Objective

2

To explore the potential mechanism of

Wenshen Tongluo Zhitong Formula

（温肾通络止痛方， WTZF） in treating comorbidity of osteoporosis （OP） and osteoarthritis （OA） from the perspectives of osteo-cartilage metabolism and the hypoxia-inducible factors （HIFs）/osteoprotegerin （OPG）/receptor activator of nuclear factor-κB ligand （RANKL） pathway.

Methods

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Thirty-five Sprague-Dawley （SD） rats were randomly divided into 5 groups， sham operation group， model group， alendronate sodium group， low- and high-dose WTZF group， 7 rats in each group. Except for the sham operation group， rats were subjected to bilateral ovariectomy combined with modified Hulth surgery to establish a rat model of comorbidity of OP and OA. Interventions were initiated 2 weeks after model establishment. The low- and high-dose WTZF groups were given WTZF by gavage at doses of 14.76 g/（kg·d） and 29.52 g/（kg·d）， respectively， once a day； the alendronate sodium group was given alendronate sodium by gavage at 6.3 mg/kg once a week； the sham operation group and the model group were given normal saline by gavage at 10 ml/（kg·d） once a day. After 10 weeks of intervention， the gross morphology of the rat knee joint was observed and scored using the Collins scoring system； micro-computed tomography （Micro-CT） was used to detect the bone mineral density of the distal femur and the subchondral bone microstructure of the tibial plateau including bone volume fraction （BV/TV） and trabecular separation （Tb.Sp）； HE staining and safranin O-fast green staining were performed to observe the pathological changes of the knee joint， and the Mankin scoring system was used for evaluation； enzyme-linked immunosorbent assay （ELISA） was used to determine the serum levels of inflammatory factors such as tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and factors related to the HIFs/OPG/RANKL signaling pathway including hypoxia-inducible factor-1α （HIF-1α）， hypoxia-inducible factor-2α （HIF-2α）， OPG， and RANKL， and the OPG/RANKL ratio was calculated. Immunofluorescence was used to detect the protein expression of HIF-1α and HIF-2α in the knee joint； Western Blotting was used to determine the levels of proteins related to knee joint osteo-cartilage metabolism， including collagen type Ⅱ α1 chain （COL2A1）， collagen type Ⅰ α2 chain （COL1A2）， matrix metalloproteinase 13 （MMP13）， matrix metalloproteinase 9 （MMP9）， and Runt-related transcription factor 2 （RUNX2）， and also the HIFs/OPG/RANKL pathway including OPG， RANKL， HIF-1α， HIF-2α， Egl nine homolog （EGLN） was determined.

Results

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Compared with the sham operation group， the model group showed significantly increased Collins score and Mankin score of the knee joint， significantly decreased BMD and BV/TV， significantly increased Tb.Sp， increased serum levels of TNF-α， IL-1β， RANKL， HIF-1α， and HIF-2α， decreased serum OPG/RANKL ratio， average immunofluorescence intensity of HIF-1α protein， increased average immunofluorescence intensity of HIF-2α protein， decreased levels of COL2A1， COL1A2， HIF-1α， and OPG/RANKL in the knee joint， and increased protein levels of MMP9， MMP13， RUNX2， RANKL， HIF-2α， and EGLN （all

P

＜0.05）. Compared with the model group， the high-dose WTZF group and the alendronate sodium group showed improvements in all the above indicators （all

P

＜0.05）. The high-dose WTZF group was superior to the low-dose WTZF group in improving serum levels of HIF-1α， HIF-2α， and OPG， the average immunofluorescence intensity of HIF-2α protein， and the levels of COL2A1， MMP9， RUNX2， COL1A2， OPG， OPG/RANKL， and EGLN （all

P

＜0.05）； the alendronate sodium group was superior to the low-dose WTZF group in reducing serum levels of TNF-α， IL-1β， HIF-1α， and HIF-2α， and the average immunofluorescence intensity of HIF-1α and HIF-2α proteins in the knee joint （all

P

＜0.05）.

Conclusion

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The

Wenshen Tongluo Zhitong Formula

may regulate the HIFs/OPG/RANKL pathway in the knee joint， balance bone metabolism， and improve cartilage degeneration， thereby alleviating the progression of comorbid osteoporosis and osteoarthritis.