OBJECTIVE To investigate whether Duyiwei improves synovial fibrosis in rats with knee osteoarthritis(KOA) by regulating the receptor for advanced glycation end-products(RAGE)-phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt) signaling pathway.

METHODS Network pharmacology was employed to predict the active components and potential targets of Duyiwei in the treatment of KOA. A total of 60 SPF-grade male SD rats were randomly divided into control group, model group, low-dose and high-dose Duyiwei group(1.0, 3.0 g·kg⁻¹·d⁻¹), high-dose Duyiwei+oe-NC group, and high-dose Duyiwei+oe-RAGE group, with 10 rats in each group. Except for the control group, rats in all other groups underwent anterior cruciate ligament transection(ACLT) to establish the KOA model. Following modeling, adeno-associated virus(AAV) was injected into the knee joints, and the rats were continuously administered Duyiwei via intragastric gavage for 4 weeks. Thermal withdrawal latency and cold withdrawal latency were measured before modeling and at various time points post-modeling to assess changes in peripheral pain sensitivity. Hematoxylin-eosin(HE) and Masson staining were performed to evaluate synovial inflammation and the degree of fibrosis. The levels of nerve growth factor(NGF), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in synovial tissues were measured by ELISA. Protein expressions levels of RAGE, p-PI3K, PI3K, p-Akt, Akt, p-p65, p65, Collagen Ⅰ, alpha-smooth muscle actin(α-SMA), tissue inhibitor of metalloproteinases-1(TIMP-1), and Vimentin in synovial tissues were detected by Western blotting. The mRNA expressions levels of CollaⅠ, Acta2, Timp1, and Vim were determined using quantitative real-time PCR(qPCR). The tissue distribution of fibrosis-related proteins was observed via immunohistochemistry(IHC).

RESULTS Network pharmacology analysis showed that Duyiwei contained 97 active components corresponding to 97 targets, with 4525 genes related to KOA, yielding 85 common drug-disease targets. There were 25 overlapping targets between the drug-disease common targets and the downstream target genes of the AGE-RAGE signaling pathway. Thermal and cold withdrawal latency tests demonstrated that Duyiwei significantly prolonged the latency periods, whereas this analgesic effect in the high-dose Duyiwei group was markedly reversed after the administration of oe-RAGE AAV. ELISA results showed that Duyiwei significantly reduced the contents of NGF, IL-6, and TNF-α in synovial tissues. Western blotting results indicated that Duyiwei significantly downregulated the expression and phosphorylation levels of key proteins in the RAGE-PI3K-Akt pathway. qPCR results revealed that Duyiwei significantly downregulated the mRNA expressions levels of CollaⅠ, Acta2, Timp1, and Vim in synovial tissues. whereas these indices were significantly elevated in the oe-RAGE group.

CONCLUSION Duyiwei can ameliorate synovial fibrosis in KOA rats by regulating the RAGE-PI3K-Akt pathway.