| Background Cancer is one of the first diseases threatening human survival.In recent years,with the improvement of cancer treatment,the five-year survival rate of cancer patients has been greatly improved.However,cancer-related pain associated with cancer seriously bothers patients and their families,which greatly reduces the quality of life.Therefore,it is particularly important to find prevention strategies and effective treatment for bone cancer pain for people to seek for a better life.Objective To screen the effective ingredients and main targets of Huachansu in the treatment of bone cancer pain with the help of network pharmacology,and to explore the mechanism of Huachansu acting on the CB2 receptor to regulate the RANKL/RANK/OPG signaling pathway to analgesia,thus to provide more theoretical basis for the clinical treatment of bone cancer pain.Methods Part 1: Using network pharmacology to analyze the target information of the effective ingredients of Huachansu on bone cancer pain:(1)Screening of effective components of Huachansu: Through CNKI and Pub Med databases,combined with oral bioavailability(OB≥30%)and pharmacokinetic(DL≥0.18)parameters,the effective components of Huachansu were screened out.(2)Screening of relevant targets for the effective ingredients of Huachansu in the treatment of bone cancer pain: using OMIM and Gene Cards database to screen out the relevant targets of Huachansu effective ingredients in the treatment of bone cancer pain.(3)Drawing the network diagram of "component-target-disease" : Cytoscape software was used to construct the "component-target-disease" network to determine the target of the effective component of Huachansu in the treatment of bone cancer pain.Part 2: to explore the mechanism of analgesia by regulating the RANKL/RANK/OPG signal pathway by Huachansu acting on the CB2 receptor:(1)Establishment of bone cancer pain model in rats: qualified rats were screened according to the pain threshold and randomly divided into normal group,sham operation group and model group,with 6 rats in each group.The rats in the normal group were not treated with any treatment.Walker-256 cel s were injected into the tibial bone marrow cavity of the left posterior limb of the model group,and the same amount of inactivated Walker-256 cel s were injected into the same part of the sham operation group.Behavioral tests(Hindpaw withdrawal mechanical threshold and Hindpaw withdrawal thermal latency)were performed on the 0th,3rd,7th,and 9th days after modeling.On the 5th,9th and 15 th days after modeling,the bone destruction of the tibia of rats was observed by the small animal in vivo imaging apparatus.(2)To explore the dose range of analgesic effect of Huachansu: The dose of high,medium and low dose groups were successively converted by referring to the dose of human body and the conversion formula of human and mouse administration.Except for sham operation group and sham operation combined with Huachansu group,the rats were randomly divided into model group,Huachansu high-dose group(5 m L/kg),Huachansu medium-dose group(2.5 m L/kg)and Huachansu low-dose group(1.25 m L/kg),with 6 rats in each group.The rats in each group were intraperitoneally injected with Huachansu or normal saline,respectively.At the 0th,5th,7th,9th,11 th,13th,15 th,and 17 th days after modeling,behavioral tests(Hindpaw withdrawal mechanical threshold and Hindpaw withdrawal thermal latency)were carried out to analyze the optimal dose.After the animals were sacrificed,HE staining was used to observe the destruction of the tibia bones of the rats.(3)Analgesic effect of continuous administration of Huachansu on bone cancer pain rats and its effect on CB2 expression: female SD rats were selected and divided into sham operation group,model group,and Huachansu group,with 6 rats in each group.The administration was started on the 7th day of model establishment,with high-dose Huachansu(5 m L/kg)as the optimal dose.The sham operation group and the model group were given the same amount of normal saline for 11 consecutive days.Behavioral tests(Hindpaw withdrawal mechanical threshold and Hindpaw withdrawal thermal latency)were performed on the 0th,5th,7th,9th,11 th,13th,15 th,and 17 th days after modeling.After the animals were sacrificed,CB2 protein expression in bone tissue was detected by Western Blot.(4)To investigate the regulation of CB2 receptor on RANKL/RANK/OPG signaling pathway: female SD rats were selected and divided into the following five groups: Sham operation group,model group,Huachansu group,AM630(CB2inhibitor)group,and Huachansu combined with AM1241(CB2 agonist)group,with 6 rats in each group.Each group was intraperitoneally injected with normal saline,normal saline,Huachansu(5 m L/kg),AM630(300 μg/kg),and Huachansu(5 m L/kg)combined with AM1241(300 μg/kg),respectively.Huachansu combined with AM1241 group was injected with Huachansu for 30 min and then given AM1241.Once a day for 11 consecutive days.Behavioral tests(Hindpaw withdrawal mechanical threshold,Hindpaw withdrawal thermal latency,and the ambulatory pain)were performed on the 0th,5th,7th,9th,11 th,13th,15 th,and17th days after modeling.After the animals were sacrificed,the protein expressions of CB2,RANKL,OPG,and TRPV1 in bone tissues were detected by Western Blot,the contents of TRACP5 b and ICTP in serum were detected by ELISA,and the expression of osteoclasts in bone tissues was observed by TRAP staining.Results Part 1: Network pharmacology analyzed 11 key active ingredients of Huachansu that have been published publicly.The "components-target-disease" network showed that Huachansu may regulate multiple targets in the treatment of bone cancer pain,and there was a certain interaction between these targets,forming a PPI network with 50 nodes and 207 edges.Some important proteins were located in the center of the network,such as C NR1,CNR2,TRPV1,etc.KEGG signal pathway enrichment analysis was conducted on this network,and multiple signal pathways related to cannabinoid receptors were found.Therefore,CNR2 with the top ranking of P values was selected as the verification target in this study.Part II: The rat bone cancer pain model was successful y constructed.Small animal in vivo imaging apparatus results showed that the bone of the rats with successful modeling was significantly damaged due to infiltration of cancer cel s,and the degree of damage gradually increased with the extension of the model time.Behavioral results showed that hindpaw withdrawal mechanical threshold and hindpaw withdrawal thermal latency of model group were significantly decreased(P < 0.01);Huachansu did not affect the hindpaw withdrawal mechanical threshold and hindpaw withdrawal thermal latency in normal rats(P < 0.05),but only increased the hindpaw withdrawal mechanical threshold and hindpaw withdrawal thermal latency in bone cancer pain rats,and the effect of high concentration of Huachansu was the most obvious(P < 0.01 or P <0.05).The results of HE staining showed that the bone marrow cavity of the sham operation group was complete and the bone trabecula was arranged neatly.In the model group,the damaged area of cancer cel s increased significantly,and the structure of the bone trabecula was disordered,thinned,and fractured,which was arranged in the shape of the trabecula.Huachansu can obviously reduce the damage of bone marrow cavity in rats with bone cancer pain.Western Blot results showed that compared with the sham operation group,the expressions of CB2,RANKL,and TRPV1 in bone tissue of the model group were significantly increased(P < 0.01).Compared with the model group,the protein expressions of CB2,RANKL,and TRPV1 in the Huachansu group,AM630 group and Huachansu combined with AM1241 group were decreased(P < 0.01 or P < 0.05).Compared with the sham operation group,the expression of OPG protein in the model group was significantly decreased(P < 0.01).Compared with the model group,the expression of OPG protein in the Huachansu group,AM630 group,and Huachansu combined with AM1241 group was increased(P < 0.01 or P < 0.05).ELISA results showed that compared with the sham operation group,the expression of TRACP5 b and ICTP in the model group was significantly increased(P < 0.01).Compared with the model group,the expressions of TRACP5 b and ICTP were significantly decreased in the Huachansu group,AM630 group and Huachansu combined with AM1241 group(P < 0.01).TRAP staining results showed that: in the sham operation group,a small number of TRAP staining positive cel s were found,the staining was shal ow and the positive staining area was smal.TRAP staining of the model group showed more positive cel s,darker staining,and larger positive staining area.Compared with the model group,TRAP staining positive cel s were decreased in the Huachansu group,AM630 group,and Huachansu combined with AM1241 group.The behavioral results showed that compared with the sham operation group,the pain threshold of the model group was significantly decreased(P < 0.01).Compared with the model group,the pain thresholds of the Huachansu group,AM630 group,and Huachansu combined with AM1241 group were gradually increased(P < 0.01 or P < 0.05).Conclusion The regulation of the RANKL/RANK/OPG signaling pathway through the CB2 receptor may be one of the mechanisms of Huachansu relieving bone cancer pain. |
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