Study Of Local Injection Huachansu On Analgesic Effects And Peripheral Mechanism In Cancer Pain Model H₂₂ Hindpaw Of Mice

Objective: In this study, we observed the effect and periphery mechanisms of Huachansuon the cancer pain behavior in model mouse based on establishing the mouse model ofhindpaw cancer pain, which is great to clinical application on the treatment of cancer pain.Methods: Experimental one: Building the mouse model of cancer pain: KunMing femalemice （18²0g） were randomly divided into normal group （the left hindpaws were injectedinto saline0.1ml） and model group (the left hindpaws of model group were injected intothe suspension of H₂₂hepatoma cells0.1ml with the concentration of6×10⁷/ml). Theweight of animals and the pain threshold before modeling and on the day of2,4,6,8aftermodeling were recorded. According to a regular interval time, animals were sacrificed toobserve the morphology of hindpaw by HE staining. Experimental two: The effect ofHuachansu Peritumoral local injection on the cancer pain behavior in model mouse:112female mice were randomly divided into normal group （n=16） and model group （n=96）.After successful modeling, the model rats were randomly sub-divided into six groups:model group, guoundmass group, morphine group, peritoneal Huachansu injection group,external Huachansu group, peritumoral Huachansu injection group. From the nineth dayafter establishing the model, the mice in each group were administered for8days,respectively. The pain behavior of each mouse were determined on the day of beforeadministration, at the time of0.5h,1h,1.5h,3h,6h after first administration and on the dayof2nd,4th,6th,8th after administration, respectively. At the last day of administration,mice were sacrificed to acquire specimens after the pain behavior of mice were measured.The spleen and thymus were weighed to calculate the index of spleen and thymus. Thetumor and its surrounding tissue specimen were reserved to acquire proper tissue slice. Thecontent of β-END, CRF, IL-1β in plasma（or blood serum）, the homogenate of the tumorand its surrounding tissue were measured by ELISA. Moreover,immunohistochemisty wasused to detect the expression of β-END, POMC, and μ-opioid receptor in the homogenateof the tumor and its surrounding tissue. The protein expressions of CD8⁺, CD8⁺, CD4⁺inthe tumor and its surrounding tissue were measured by immunofluorescence.Results: Experimental one: making the mouse model of hindpaw cancer pain: The mice inmodel group showed decreased active following the increasing of hindpaw tumor. Duringthe time, the behavior of spontaneous foot raise, claudication and scrunch can be seen.Cancer cells were gradually infiltrated into the bone of left hindpaw by morphology. The achievement ratio of hindpaw tumor-bearing mice is100%, the tool can be used as theresearch of cancer pain. Compared with the measurements before making model, thethreshold of thermal and mechanical of the mice model of hindpaw cancer pain decreasedgradually. Two days after making model, the threshold of thermal hyperalgesia decreasedwith statistical differences （P<0.05）. Four days after making model, the threshold ofmechanical hyperalgesia decreased noticeably with statistical differences （P<0.01）.Compared with normal group, measurements on the2nd,4th,6th,8th days after makingmodel showed that the threshold of thermal and mechanical hyperalgesia of mice in modelgroup both decreased （P<0.01）. Experimental results showed that Experimental two:（1）The measurements of pain behavior after administration: The thermal and mechanical painthreshold of mice in each treated group were significance decreased before administrationand at the time of0.5h,1h,1.5h,3h,6h after first administration（compared with the groupbefore administration and model group: P<0.05or P<0.01）; After eight days of successiveadministration, the threshold of thermal and mechanical of mice in each treated groupdecreased significantly on the2th,4th,6th,8th day after administration （compared with thegroup before administration: P<0.01, compared with the model group: P<0.01）.（2） Theresults of spleen and thymus index: Compared with mice in normal control group, thespleen and thymus index of model mice were significantly reduced （P<0.01）. Huachansuand morphine can significantly raised the spleen index of model mice （P<0.05or P<0.01）.The thymus index of each experimental group has no statistically significant.（3） Thecontent of β-END, CRF, IL-1β in the plasma （or blood serum） and homogenate of the lefthindpaw pathological tissue of tumor:①In the tissues: Compared with normal group, thecontent of CRF, IL-1β in homogenate of mice in model group can be significantly raised（P<0.01）. The content of β-END significantly reduced （P<0.05or P<0.01）. Compared withmodel group, group of Huachansu by Peritumoral injection, group of Huachansu byexternal and group of Huachansu by ip can significantly increase the content of β-END,CRF in homogenate of model mice （P<0.05or P<0.01）, but decrease the content of IL-1βin homogenate.②In the blood: Compared with control group, the content of CRF, IL-1βin the plasma increased dramatically, while the content of β-END decreased remarkably（P<0.01）. Compared with model group, external Huachansu group, peritoneal Huachansuinjection group and the morphine group can increase the content of β-END in plasma ofmodel mice （P<0.01）,the content of CRF decreased remarkably. The content differences ofCRF and IL-1β in in other groups have no statistical significance （P>0.05）.（4） The expression of β-END, POMC, and μ-opioid receptor in the pathological tissue of tumor:Immunohistochemistry showed that, compared with normal group, the expression ofβ-END, POMC, and μ-opioid receptor in the left hindpaw pathological tissue of tumorwere significantly lower with statistical differences（P<0.01）. The expressions of μ-opioidreceptor in morphine group were very high with statistical differences（P<0.01）, but theexpression differences of β-END and POMC have no statistical significance. Theexpression of β-END, POMC, and μ-opioid receptor in other groups were very high withstatistical differences （P<0.05or P<0.01）.（5） The expressions of CD8⁺, CD8⁺, CD4⁺cellprotein in the pathological tissue of tumor: Immunofluorescence showed that, comparedwith model group, the expressions of CD4⁺in the peritoneal Huachansu injection groupand external Huachansu group were increased（P<0.05or P<0.01）,that of morphine groupand intraperitoneal injection group have no statistical significance （P>0.05）. The CD8⁺,CD8⁺protein expressions in the pathological tissue of tumor in peritoneal Huachansuinjection group, external Huachansu group and peritumoral Huachansu injection groupwere increased（P<0.01）. The expressions of CD8⁺, CD8⁺, CD4⁺cell protein in morphinegroup have no statistical significance.（6） Huachansu three administrations （peritumoralinjection, external smear, intraperitoneal injection） pair-wise comparison positive results:①pain behavior: the initial administration, each group the threshold of mechanicalhyperalgesia with statistical difference （P<0.05or P<0.01）, the threshold of mechanicalhyperalgesia increased significantly in peritoneal Huachansu injection group,followed byperitumoral injection group,that of external group at last.The threshold of thermal andmechanical of mice in peritumoral injection group increased significantly on the0.5h afteradministration compared with external group andperitumoral Huachansu injection group（P<0.01）.②the pathological tissue of tumor: the content of β-END in Huachansu peritumorinjection group compared with that of intraperitoneal group and external group weresignificance decreased（P<0.01）, the expression of CD4⁺cell protein in Huachansu externalgroup compared with that of intraperitoneal group and peritumor injection group weresignificance decreased（P<0.01）, the expression of CD8⁺cell protein in Huachansuperitumor injection group compared with that of intraperitoneal group was significanceincreased（P<0.01）.Conclusion:（1） In this study we focus on the hindpaw cancer painmodel with H₂₂ hepatoma carcinoma cell, which is simple, easily reproducible and has ahigh achievement ratio. This have confirmed the successful model, and provided a hardfoundation for the followed experiments about the effect and periphery mechanisms of Huachansu on the cancer pain.（2） Huachansu could relive cancer pain of model micethrough topical administration, whose mechanism may involve the increased content ofβ-END in the hindpaw tumor and its surrounding tissue. Cinobufotalin have a certain rolein promoting TIL. The relative detections of index were validated by external group（containing groundmass group） and compared with intraperitoneal injection group, whichcan further illustrated the analgesic effect of Huachansu is developed by peripherallocation; whether the β-END of local neoplastic lesion tissue come from the infiltration ofTIL（or which celltypes） required further corroboration.