Mechanism Of Bone Cancer Pain In BALB/c Mice Based On PI3K/Akt/WNK1 Pathway

Objective:In this study,the mouse model of bone cancer pain was used to explore the mechanism of PI3K/Akt/WNK1 signal pathway in bone cancer pain in mice.Methods:6-8-week-old female BALB/c mice were selected for the experiment.The mice were randomly divided into normal(N)group,sham operation(Sham)group and bone cancer pain(BCP)group with 6 mice in each group.The cultured 4T1 mouse breast cancer cell suspension(1×10~7/m L)was injected into the bone marrow cavity(10μL)from the tibial joint to establish the model of bone cancer pain.The threshold of mechanical stimulation foot contraction in mice was measured by Von Frey fiber pain meter,and the pathological analysis of tibia tissue was carried out by hematoxylin-eosin staining to verify the successful establishment of bone cancer pain model.The mice were randomly divided into three groups:normal(N)group,bone cancer pain(BCP)group and Akt inhibitor(Akt-i)group,with 6 mice in each group.Fourteen days after inoculation of tumor cells,Akt-i group was intraperitoneally injected with GSK690693 at the dose of 20 mg/kg for 5 times(2 days off for2 days),and other groups were given the same volume of solvent.The threshold of mechanical foot contraction was measured by Von Frey filament pain meter,the expression levels of interleukin-17,tumor necrosis factor-αand WNK1 in serum were detected by enzyme-linked immunosorbent assay,and the expression levels of PI3K,p-PI3K,Akt,p-Akt,WNK1 and p-WNK1 were detected by Western blot.So as to study the mechanism of PI3K/Akt/WNK1 signal pathway and related inflammatory factors in bone cancer pain.Results:The paw withdrawal mechanical threshold of the BCP group decreased with time,and there was a significant difference between the BCP group and N group and the Sham operation group on the 6th day(P<0.001),and the decrease was the most obvious on the 14th day.he pathological film of tibia tissue in BCP group showed that cancer cells eroded the whole bone tissue,engulfed bone trabeculae,destroyed bone cortex,and cancer cells expanded continuously and infiltrated the surrounding muscle tissue.The above results prove that the modeling is successful.in the BCP group,compared with the N group,the cancer cells eroded the whole bone tissue,swallowed the bone trabecula,destroyed the bone cortex,and the cancer cells expanded continuously and infiltrated the surrounding muscle tissue.The above results prove that the modeling is successful.Paw withdrawal mechanical threshold:There was no significant difference in the threshold of mechanical foot withdrawal between the BCP group and the Akt-i group at D0 and D1(P<0.001).On the 3rd day,the mechanical foot withdrawal threshold in the Akt-i group was higher than that in the BCP group(P<0.05).There was no difference in paw withdrawal mechanical threshold between the Akt-i group and the N group on the 7th day.Enzyme-linked immunosorbent assay:Before the beginning of the experiment,the expressions of tumor necrosis factor-alpha,interleukin-17 and WNK1 in the serum of mice in the BCP group were significantly higher than those in the N group,and showed an upward trend with the passage of time.The levels of serum tumor necrosis factor-alpha,interleukin-17 and WNK1 in the inhibitor group were significantly lower than those in the BCP group(P<0.001),but there was no significant difference between the inhibitor group and the N group.Western blot:In the BCP group,the expression of PI3K/p-PI3K,Akt/p-Akt,WNK1/p-WNK1 in spinal cord tissue was increased significantly,while the expression level of Akt,WNK1 protein decreased significantly in Akt-i group,and the corresponding phosphorylated protein was inhibited.Conclusion:The PI3K/Akt pathway in the spinal dorsal horn of mice activates downstream WNK1 protein,which increases the release of inflammatory cytokines and leads to the formation of bone cancer pain in mice.Inhibition of Akt can reduce the level of interleukin-17and tumor necrosis factor-alpha,cut off the downstream WNK1 signal reception pathway,increase the mechanical foot withdrawal threshold and relieve bone cancer pain in mice.