A novel therapeutic strategy for Mycoplasma synoviae infection: Elucidating the synergistic effects and underlying mechanisms of Tilmicosin and Sinomenine.

Mycoplasma synoviae (MS) is an important poultry pathogen that causes multisystem pathological damage, particularly infectious synovitis, resulting in significant economic losses to the poultry industry. Tilmicosin (TMS) is a first-line antimicrobial for mycoplasma infections; however, its limited penetration into joint tissues of MS-infected chickens compromises clinical efficacy. Sinomenine (SIN), an alkaloid extracted from Stephania tetrandra, exhibits specific anti-arthritic and anti-inflammatory effects. Previous studies have shown that combined administration of TMS and SIN produces synergistic effects against MS infection, although the underlying mechanism remains unclear. This study aimed to elucidate the synergistic mechanism of TMS and SIN by analyzing pharmacokinetic changes and anti-inflammatory actions in MS-infected chickens. Microdialysis was used to compare the pharmacokinetics of TMS and SIN administered alone or in combination. Knee joints were collected for histopathological examination using hematoxylin-eosin and safranin O-fast green staining. Inflammatory cytokine mRNA levels in synovial tissues were quantified by RT-PCR, while the expression and subcellular localization of JAK1, p-JAK1, STAT3 and p-STAT3 were evaluated by Western blotting and four-color immunofluorescence. Combined treatment significantly increased the area under the concentration-time curve of TMS during the first three days, while reducing SIN exposure during the early stage. The bacterial load of MS was significantly reduced in the MIX group compared with monotherapy. SIN markedly alleviated synovial and cartilage damage, downregulated pro-inflammatory cytokines, and inhibited activation of the JAK/STAT signaling pathway. These findings indicate that SIN enhances TMS accumulation at joint sites and exerts synergistic anti-inflammatory effects, providing a scientific basis for optimized clinical treatment of MS infection.