Objective

2

To observe the effect of acupotomy on heat shock protein A family member 5 (HSPA5)/glutathione peroxidase 4 (GPX4) signaling pathway in the chondrocytes of the rabbits with knee osteoarthritis (KOA) and explore the mechanism of acupotomy on chondrocyte ferroptosis in KOA.

Methods

2

Twenty-seven New Zealand rabbits were randomly divided into a normal group

a model group and an acupotomy group

with 9 rabbits in each group. The left hind limb was fixed by the modified Videman method for 6 weeks to establish KOA model. After modeling

acupotomy was given in the acupotomy group

once a week and for consecutive 3 weeks. Using Lequesne MG score

the local symptoms

physical signs and functions of knee joint were evaluated. With HE staining and saffrane-solid green staining adopted

the morphology of chondrocytes and cartilage tissue was observed. Under transmission electron microscope

the mitochondrial structure of chondrocytes was observed. The iron content of cartilage tissue was detected by iron ion kit. The mitochondrial membrane potential (Δψm) and the reactive oxygen species (ROS) level in cartilage tissue were determined by flow cytometry

and the mitochondrial damage rate was calculated. The mRNA expression of HSPA5

GPX4

type Ⅱ collagen α1 chain (COL2A1)

matrix metalloproteinases (MMP) 3 and MMP13 was detected by the real-time quantitative PCR; and the protein expression of HSPA5

GPX4

type Ⅱ collagen (COL-Ⅱ)

MMP3 and MMP13 was detected by Western blot. The mean flourscence intensity of HSPA5 and GPX4 in cartilage tissue was determined by immunofluorescence.

Results

2

Before intervention

compared with the normal group

the Lequesne MG scores were increased in the model group and the acupotomy group (

P

<

0.01). After intervention

the Lequesne MG score in the acupotomy group was decreased when compared with that in the model group. In comparison with that in the normal group

the number of chondrocytes was reduced and the cells were disarranged; the layers of cartilage structure were unclear

the tide lines disordered and blurred; the mitochondria were wrinkled a

nd the mitochondrial crista decreased or even disappeared in the model group. Compared with the model group

the number of chondrocytes was increased

the layers of cartilage structure were clear

the tide lines recovered

the number of mitochondria elevated

with normal structure and more crista in the acupotomy group. The iron content of cartilage tissue was increased (

P

<

0.01)

the Δψm of chondrocytes was declined

the mitochondrial damage rate was increased (

P

<

0.01)

the average fluorescence intensity of ROS was increased (

P

<

0.01); the mRNA and corresponding protein expression of HSPA5

GPX4 and COL2A1 was decreased (

P

<

0.01)

the mRNA and protein expression of MMP3 and MMP13 was increased (

P

<

0.01) and the average fluorescence intensity of HSPA5

GPX4 was decreased (

P

<

0.01) in the model group when compared with those in the normal group. Compared with the model group

the iron content in cartilage tissue was reduced (

P

<

0.01)

the Δψm of chondrocytes was increased

the mitochondrial damage rate was decreased (

P

<

0.01)

and the average fluorescence intensity of ROS was decreased (

P

<

0.01); the mRNA and corresponding protein expression of HSPA5

GPX4 and COL2A1 was higher (

P

<

0.01)

and the mRNA and protein expression of MMP3 and MMP13 was lower

and the average fluorescence intensity of HSPA5

GPX4 was increased (

P

<

0.01) in the acupotomy group.

Conclusion

2

Acupotomy can alleviate cartilage injury of KOA rabbits

and its mechanism may be related to the regulation of HSPA5/GPX4 signaling pathway to maintain iron homeostasis in articular cartilage

thus inhibiting chondrocyte ferroptosis and relieving extracellular matrix degradation.