| Objective By observing the expression of P2X3R and CASK proteins and their interactions in the dorsal root ganglia(DRG)of rats with bone cancer pain and the effect of intrathecal injection of CASK siRNA on the expression of P2X3R in the DRG to clarify the role of P2X3R membrane transport in DRG in peripheral sensitization of bone cancer pain and the role of CASK protein in maintaining P2X3R membrane anchorage.By observing the effects of electroacupuncture on the paw withdrawal thresholds(PWTs)of rats with bone cancer pain,the P2X3R activity in DRG and its protein and positive cell expression,as well as the effect of CASK protein expression to investigate the possible mechanism of electroacupuncture on bone cancer pain in rats.Methods Healthy female Sprague-Dawley(SD)rats were injected with MRMT-1 breast cancer cells(3 ×104 cells/3μl)into the left tibial cavity to construct a rat model of bone cancer pain.Part 1:107 SD rats were randomly divided into a control group(Con,n=50)and a bone cancer pain group(BCP,n=57),of which the rats in the BCP group were treated with modeling,the rats in the Con group were injected with the same amount of sterile PBS;30 rats underwent intrathecal tube implantation to bury PE-10 tubes to L4-5 DRG level.23 successful rats were randomly divided into BCP+CASK siRNA group(n=11)and BCP+scrambled siRNA group(n=12).They were injected 10μl of CASK siRNA(2μg/10μl)or an equivalent amount of scrambled siRNA was injected intrathecally at 11-13d,15 and 17d after cancer cell inoculation.Von Frey hairs were used to measure changes in Paw withdrawal thresholds(PWTs)in rats;immunoblotting and co-immunoprecipitation techniques were used to detect the expression of P2X3R and CASK proteins in DRG and their interactions.Part 2:72 SD rats were randomly divided into control group(Con,n=21),bone cancer pain+sham electroacupuncture group(BCP+s-EA,n=25),bone cancer pain+electroacupuncture group(BCP+EA,n=26).Rats in the Con group were injected with the same amount of sterile PBS only.On the 11th day after the cancer cells inoculated,the BCP+s-EA group were treated with sham electroacupuncture.The BCP+EA group were treated with electroacupuncture on the 11th day after the cancer cells were inoculated.The stimulation parameters were bilateral"Zusanli" and "Kunlun" points,dense waves,frequency of 2/100Hz,intensity of 0.5-1.5mA(in 0.5mA steps,each stimulation forl0 min),30 min for each stimulation,once a day,and the treatment was continued for 7 days.Immunoblotting and fluorescent immunohistochemistry were used to detect the expression of P2X3R protein and positive cells in DRG,and intracellular calcium imaging was used to detect changes in calcium influx in DRG neurons induced by α,β-meATP.Results Part 1:(1)There was no difference in PWTs between the groups of rats before cancer cell inoculation(P>0.05);PWTs of BCP rats were significantly lower than those of Con rats during the same period(P<0.01)at 10-17 days after cancer cell inoculation.(2)On the 17th day after cancer cell inoculation,the expression of P2X3R membrane protein and total protein in L4-6 DRG on the affected side of BCP rats was significantly higher than that of Con rats in the same period(P<0.05 or P<0.01);However,CASK between the two groups,there was no significant difference in protein expression(P>0.05);Co-immunoprecipitation results showed that there was an interaction between P2X3R and CASK protein.(3)Compared with scrambled siRNA,intrathecal injection of CASK siRNA can significantly increase PWTs in rats with bone cancer pain(P<0.01),and significantly inhibit the expression of P2X3R protein in the affected side L4-6 DRG(P<0.05).Part 2:(1)There was no difference in the basic PWTs of the rats in each group before modeling(P>0.05);The PWTs on the affected side of the rats in the BCP+EA group were significantly higher than those in the BCP+s-EA group at all observation points.(P<0.01),which was lower than the Con group in the same period(P<0.01).(2)The amplitude and positive rate of Ca2+response induced byα,β-meATP by DRG neurons in BCP+EA group were significantly lower than those in BCP+s-EA group(P<0.01),but higher than those in Con group(P<0.05 or P<0.01).(3)Compared with the Con group,the total protein,membrane protein,and number of positive cells of P2X3R in DRG in the BCP+s-EA group were significantly increased(P<0.05 or P<0.01);the BCP+EA group was significantly lower than BCP+s-EA group(P<0.05 or P<0.01);(4)There was no significant difference in total CASK protein expression in DRG of the three groups of experimental rats(P>0.05).Conclusion CASK-mediated increase of P2X3R protein expression in DRG neurons mediates peripheral sensitization of bone cancer pain.Electroacupuncture can effectively alleviate bone cancer pain in rats.Inhibition of P2X3R activity in DRG of bone cancer pain rats,reduction of P2X3R-positive cells and protein expression,and membrane transport may be one of the key peripheral mechanisms of electroacupuncture against bone cancer pain in rats.CASK protein may be involved in P2X3R membrane anchoring,but it is not involved in electroacupuncture analgesia. |
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