Objective

2

To observe the effect of tuina plus

Chenyuangao

（陈元膏） in the treatment of knee osteoarthritis （KOA） with yang deficiency and congealing cold syndrome， and to explore its potential mechanism.

Methods

2

Sixty New Zealand white rabbits were randomly divided into six groups， namely the normal group， model group， tuina group，

Chenyuangao

group， tuina plus

Chenyuangao

group and voltaren group， with ten rabbits per group. The model group and all intervention groups were treated by polymer bandage fixation at flexion angle combined with peppermint oil and climate box to establish rabbit model of KOA with yang deficiency and congealing cold syndrome. After eight weeks of modeling， the normal group and the model group were given no intervention. Tuina group was administered with tuina manipulation only， and

Chenyuangao

group was applied with ointment only， while tuina plus

Chenyuangao

group performed tuina based on the ointment

Chenyuangao

. The voltaren group was treated with voltaren ointment. Treatment was given once a day for 6 weeks. On the second day after the last intervention， samples were collected. Using HE staining to observe histopathological changes of cartilage and synovial membrane microscope， and the pathological Mankin score of the sections was assessed. Serum levels of inflammatory factors interleukin 1β （IL-1β）， interleukin 6 （IL-6）， interleukin-10 （IL-10） and tumor necrosis factor α （TNF-α） were detected by ELISA. Western Blot was used to detect the levels of B-lymphoma 2 （Bcl-2） and B-lymphoma 2-associated X protein （Bax）. The protein and mRNA expression levels of metalloproteinase inhibitor 1 （TIMP-1）， matrix metalloproteinase 13 （MMP-13） and matrix metalloproteinase 3 （MMP-3） in articular cartilage tissue were determined by immunohistochemistry and qRT-PCR.

Results

2

The pathological results showed that the articular chondrocytes of the rabbits in the normal group were arranged in an orderly manner， and the matrix was uniform， with normal synovial tissue structure and neatly arranged synovial cells. In the model group， the cartilage matrix lost its homogeneity， and the chondrocytes disintegrated， disappeared and disordered in certain areas， with increased synovial cells and number of layers， interstitial fibrous tissue proliferated with partial hyaline degeneration， and more chronic inflammatory cells infiltrated. The injury of cartilage cells in the tuina group， the

Chenyuangao

group and tuina plus

Chenyuangao

group were improved to varied degrees， and more changes were seen in the latter two groups. The model group had higher articular cartilage tissue Mankin score， higher levels of IL-1β， TNF-α， and IL-6， lower expression of Bcl-2 protein and Bcl-2/Bax value in the cartilage tissue， higher protein expressions of Bax and MMP-13， higher mRNA expressions of MMP-3 and MMP-13， and lower mRNA expression of TIMP-1 than the normal gorup （

P

<

0.05）. Compared to those in the model group， the Mankin score in the tuina plus

Chenyuangao

group significantly decreased； the serum IL-1β level and mRNA levels of MMP-3 and MMP-13 in cartilage tissue in other intervention groups significantly decreased， while the mRNA level of TIMP-1 increased； the IL-10 level， the expression of Bcl-2 protein and the Bcl-2/Bax value in the tuina plus

Chenyuangao

group and the voltaren group significantly increased， while the protein expression of Bax， MMP-13 and TIMP-1 decreased （

P

＜0.05）. Compared to those in the voltaren group， the Mankin score in the tuina plus

Chenyuangao

group was reduced， while the expressions of Bax protein and MMP-13 mRNA increased （

P

＜0.05）， and there was no significant difference between groups in the levels of inflammatory factors （

P

＞0.05）.

Conclusion

2

Tuina plus

Chenyuangao

can improve the histopathology of cartilage and synovial membrane， and has the functions of anti-inflammatory， anti-cartilage matrix degradation and anti-chondrocyte apoptosis， thereby reducing the damage of articular cartilage and delaying the progression of KOA with yang deficiency and congealing cold syndrome.