the
regulation of TNF-\selectlanguage{greek}α \selectlanguage{english}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
pathway\textsuperscript{21}. Mir-99a regulates the formation and
differentiation of rat mesenchymal stem cells by targeting bone
morphogenetic protein receptor (BMPR2) gene\textsuperscript{22}.

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
diseases\textsuperscript{23-25}, as well as in the regulation of the
proliferation, differentiation and apoptosis of osteoclasts and
osteoblasts\textsuperscript{{[}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.

\textbf{2 Materiasl and methods}

\textbf{2.1 Experimental animals and} \textbf{Cartilage damage model}

New Zealand white rabbits(A total of 20 healthy adult New Zealand white
rabbits with body weight in 3.0-3.5kg,male and female unlimited)were
obtained from the Experimental Animal Center of Anhui Medical
University.All procedures were approved by the Anhui Medical University
Laboratory Animal Ethics Committee(approval number:LLSC20210237).We used
Borrelli's method to modify the animal model of impingement cartilage
injury in rabbit knee joint.After adaptive feeding for 1 week, rabbits
were modeled. The specific method was as follows: the supine position
was taken, and the pelvis and thigh were fixed. Anesthesia with 10\%
chloral hydrate and hair removal at both knees. A longitudinal incision
of about 3 cm was made on the medial side of one knee joint. The rabbit
patella was everted, the knee joint was flexion and extension 120\selectlanguage{ngerman}°, and
the femoral shaft was 90°. A 1kg impingent was used to fall down from a
height of 50 cm along the guide rod and impinged on the medial femoral
condyle, causing cartilage damage on the medial femoral condyle.All
rabbits were sacrifificed in a CO\textsubscript{2} chamber at 4 weeks
post-surgery and knee joints were collected for MRI imaging and
immunohistochemistry (IHC) staining.

\textbf{2.2 Histological and Immunohistochemical}

All the joint tissues were demineralized in 10\% EDTA for 6 weeks and
embedded in paraffin after gradient dehydration. Series mid-sagittal
sections of 5\selectlanguage{greek}μ\selectlanguage{english}m were cut parallel to the condyle. After dewaxing in
xylene and hydration in gradient alcohol, H\&E, Safranin O staining were
carried out according to themanufacturer's protocol. Cartilage thickness
was determined from H\&E staining on the basis of the predecessors'
methods. A modified Mankin scoring system was used for cartilage OA
score.

After dewaxing and hydration, the tissue sections were microwave
antigen-retrieved in citrate solution. The sections were endogenous
peroxidase activity blocked, following by serum blocking unspecific
ligations. Rabbit anti-TLR2, TLR4, TLR8 were incubated overnight at 4\selectlanguage{ngerman}°C
severally, then reacted with anti-rabbit UltraSensitive S-P kit
according to manufacturer's protocol. For calculating the rate of
positive cells, Image-Pro Plus 6.0 software was used to quantify the
number of positive cells and total cells on selected cartilage area.
Anterior, middle and posterior, 3 fields of each section were counted
and averaged by two observers independently.

\textbf{2.3 RT-PCR}

After total mRNA of all the rabbits cartilage samples was extracted
using TRIzol Kit and reversely transcribed, polymerase chain reactionwas
manipulated to cDNA, with the SYBR Premix Ex Taq. Then, gene expression
of mRNA was calculated using the cycle threshold method
(2\selectlanguage{english}\textsuperscript{-ΔΔCt}). The primer sequences were as follows:

TLR1 F CTTGATCTGCACAATAACCG R GAAGATCCACCAAGGAGT

TLR2 F TGCCCTGTTCATTTTCCC R TTGCCTTAAAATCAACGTGTG

TLR3 F TTTCAAGGGCTTAGCTCA R AAACATTCTTGTCAACGG

TLR4 F ACAAGGCTTACTTTCACT R TCTACGCTCAGAACAGCA

TLR5 F TCTGATTCTCGCCTACCACA R TCAGGGAGAAGATAAATCCGT

TLR6 F CTGCACACATCATGAAGC R TATGTTCAATGATCAGCACAGA

TLR7 F ACAACGATATCTCCACCT R CATCTCGCCATAAAACGTCT

TLR8 F CTTTATTGGGGAAAAGCA R CAAATATTTAACATGAGGCACA

TLR9 F GGAAACCAACTGAAGAGCCTGA R TTGAGCTGTCGCAACTCCC

The gene of GAPDH was used as an internal control.

\textbf{2.4 Western blotting}

The collected and measured proteins were separated by SDS-PAGE
electrophoresis and then electro-transferred onto a PVDF membrane. After
blocking with non-fat milk, the membrane was incubated with different
primary antibodies at 4\selectlanguage{ngerman}°C overnight, respectively, including rabbit
anti-TLR2, TLR4, TLR8(MDL,USA);Then, HRP-conjugated secondary antibody
was incubated with PVDF membrane, and a chemiluminescence ECL
system(Bio-rad,USA) was utilized to detect the immunoreactive proteins.

\textbf{2.5 Double luciferase assay}

The circRNAs were built into PSI\selectlanguage{english}-CHECK2 vector, then circRNA and miRNA
were cotransfected into 293T cells.Luciferase activities were measured
at 48\selectlanguage{english}-hours after transfection using the Dual\selectlanguage{english}-Luciferase Reporter
AssaySystem (Promega, Madison, WI) according to themanufacturer's
protocol.Renilla luciferase activity wasnormalized to firefly luciferase
activity and expressed as apercentage of the control. All transfection
experiments were performed in triplicate.

\textbf{2.6 RNA pull-down assay}

The RNA was first marked by Pierce Magnetic RNA-protein pull-down Kit
(Thermo), and then the RNA precipitated overnight was removed and
centrifuged at 4\selectlanguage{english} for 30min at maximum speed. Add 20\selectlanguage{greek}μ\selectlanguage{english}L nuclease
free-water to each tube to dissolve RNA. Take 400\selectlanguage{greek}μ \selectlanguage{english}L beads from each
tube, place them on a magnetic frame, and remove the supernatant. Wash
with 800\selectlanguage{greek}μ \selectlanguage{english}L 1X Binding \& Washing buffer for 3 times and suck out the
supernatant. Add 400\selectlanguage{greek}μ \selectlanguage{english}L 2X Binding \& washing buffer; Add 20\selectlanguage{greek}μ\selectlanguage{english}L RNA and
380\selectlanguage{greek}μ\selectlanguage{english}L DEPC water to make the final volume 800\selectlanguage{greek}μ\selectlanguage{english}L, Rotate the beads slowly
at room temperature for 20min to fully combine with RNA. Add 500\selectlanguage{greek}μ \selectlanguage{english}L cell
lysate to each tube. Meanwhile, add RNase Inhibitor (1U/\selectlanguage{greek}μ\selectlanguage{english}L) to each
tube. Rotate slowly at 4 for 2h to fully combine beads with cell lysis
fluid. QPCR was performed after reverse transcription.

\textbf{2.7 Plasmid DNA transfection and construction of overexpression
vector} .

One day before transfection, plate 0.5-2\selectlanguage{ngerman}×10\textsuperscript{5} cells in
500 ul of growth medium without antibiotics so that cells will be
90-95\% confluent at the time of transfection. Suspension cells: Just
prior to preparing complexes, plate 4-8 ×10\textsuperscript{5}cells in
500ul of growth medium without antibiotics.For each transfection sample,
prepare complexes as follows:a.Dilute DNA (pCDNA3.1-EGFP-T2A-Puro) in 50
ul of Opti-MEN Reduced Serum Medium without serum, mix gently.b.Mix
Lipofectamine 3000 gently before use, then dilute the appropriate amount
in 50 ul of Opti-MEN Medium. Incubate for 5 minutes at room
temperature.c.After the 5 minutes incubation, combine the diluted DNA
with diluted Lipofectamine 3000 (total volume=100ul).Mix gently and
incubate for 20 minutes at room temperature.Complexes are stable for 6
hours at room temperature. Add the 100 ul of complexes to each well
containing cells and medium.Incubate cells at 37°C in a
CO\textsubscript{2} incubator for 18-48 hours prior to testing for
transgene expression. Medium may be changed after 4-6 hours.Passage
cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours
after transfection. Add selective medium the following day.

\textbf{2.8 Cell culture was transfected with SiRNA.}

TLR2SiRNA,TLR4SiRNA and TLR8SiRNA were purchased from Huzhou Hippo
Biotechnology Co., LTD, the specific sequence is shown in S1. 6cm cell
dishes were taken and 2mL
(1\textasciitilde{}2×10\textsuperscript{5}cells) culture medium was
added into each well. The culture density reached 70-90\% at 37\selectlanguage{english} and
5\%CO\textsubscript{2}; Dilute Lipofectamine 3000 (Invitrogen,
No.3000015) with Opti-MEM and mix well; Dilute the carrier with
Opti-MEM, add P3000, and mix well; Diluted carrier (1:1) was added into
the diluted Lipofectamine 3000 and incubated at room temperature for 5
minutes; Add the mixture into the culture medium, shake well and
incubate at 37\selectlanguage{english} for 3 days.

\textbf{2.9 Statistical analysis}

All results were the averages of at least 3 independentexperiments from
separately treated and transfected cultures. Data were expressed as the
mean +- SD. Statistical comparisons were performed using 1\selectlanguage{english}-way analysis
of variance. P \textless{}0 .05 was considered to indicate a
statistically significant difference.

\textbf{3.Result}

\textbf{3.1 In a rabbit model of impingement knee cartilage injury, we
found an innate immune response mechanism at the early stage of injury.}

As described above, We applied Borrelli's method in rabbits to verify
the pathological changes of impingement knee cartilage injury. According
to the experimental plan, after the surgery of rabbit, we have selected
1, 2 and 4 weeks as the detection time points for research. At 1 week
after injury, the articular cartilage was significantly degenerated in
rabbits with impingement knee cartilage injury ,as shown by HE and
safranin-O \& fast green staining(Fig. 1A,B) and the Mankin's scores
were also signifificantly increased compared with those of control
rabbits in 0 week (Fig. 1C). All joint specimens obtained were
photographed for observation prior to histological examination\selectlanguage{english},We can
observe the repair of articular cartilage injury at different time
points, the cartilaginous surface of the joint after injury is repaired
to varying degrees over time (Fig.S1).As time went on, the degree of
cartilage damage gradually decreased, and the cartilage damage was
significantly improved four weeks later. We also performed MRI scanning
and marked imaging on the articular surface of different groups of
rabbit joints, and observed that the normal articular cartilage surface
was clear and smooth, with complete morphology and uniform thickness.
Form the result,1 week after the injury, the cartilage injury site was
obvious and clear, as it is showed by the red arrow marks. Cartilage
damage was still visible 2 weeks later, however, after 4 weeks the
cartilage basically returned to normal (Fig.1D).

Meanwhile, one week after the injury, the expression of MMP13 and MMP9,a
molecule directly involved in cartilage degradation, was significantly
increased compared with control rabbits of 0 week ,and the expression of
MMPs decreased gradually with the extension of injury time. To further
quantitatively detect the expression of genes related to cartilage
repair, molecular markers COL1A1, COL2A1, MMP9, MMP13 and ADAMTS-5 were
selected to be observed at different time points after cartilage injury.
The results indicated that the expression levels of COL1A1, COL2A1 and
MMP9 gradually increased 1 week after impaction, and reached a peak at 2
weeks after injury. Meanwhile, the expressions of MMP13 and ADAMTS-5
peaked in the first week after injury, and then decreased gradually
(Fig.1E-I). In order to further verify damage, we further tested the
protein level of the cartilage, result of western blot showed that the
expressions of COL1A1, COL2A1 and MMP9 were the highest in the second
week, while the expressions of mmp13 and ADAMTS5 were the highest in the
third week (Fig.1H,S2).

Based on these results, we successfully constructed an animal model of
cartilage impact injury, and further verified the changes of different
injury factors during the injury process through histology and molecular
studies.

\textbf{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.

\textbf{3.3 TLR8 localizes in lysosomes mediating innate immune
responses during chondrocyte injury.}

Previous studies have shown that TLRs may have different subcellular
distribution in different cells. TLR8 in the immune system is mainly
located in the cytoplasmic endosomes, while its location in cartilage
organelles remains unclear. In order to determine the expression
distribution of TLR8 in chondrocytes, the co-labeling of TLR8 with
lysosomal marker LAMP, endoplasmic reticulum marker Calnexin, and
endosomal marker EEA1 was detected by the double-target
immunofluorescence method, respectively. The results showed that TLR8
was mainly co-labeled with the lysosomal marker LAMP (Fig.3A) and the
endosomal marker EEA1(Fig.3C) in rabbit chondrocytes with impact knee
joint injury, but less co labeled with the endoplasmic reticulum marker
calnexin(Fig.3B).Therefore, we concluded that TLR8 was mainly expressed
in lysosomes and endosomes, and there was no intracellular metastasis of
TLR8 after chondrocyte injury.

\textbf{3.4 RNA sequencing of sample of impingement knee cartilage
injury in rabbits and TLR8 target prediction.}

Related studies have reported that the TLRs receptor-mediated innate
immune responses are mostly mediated by miRNAs. It has been reported
that TLR8 and its endogenous ligand miR-21 contribute to neuropathic
pain in murine DRG\textsuperscript{26}.There is no literature report
that the congenital immune response of TLR8-mediated cartilage injury is
mediated by which miRNA species. Therefor we performed transcriptome
sequencing of specimens from the rabbit cartilage damage model. The
expression level of miRNA was detected in samples of control cartilage
and in cartilage 1 week after injury. DESeq (Version 1.18.0, Anders S
and Huber W, 2010) was used to analyze the differential expression of
miRNA (Fig.4A). According to the expression quantity ratio difference
(\textbar{} log2FoldChange \textbar{} \& gt; 1) and significant
difference in expression (p-value; 0.05) Differential conserved mi-RNAs
were screened. The results showed that, 21 miRNA down-regulated and 26
mRNA up---regulated, among which mir-12093-3p, mir-99a-5p, mir-873-5p,
mir-452-5p, and mir-1-3p were the top 5 miRNAs with the most
significantly down-regulated expression, mir-12093-3p and mir-133-3p had
no relevant information in the database. Therefore, mir-99a-5p with the
highest degree of difference was selected for verification. Then through
the NCBI database search and blast comparison found that mir-99a-5p may
bind to TLR8. We can see that there is a conserved binding site in both
rabbits and humans (Fig. 4B). To further understand the changes of in
the mir-99a-5p human body, the blood samples were collected from
patients who diagnosed with cartilage injury and normal patients
respectively, and then analyze the level of mir-99a-5p. Thirty-two
patients were included in each group, and their clinical data can be
seen in Table1(Fig. S5). The blood level of mir-99a-5p was 1.03 +- 0.04
and 0.31 +- 0.02 in group of normal and injury respectively,
p\textless{}0.05(Fig. 4C).

In order to verify the interaction between mir-99a-5p and TLR8, we first
using RNA pull-down techniques find when mir-99a-5p as the probe, it
could be seen that TLR8 in mir-99a-5p probe group was significantly
enriched and the average CT values was 17.93, significantly decreased
compared with the cell lysis buffer(Fig.S6). After taken agarose gel
electrophoresis, and gray values of electrophoresis bands were
calculated. The results showed that the amplification bands of
Mir-99a-5p probe group were significantly stronger than cell lysates
(Fig.4.D,E). Then the TLR8 probe was labeled with the same method, and
mir-99a-5P in the TLR8 probe group was significantly enriched, and the
gray value of the amplified band in the TLR8 probe group was
significantly stronger than that in the cell lysate (Fig.4.F,G).Therefor
through the positive and negative RNA pull down experiments of
mir-99a-5p and TLR8, it is likely that mir-99a-5p binds and interacts
with TLR8.

To further clarify the relationship between them, we performed a double
luciferase assay. The results show that the average fluorescence value
ratio of Psichec2-TLR8-WT+miR-99a group is 0.33, much lower than the
other groups(P\textless{}0.05) (Fig.4H),which suggested that the
activity of luciferase decreased after TLR8 was combined with
mir-99a-5p, indicating the interaction of the 3-terminal non-coding
region of mir-99a-5p and TLR8. At the same time, we also carried out
double fluorescence on cells, found that the expression of TLR8
increased and mir-99a-5p decreased at the same time, and the position of
the cells was similar(Fig.4I).To further illustrate the changes of
related immune-involved factors in cells after cartilage injury, we
selected NF-\selectlanguage{greek}κ\selectlanguage{english}B factor for detection and found that it is activated after
injury and transferred to the nucleus (Fig.4J).This suggests that TLR8
activation in cartilage injury can mediate inflammatory molecular
pathways.

\textbf{3.5} \textbf{miR-99a-5p negatively regulates TLR8 in the
occurrence of impingement cartilage injury of rabbit knee joint.}

To gain further understanding of the downstream molecular mechanisms of
cartilage damage underlying TLR8 activation, we injected mir-99a-5p as
well as the overexpression and inhibitory vector of TLR8 in the
auricular vein of model rabbits, related inflammatory properties and
apoptosis-related indicators were detected one week later(Fig.5A).Animal
models were divided into six groups, depending on the rabbit treatment
pattern,from no treatment in the control group to overexpression of
mir-99a-5p mimic and TLR8 after cartilage injury(Fig.5B).All groups of
animals underwent further testing one week after receiving the
corresponding treatment, PCR analysis of cartilage tissue in each group
showed when the TLR8 overexpression or mir-99a-5p decline, the
MyD88(Fig.5C),IRF7(Fig.5D),NF-\selectlanguage{greek}κ\selectlanguage{english}B(Fig.5E) also increased expression, but
I\selectlanguage{greek}κ-\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}is decrease when TLR8 overexpression(Fig.5F). They are accompanied
by TLR8 or mir-99a-5p changes, suggesting them involved in the
activation of the signaling pathway during cartilage injury. We further
detected the apoptosis related factors of IL-6, TNF, caspase-9 and BCL2,
and found that with the TLR8 overexpression or mir-99a-5p decline, IL-6,
TNF and caspase-9 is become higher expression (Fig.5G-I), however the
BCL2 expression is opposite (Fig.5J). As above to the changes of TLR8
and mir-99a-5p, indicating that they may involve in various regulation
and effects in cartilage injury. At the same time, WB test was used to
further confirm the protein expression levels of MyD88,IRF7,NF-\selectlanguage{greek}κ\selectlanguage{english}B ,
I\selectlanguage{greek}κ-\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and the results were basically consistent with the PCR
results.(Fig.5K). Base on this study, we can infer that in the cartilage
repair model, mir-99a-5p negatively regulates the expression of TLR8 and
affect the occurrence of chondrocyte innate immune response(Fig.5L).

\textbf{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.

\textbf{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
osteoarthritis\textsuperscript{27}. 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 apoptosis\textsuperscript{28}.

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 OA\textsuperscript{29}, 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)\textsuperscript{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
disease\textsuperscript{32}. 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 components\textsuperscript{32}. 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-\selectlanguage{greek}α\selectlanguage{english}and NO were found in the surrounding
tissues and synovial fluid, indicating that many inflammatory factors
were involved in cartilage injury\textsuperscript{28, 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 repair\textsuperscript{34}. 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-\selectlanguage{greek}κ\selectlanguage{english}B 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
injury\textsuperscript{35}.

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, I\selectlanguage{greek}κ-\selectlanguage{english}B\selectlanguage{greek}α
\selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B, 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-\selectlanguage{greek}κ\selectlanguage{english}B occurred after
cartilage injury, which confirmed the effect of TLR8 on NF-\selectlanguage{greek}κ\selectlanguage{english}B.

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.

\textbf{5.} \textbf{Declarations}

\textbf{Ethics approval statement}

Clinical specimen experiments and animal experiments were reviewed and
approved by the Ethics Committee of the First Affiliated Hospital of
Anhui Medical University. The procedures used in this study adhere to
the tenets of the Declaration of Helsinki.

\textbf{Author contributions}

Cong Sui designed this study; Jiebin Zhang and Ke Zheng completed
specimen experiments; Shengting Zhang and Ao Guo performed cell culture
studies; Cong Sui and Ke Zheng performed in vivo studies; Jiebin Zhang
collected data; Cong Sui and Yichao Wu analyzed data; Cong Sui revised
the article.

\textbf{Acknowledgments}

Thanks to Life Science laboratory teachers of Anhui medical university
for their technical support and assistance.

\textbf{Funding statement}

This study was supported by the fundamental and clinical research
collaboration cooperation project of Anhui Medical
University(2022xkjT019), Natural Project of Universities in Anhui
Province (2020ZR12925B009), Postdoctoral Foundation of the First
Affiliated Hospital of Anhui Medical University(BSKY2022061), Natural
Science Foundation of Anhui Medical University(2020xkj169), Natural
Project of Universities in Anhui Province (2023AH053329).

\textbf{Conflict of interest disclosure}

The authors declare no conflict of inte.

\textbf{Data availability statement}

The data used to support this study are included withinthe articl.

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