| Objective To explore the underlying mechanism of lappaconitine(LA)in treatment on bone cancer pain(BCP),we observed the number of M1 microglia and the expression of pro-inflammatory factors linked with it in the spinal cord during the development of bone cancer pain,which by constructing a rat model of bone cancer pain.Methods Experiment one:A total of 18 female Sprague-Dawley rats were randomly divided into three groups of six.Normal group(Ctrl group):no treatments needed;Sham-operated group(Sham group):PBS suspension of heat-killed cancer cells was injected into the right tibial cavity;Bone cancer pain group(BCP group):BCP model was prepared by inoculating freshly extracted Walker 256 ascites cancer cell PBS suspension into the right tibial cavity.The paw withdrawal threshold(PWT),limb use score,tibia plain film,and bone tissue HE staining were used to comprehensively evaluate the construction of rat tibia BCP model.Experiment two:A rat BCP model was established,then detected the number of M1 microglia(Iba-1~+CD68~+)and the expression of pro-inflammatory factors linked with it(iNOS,IL-1β)in the spinal cord at day 0,6,12 and 18,which by immunofluorescence double-labeling staining and Western Blot.Experiment three:At the day 12 after the establishment of the BCP model,4 mg/kg of LA or an equal amount of normal saline was intraperitoneally injected for 7consecutive days.The pain behavior of rats in the control group(Naive group),saline treatment group(BCP-NS group),and lappaconitine treatment group(BCP-LA group)was observed by PWT and limb use scores.Immunofluorescence staining and Western Blot were used to detected the number of M1microglia(Iba-1~+CD68~+)and the expression of pro-inflammatory factors linked with it(iNOS,IL-1β)in the spinal cord of each group rats at day 18.Results(1)The pain behavior changes:the PWT and limb use scores of the rats in the BCP group gradually decreased(P<0.05)during the entire observation period,while the changes in the rats in the Ctrl group and Sham group were not statistically significant(P>0.05).Tibia plain film:the degree of bone destruction of tibia in rats in the BCP group increased over time,while the tibial cortex of the rats in the Ctrl group and the Sham group continued without bone destruction.Bone tissue section HE staining:at day 18 after modeling,the trabeculae of the Ctrl group and the Sham group were intact and the bone tissue structure was normal.Normally distributed bone marrow cells were seen in the medullary cavity,while the bone structure of the BCP group was significantly damaged,and a large number of deeply stained tumor cells infiltrated.(2)The immunofluorescence double-labeling staining results showed that the number of M1 microglia(Iba-1~+CD68~+)in the spinal cord gradually increased over time.Compared with the day 6after the cancer cells were inoculated,the number of M1 microglia(Iba-1~+CD68~+)in the spinal cord of BCP rats were increased at the day 12 and day 18,and the difference was statistically significant(P<0.01).The Western Blot results showed that the expression level of pro-inflammatory factors linked with M1microglia(iNOS,IL-1β)were significantly increased at day 6,day 12,and day 18after inoculation of cancer cells into the tibia of rats,which was most significantly up-regulated at day 18(P<0.05).(3)From the day 12 to the day 18 after the establishment of the BCP model,after daily intraperitoneal injection of saline or LA into the rats,compared with the BCP-NS group,the pain behavior of the BCP-LA group was significantly improved(P<0.05),which indicates LA has a analgesic effect on BCP.The immunofluorescence double-labeling staining and Western Blot results showed that compared with the Naive group,the number of M1 microglia(Iba-1~+CD68~+)and the expression level of its pro-inflammatory factors(iNOS,IL-1β)in the spinal cord of BCP-NS group rats were significantly increased(P<0.05),while LA can inhibit M1 microglia proliferation(P<0.05)and down-regulate the expression of pro-inflammatory factors linked with M1microglia(iNOS,IL-1β).Conclusion lappaconitine may inhibit the proliferation and activation of M1microglia in the spinal cord of bone cancer pain rats,and then reduce the expression of its related pro-inflammatory factors iNOS and IL-1β,thereby producing analgesic effect. |
