| Objective:This study aimed to explore the action mechanism of Wu-Tou Decoction (WTD) treating for rheumatoid arthritis (RA) by network pharmacology and perform further verification by experiments.Methods:1. Detailed steps for exploration of the action mechanism of Wu-Tou Decoction (WTD) treating for rheumatoid arthritis (RA) by network pharmacology were as follows:Firstly, chemical compounds in herbs forming WTD were obtained in TCM Database @ Taiwan. Human Targets interacting with these chemical compounds were searched on PubChem. RA-related human genes were searched on GenBank. Then, targets of WTD and RA-related genes were uploaded to bioinformatic analysis software Ingenuity Pathway Analysis (IPA). Molecular networks and canonical pathways constructed by targets of WTD and RA-related genes were obtained by core analysis of IPA, respectively. Shared canonical pathways of targets of WTD and RA-related genes were obtained by comparison analysis of IPA. Finally, the results obtained by network pharmacology were analysed.2. Ex vivo texts were designed to explore the effect of WTD on CCR5 Signaling in Macrophages based on network pharmacology analysis results. Protocols were as follows: Normal group, model group (MIP-1β,25 nM), WTD group (20μg/mL and 40μg/mL) and positive control group (Maraviroc,1μM) were designed. Maraviroc (MVC) is a CCR5 inhibitor. Cell suspensions with the concentration of 5×105 cells/mL and 10 ng/mL PMA were added into 6-well cell culture plate with the volume of 2 mL/well. After 48 h, old culture medium was discarded and cells were washed twice gently with PBS. Then, cells in normal group and model group were incubated with fresh culture medium. Cells in WTD group were incubated with fresh culture medium containing 20μg/mL and 40μg/mL WTD. Cells in positive control group were incubated with fresh culture medium containing 1μM MVC. After incubation for 2 h, MIP-1β was added into culture medium in each group except normal group and the final concentration of MIP-1β was 25 nM. After stimulation for 10 min, cells in each group were collected to detect levels of CCR5, p-pkc δ, pkc δ, p-p38 and p38 by western blot. Another batch of cells were treated with same protocol and cell supernantants were collected to detect RANTES level by ELISA after stimulation with MIP-1β for 24h.3. In vivo texts were designed to further validate the effect of WTD on CCR5 Signaling in Macrophages. Protocols were as follows:SD rats with Collagen induced arthritis (CIA) model were used in this study. Rats were grouped into normal group (N), model group (M), WTD low dose group (WL,3.75g/kg/d), WTD high dose group (WH,7.5g/kg/d) and methotrexate group (MTX, lmg/kg/w). All agents were intragastric administered in a volume of 1 mL/100 g for twenty-eight days. We observed the general status of rats and recorded rats’weight as well as arthritis index (AI) score every three days. After the end of treatment, histopathological changes of ankle joints and histological scores were observed and assessed. Sera levels of MIP-la, MIP-2, RANTES, IP-10 and MCP-1 were detected by ELISA. Relative expression of mRNA of MIP-1α, RANTES and IP-10 in rats’ankle joints were determined by real-time PCR. Levels of CCR5, pkc 8, p38 and phosphorylation forms of pkc δ and p38 were measured by immunohistochemistry and western blot.Results:1. Network pharmacology analysis results:One hundred and seventy-four chemical compounds, one hundred and eighty-six human protein targets of WTD and eight hundred and thirty-one RA-related human genes were obtained. Twelve molecular networks were constructed by targets of WTD and twenty-five molecular networks were constructed by RA-related genes. The biofunctions regulated by targets of WTD and RA-related genes in common indicated that WTD treating for RA might be related to the regulation of inflammatory response, hematological system development and function, cell-to-cell signaling and interaction, etc. Three hundred and fourty-six and three hundred and ninety-four signaling pathways regulated by targets of WTD and RA-related genes were obtained, respectively. And three hundred and fourteen signaling pathways were regulated by targets of WTD and RA-related genes in common. Top ten shared canonical pathways related to cellular immune response and cytokine signaling were as follows:CCR5 Signaling in Macrophages, Prolactin Signaling, Calcium-induced T Lymphocyte Apoptosis, IL-8 Signaling, fMLP Signaling in Neutrophils, Natural Killer Cell Signaling, NF-κB Activation by Viruses, IL-12 Signaling and Production in Macrophages, Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes, Leukocyte Exstravasation Signaling. We focused on CCR5 Signaling in Macrophages (Top one). This signaling pathway was related to inflammatory response and could regulate the expression of chemoattractants and proinflammatory mediators. In this signaling pathway, pkc and p38 were targets of WTD as well as RA-related genes.2. Ex vivo test results:ELISA results showed that RANTES level in cell supernatant in model group increased markedly compared to normal group (P<0.05). And RANTES level in WTD group and positive control group decreased significantly in comparision with model group (P<0.05). Western blot results showed that levels of p-pkc 8 and p-p38 increased significantly in model group compared to normal group (P<0.01). Levels of p-pkc 8 and p-p38 in WTD group and positive control group reduced apparently in comparision with model group (P<0.05 or P<0.01). But, the level of CCR5 in each group showed no significant difference (P>0.05).3. In vivo test results:AI score and histopathological results showed that WTD effectively alleviated arthritis severity in CIA rats. ELISA results indicated that WTD decreased the levels of MIP-la, MIP-2, RANTES, IP-10 and MCP-1 in sera of CIA rats significantly (P<0.05 or P<0.01). Real-time PCR results demonstrated that relative expression of mRNA of MIP-1α, RANTES and IP-10 in rats’ankle joints were decreased prominently in WTD group compared to model group (P<0.05 or P<0.01). Immunohistochemistry and western blot results showed that CCR5, p-pkc 8 and p-p38 levels in rats’ankle joints were decreased apparently in WTD group compared to model group (P <0.05 or P<0.01).Conclusions:1. This study obtained CCR5 Signaling in Macrophages that is important signaling pathway for WTD on RA by network pharmacology analysis.2. In vivo test verified the therapeutic effect of WTD on CIA rats.3. In vivo and in vitro experiments indicated that therapeutic effect of WTD on RA might be associated with down-regulating the phosphorylation levels of pkc 8 and p38 and the expression of chemokines in CCR5 Signaling in Macrophages. |
