Abstract:Objective To explore the potential mechanism of Gutongling prescription in the treatment of pain with bone metastases from lung cancer. Methods 50 SPF grade C57BL/6 male mice were randomly divided into 5 groups by random number table method, sham operation group, model group, Gutongling group, zoledronic acid group, and Gutongling combined with zoledronic acid group, with 10 mice in each group.Except for the sham operation group, which was inoculated with PBS solution, the other groups were inoculated with Lewis-luc suspension of mouse-derived lung adenocarcinoma cells. The sham operation group and the model group were given 0.9% NaCL solution by gavage and intraperitoneal injection;The Gutongling group was given Gutongling Decoction (12g/kg) by gavage, and the zoledronic acid group was given zoledronic acid (50μg/kg) by intraperitoneal injection;Gutongling combined with zoledronic acid group was given Gutongling prescription (12g/kg) by gavage and intraperitoneal injection of zoledronic acid (50μg/kg) ; All groups were treated for 21 days .The mechanical pain threshold of mouse plantar was detected by Von Frey filament pain meter;the morphology and number of F-actin rings of osteoclast pseudopodia were observed under immunofluorescence staining and confocal fluorescence microscope; The mRNA and protein expression levels of αvβ3, proline-rich tyrosine kinase 2 (PyK2), Tyrosine kinase Src, Casitas B-cell lymphoma family protein (Cb1)in the tibia bone tissue of the left hindlimb of mice in each group were detected by RT-PCR and immunohistochemistry. Results: (1) The mechanical pain threshold of mice in the sham-operated group showed a stable trend with no obvious fluctuation. The other four groups showed a downward trend from the 7th day, especially in the model group. Compared with the sham operation group, the mechanical pain threshold of mice in the model group decreased after modeling (P<0.05). On the 14th and 21st days after modeling: Compared with the model group, the mechanical pain threshold of each intervention group increased (P<0.05); The mechanical pain threshold of the zoledronic acid group was comparable to that of the Gutongling group (P>0.05); On the 21st day after modeling, the mechanical pain threshold in the Gutongling combined with zoledronic acid group was significantly higher than that in the Gutongling group and the zoledronic acid group (P<0.05). (2) Compared with the sham-operated group, the osteoclast pseudopodia F-actin ring structure in the model group was complete; Compared with the model group, the number of intracellular F-actin rings in each administration group was decreased, and the ring shape was incomplete, fractured, and a small amount of stress fibers were formed around it;Compared with the zoledronic acid group, the damage degree of the F-actin ring in the Gutongling group was similar. (3) Compared with the sham operation group, the mRNA expression and protein staining intensity of αvβ3, PyK2, Scr and Cbl in the model group were increased (P<0.05);Compared with the model group, the mRNA expression and protein staining intensity of αvβ3, PyK2, Scr and Cbl decreased in each administration group (P<0.05);Compared with zoledronic acid group, the mRNA expression and protein staining intensity of αvβ3, PyK2 and Scr in Gutongling group were increased;Compared with Gutongling group and zoledronic acid group, the mRNA expression and protein staining intensity of αvβ3, PyK2, Scr and Cbl in the Gutongling combined with zoledronic acid group were significantly decreased (P<0.05). Conclusion Gutongling Prescription can increase the mechanical pain threshold of bone metastases cancer pain, destroy the F-actin ring structure in bone tissue, and it has a synergistic effect when used together with zoledronic acid.Its mechanism may play a role in bone protection by regulating the αvβ3/PyK2/Src/Cbl signaling pathway in bone tissue and inhibit the formation of osteoclast pseudopodia, thereby treating the pain of bone metastases from lung cancer.
