Molecular Mechanism Of Spatholobi Caulis In Treatment Of Microglia-mediated Bone Cancer Pain Based On Network Pharmacology

Objective To explore the mechanism of Spatholobi Caulis on microglia-mediated bone cancer pain by network pharmacology and establishment of cell inflammation model.Methods Part I: Using network pharmacology and molecular docking methods to screen the targets of Chinese medicine Spatholobi Caulis and bone cancer pain,and construct drug-component-target-disease network map.Furthermore,key target analysis was conducted through GO and KEGG analysis to obtain relevant signal pathways.Molecular docking of the active ingredients and key targets was performed to evaluate the binding effect.Part II:According to the results of pre-network pharmacological analysis,luteolin(LUT)was predicted to be the main compound of Spatholobi Caulis for the treatment of bone cancer pain.BV2 microglia were stimulated with lipopolysaccharide(LPS)to establish an inflammatory cell model.The cells were then divided into the following groups: control group,LPS Group,LPS + Lut(7.5 μmol/L)group,LPS + LUT(15 μmol/L)group,LPS + LUT(30 μmol/L)group,and LPS + LUT(60 μmol/L)group.The control group and LPS group were added with serum-free medium for 2 hours;LPS + Lut(7.5 μmol/L)group,LPS + LUT(15 μmol/L)group,LPS + LUT(30 μmol/L)group and LPS + LUT(60 μmol/L)group were pretreated with Lut(7.5 μmol/L,15 μmol/L,30 μmol/L,60 μmol/L)for 2 hours;The control group was treated with serum-free medium;The other groups were added with serum-free medium containing 1 μg/m L LPS.Cells in each group were activated for 24 h,the cells and supernatants were collected for subsequent experiments.BV2 microglia activity was evaluated using the CCK-8 method,while RT-q PCR was utilized to assess the gene expressions of TNF-α、IL-6、NF-κB and HIF-1α in the signaling pathway.Additionally,the contents of TNF-α、IL-6 and HIF-1α in the supernatant were measured using ELISA.Results Part I:The results of network pharmacology showed that there were 102 overlapping genes between Spatholobi Caulis and Bone Cancer Pain.The analysis results showed that there were 22 active ingredients that Spatholobi Caulis could act on bone cancer pain through common targets,among which luteolin was the ingredient with the largest number of enriched targets Using PPI protein network analysis,10 core target proteins were identified: AKT1,EGFR,MAPK1,JUN,MYC,VEGFA,CASP3,IL-6,STAT3,and PTGS2;GO enrichment analysis revealed the involvement of 141 molecular functions,1630 biological processes,and81 cellular components;KEGG analysis identified 166 pathways,among which the HIF-1αsignaling pathway was selected for further investigation;The main enrichment factors in the signal pathway map were IL-6,NF-κB and HIF-1α.Molecular docking results showed that AKT1,EGFR,MAPK1,JUN,MYC,VEGFA,CASP3,IL-6,STAT3 and the first seven primary compounds exhibite good binding properties for the treatment of bone cancer pain by Spatholobi Caulis.Part II: Based on the results of network pharmacological analysis,luteolin was selected to act on LPS-induced mouse BV2 microglia inflammation model in vitro for preliminary verification of core targets and pathways,and the following results were obtained:RT-q PCR and ELISA results showed that compared with the control group,the m RNA and protein levels of TNF-α、IL-6 and HIF-1α in LPS group were significantly increased,and the m RNA level of NF-κB was significantly increased,with statistical significance(P < 0.05).Compared with LPS group,the m RNA and protein levels of TNF-α、IL-6 and HIF-1α in LPS+LUT groups were significantly decreased,and the m RNA level of NF-κB was significantly decreased,with statistical significance(P < 0.05).There was no significant difference between LPS+LUT(30μmol/L)group and LPS+LUT(60μmol/L)group(P >0.05).Conclusion 1.In this study,the core component of Spatholobi Caulis alleviating BCP was identified as LUT by network pharmacology,and the typical targets of IL-6,NF-κB,HIF-1αand TNF-α in HIF-1α signaling pathway could be regulated.2.The inhibitory effect of LUT in the range of 7.5-30μmol/L on LPS-induced inflammatory response of BV2 cells was confirmed by RT-q PCR and ELISA.