compound-target network embodied 207 nodes(17 potential compounds and 190 potential targets). The mean degree value(the number of associated targets)of candidate components was 18.1, and 1l components possessed a mean degree value greater than 12, suggesting that most components associated with multiple targets to exert different biological and pharmacological effects. The major components and its target numbers are shown in Table S3. Specifically, components such as caffeine, beta-sitosterol(Sito) and arachidonic acid(AA), which acted on 54, 38 and 38 targets respectively, were the crucial active components for O. sinensis in this network. The second major components are oleic acid, cordycepin and nicotinic acid mmune regulation mode. Previous data showed that the targets of O. sinensis were involved in immune function that included positive regulation of the immune system to bacterial infection, and T and B cell function. Cytokines also play an important role in the activation and regulation of immune responses. As can be seen from Figure 3, marked target genes make a contribution to the production of interferon which are detected to connect with anti-virus or bacterial responses. On the other hand, components in O. sinensis also possess anti-inflammatory effects during both our prediction and some other studies. For instance, cordycepin was observed to suppress LPS-stimulated release of pro-inflammatory cytokines TNFa and IL-1B through NF kB pathway, Sito was observed to induce cancer cell line apoptosis through decrease the expression of apoptosis regulator Bcl-2 which makes it a candidate for cancer chemotherapy. Besides, cordycepin was predicted to interact with ROCK which related to leukocyte migration and this may contribute to the formation of germinal
6 compound-target network embodied 207 nodes (17 potential compounds and 190 potential targets). The mean degree value (the number of associated targets) of candidate components was 18.1, and 11 components possessed a mean degree value greater than 12, suggesting that most components associated with multiple targets to exert different biological and pharmacological effects. The major components and its target numbers are shown in Table S3. Specifically, components such as caffeine, beta-sitosterol (Sito) and arachidonic acid (AA), which acted on 54, 38 and 38 targets, respectively, were the crucial active components for O. sinensis in this network. The second major components are oleic acid, cordycepin and nicotinic acid. Immune regulation mode. Previous data showed that the targets of O. sinensis were involved in immune function that included positive regulation of the immune system to bacterial infection, and T and B cell function. Cytokines also play an important role in the activation and regulation of immune responses. As can be seen from Figure 3, marked target genes make a contribution to the production of interferon which are detected to connect with anti-virus or bacterial responses. On the other hand, components in O. sinensis also possess anti-inflammatory effects during both our prediction and some other studies. For instance, cordycepin was observed to suppress LPS-stimulated release of pro-inflammatory cytokines TNFα and IL-1β through NF- κB pathway31; Sito was observed to induce cancer cell line apoptosis through decrease the expression of apoptosis regulator Bcl-2 which makes it a candidate for cancer chemotherapy32. Besides, cordycepin was predicted to interact with ROCK which related to leukocyte migration and this may contribute to the formation of germinal
center or other cellar immune responses There have been many studies in the 3 key components in O. sinensis, for example caffeine was used to defense depression and AA could promote secretion of pro-Inflammatory leukotrienes 33, 34 After screening the major components of o sinensis, the nucleoside antibiotic cordycepin was selected for further experimental alidation based on our ad has d much attention, mostly because of its anti-tumor and antiviral activities in the inhibition of viral DNA/RNA synthesis 5-37. For example, cordycepin induces apoptosis of tumorigenic Leydig cells in the mouse via several signaling pathways. It also plays roles in platelet aggregation and inflammation, 142, however, there are few reports on the use of cordycepin as an adjuvant to enhance the immune response. We here in combined cordycepin with HBsAg to generate a new vaccine and tested its immunologic activity in mice. Cordycepin improves HBsAg-specific antibody production without side effects Serum HBV antibodies were measured by elisa to determine the effects of the adjuvant on the humoral immune response. As shown in Figure 4, cordycepin(0. 2, 1 and 2 mg/kg)adjuvanted with HBsAg led to an increase in the serum antibody level in a dose-dependent manner. The IgM level remained high on day 21 in the 2 mg/kg cordycepin group but not in the other groups(Figure 4a). On day 14, the IgG titers nduced by 2 mg/kg cordycepin adjuvanted with HBsAg were similar to the vaccine group on day 21 after the last injection(Figure 4b). However, there was no difference in the IgG level in this group on days 14 and 21. Furthermore, there was no significant
7 center or other cellar immune responses. There have been many studies in the 3 key components in O. sinensis, for example, caffeine was used to defense depression and AA could promote secretion of pro-inflammatory leukotrienes33,34. After screening the major components of O. sinensis, the nucleoside antibiotic cordycepin was selected for further experimental validation based on our adjuvant screen purpose. Cordycepin has received much attention, mostly because of its anti-tumor and antiviral activities in the inhibition of viral DNA/RNA synthesis35-37. For example, cordycepin induces apoptosis of tumorigenic Leydig cells in the mouse via several signaling pathways38-40. It also plays roles in platelet aggregation and inflammation36,41,42; however, there are few reports on the use of cordycepin as an adjuvant to enhance the immune response. We here in combined cordycepin with HBsAg to generate a new vaccine and tested its immunologic activity in mice. Cordycepin improves HBsAg-specific antibody production without side effects. Serum HBV antibodies were measured by ELISA to determine the effects of the adjuvant on the humoral immune response. As shown in Figure 4, cordycepin (0.2, 1 and 2 mg/kg) adjuvanted with HBsAg led to an increase in the serum antibody level in a dose-dependent manner. The IgM level remained high on day 21 in the 2 mg/kg cordycepin group but not in the other groups (Figure 4a). On day 14, the IgG titers induced by 2 mg/kg cordycepin adjuvanted with HBsAg were similar to the vaccine group on day 21 after the last injection (Figure 4b). However, there was no difference in the IgG level in this group on days 14 and 21. Furthermore, there was no significant
different in body weight in mice from all groups(Figure Sla). There was also no apparent histological change in the liver and spleen of mice from all groups on day 28 by haematoxylin-eosin staining and routine light microscopy(Figure SIb) Cordycepin adjuvanted vaccine promotes lymphocyte proliferation and cytokine production in vivo and in vitro. The enhancement of cellular immunity is also important barrier for HBV infection. HBSAg adjuvanted with I or 2 mg/kg cordycepin resulted in significantly higher proliferative activity than that in the other group. The cell activity of the 2 mg/kg cordycepin group was similar to that of the positive control (Figure 4c), indicating that it elicits an effective cellular immune response. The increased cell supernatant cytokine levels(Figure 4d) suggest that both Thl and Th2 cell responses were activated by cordycepin Lymphocyte differentiation in the spleen after immunization. To assess t and b cell differentiation after immunization, spleen cells were collected and analyzed by flow cytometry( Figure 5). The percentage of CD3 CD4 Th cells and CD3 CD8 CTL cells were all increased by the supplementary of cordycepin. The CD80 and CD86 expression on CD19+ B cells were also enhanced in the adjuvant groups, indicating hat b cells were activated more effectively. The increase in Th and B cell number indicates that the increase in antibody production and CTL cell number enhanced the cytotoxic effect, which might associate with IFN-Y Discussion Complex composition of TCMs makes it hard to understand its therapeutic mode from a molecular level. In recent years, several herbal components with different
8 different in body weight in mice from all groups (Figure S1a). There was also no apparent histological change in the liver and spleen of mice from all groups on day 28 by haematoxylin-eosin staining and routine light microscopy (Figure S1b). Cordycepin adjuvanted vaccine promotes lymphocyte proliferation and cytokine production in vivo and in vitro. The enhancement of cellular immunity is also an important barrier for HBV infection. HBsAg adjuvanted with 1 or 2 mg/kg cordycepin resulted in significantly higher proliferative activity than that in the other group. The cell activity of the 2 mg/kg cordycepin group was similar to that of the positive control (Figure 4c), indicating that it elicits an effective cellular immune response. The increased cell supernatant cytokine levels (Figure 4d) suggest that both Th1 and Th2 cell responses were activated by cordycepin. Lymphocyte differentiation in the spleen after immunization. To assess T and B cell differentiation after immunization, spleen cells were collected and analyzed by flow cytometry (Figure 5). The percentage of CD3+ CD4+ Th cells and CD3+ CD8+ CTL cells were all increased by the supplementary of cordycepin. The CD80 and CD86 expression on CD19+ B cells were also enhanced in the adjuvant groups, indicating that B cells were activated more effectively. The increase in Th and B cell number indicates that the increase in antibody production and CTL cell number enhanced the cytotoxic effect, which might associate with IFN-γ. Discussion Complex composition of TCMs makes it hard to understand its therapeutic mode from a molecular level. In recent years, several herbal components with different
pharmacological activities have been identified as the development of biological analysis, these components constitute a substantial percentage of todays new drugs#4. One of the most commonly used method to investigate new drugs is to start from a disease and search from natural products which could treat it, however, the limitation of technical difficulties and suitable animal models makes it difficult to understand their mode of action 5,46. Recently, systems pharmacology offers a new approach to study bioactive components of TCM agents, as well as their drug-target interactions. Herein, we try to selective adjuvant from O. sinensis for its remarkable immunoregulation effects. Although there have been many studies about effective components in O. sinensis, the exact action mode was not fully elicited at a system level In the present study, with the help of OB, DL and HL screening, 17 active compounds with 190 potential targets were identified in O. sinensis. The target enrichment analysis shows that these targets take part in many biological pathways such as cytokine secretion, drug binding, blood circulation and so on. Several targets were lso involved in different types of cancer, supporting an earlier study which showed O sinensis extracts to exhibit anti-cancer effects"-49. It was not a surprise that several ts have been studied clearly in ches. Caffeine is well-known phosphodiesterase 3(PDE3)inhibitor and antagonist of adenosine receptors. Caffeine can inhibit the production of tumor necrosis factor alpha(tNF-a in lipopolysaccharide-stimulated human whole blood and modulate specific biological parameters associated with depression, thereby preventing the disease 2. 53
9 pharmacological activities have been identified as the development of biological analysis; these components constitute a substantial percentage of today’s new drugs43,44. One of the most commonly used method to investigate new drugs is to start from a disease and search from natural products which could treat it, however, the limitation of technical difficulties and suitable animal models makes it difficult to understand their mode of action45,46. Recently, systems pharmacology offers a new approach to study bioactive components of TCM agents, as well as their drug-target interactions. Herein, we try to selective adjuvant from O. sinensis for its remarkable immunoregulation effects. Although there have been many studies about effective components in O. sinensis, the exact action mode was not fully elicited at a system level. In the present study, with the help of OB, DL and HL screening, 17 active compounds with 190 potential targets were identified in O. sinensis. The target enrichment analysis shows that these targets take part in many biological pathways such as cytokine secretion, drug binding, blood circulation and so on. Several targets were also involved in different types of cancer, supporting an earlier study which showed O. sinensis extracts to exhibit anti-cancer effects47-49. It was not a surprise that several major components have been studied clearly in previous researches. Caffeine is a well-known phosphodiesterase 3 (PDE3) inhibitor and antagonist of adenosine receptors50. Caffeine can inhibit the production of tumor necrosis factor alpha (TNF-α) in lipopolysaccharide-stimulated human whole blood51 and modulate specific biological parameters associated with depression, thereby preventing the disease52,53
Interestingly, caffeine can also protect against the production of free radicals 4. Sito is a functional phytosterol with a chemical structure similar to that of cholesterol. Sito alone or combined with other plant sterols is known to reduce blood cholesterol levels by blocking cholesterol absorption 6,7 Sito has also been reported to possess antimicrobial activity". AA, an unsaturated fatty acid that modulates the activities of various ion channels, functions as a second messenger. As the adjuvant selection purpose, we choose all immune related function to get a component-target -function network. Results show that all components participate in these processes. Then,an immune-related pathway was constructed to further dissect the potential immunoregulation activities of components in O. sinensis. Cordycepin was finally selected because of its significant functions to suppress inflammation and regulate leukocyte migration as well as cell proliferation in the networks. Although it was not the only key component in O. sinensis, other components may interference with nutrient absorption or inhibit cytokine secretion, may have an adverse effect on healthy people or just suppress immune responses There have been many studies on cordycepin these days, mainly concentrate on its anti-tumor and immunoregulation effects, but few reports pay attention to its adjuvanticity to vaccines In the experimental validation part, cordycepin triggered IgM and Igg antibody responses in mice by 7 days in a dose dependent manner after combination with a hepatitis b vaccine. The high cordycepin group showed no difference in IgG titers between days 14 and 21 suggesting that the third injection might be unnecessary. Certainly, this would reduce the overall cost and the immune
10 Interestingly, caffeine can also protect against the production of free radicals54. Sito is a functional phytosterol with a chemical structure similar to that of cholesterol55. Sito alone or combined with other plant sterols is known to reduce blood cholesterol levels by blocking cholesterol absorption56,57. Sito has also been reported to possess antimicrobial activity58. AA, an unsaturated fatty acid that modulates the activities of various ion channels, functions as a second messenger59. As the adjuvant selection purpose, we choose all immune related function to get a component-target-function network. Results show that all components participate in these processes. Then, an immune-related pathway was constructed to further dissect the potential immunoregulation activities of components in O. sinensis. Cordycepin was finally selected because of its significant functions to suppress inflammation and regulate leukocyte migration as well as cell proliferation in the networks. Although it was not the only key component in O. sinensis, other components may interference with nutrient absorption or inhibit cytokine secretion, may have an adverse effect on healthy people or just suppress immune responses. There have been many studies on cordycepin these days, mainly concentrate on its anti-tumor and immunoregulation effects60-62, but few reports pay attention to its adjuvanticity to vaccines. In the experimental validation part, cordycepin triggered IgM and IgG antibody responses in mice by 7 days in a dose dependent manner after combination with a hepatitis b vaccine. The high cordycepin group showed no difference in IgG titers between days 14 and 21 suggesting that the third injection might be unnecessary. Certainly, this would reduce the overall cost and the immune