ed receptor gamma (PPAR), CCAAT/enhancer-binding c-Rel Inhibitor custom synthesis protein alpha (C/EBP), C/EBP, glucocorticoid receptor (GR), and and CCAAT/enhancer-binding protein alpha (C/EBP), C/EBP, glucocorticoid receptor (GR), glyceraldehyde3-phosphate dehydrogenase (GAPDH) in differentiated D3 Receptor Inhibitor Compound 3T3L-1 cells cells on8day 8 soon after glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in differentiated 3T3L-1 on day just after treatment with hispidulin and/or p-synephrine. (B) Evaluation on the the ratios of band intensities of treatment with hispidulin and/or p-synephrine. (B) Evaluation of ratios of the the band intensities of P-ERK, P-JNK, P-P38, PPAR, C/EBP, C/EBP, and GR in thethe treated cells compared with those in P-ERK, P-JNK, P-P38, PPAR, C/EBP, C/EBP, and GR in treated cells compared with those in the untreateddifferentiated 3T3L-1 cells (n (n3 independent experiments, p 0.05,0.05, Kruskal allis the untreated differentiated 3T3L-1 cells = = three independent experiments, p Kruskal allis nonparametrictest). Information are presented as because the mean SEM. nonparametric test). Information are presented the imply SEM.Biomolecules 2021, 11,16 of4. Discussion In this study, we applied a network pharmacology analysis to predict the anti-obesity mechanism of action of hispidulin and p-synephrine. Through a network pharmacology evaluation, the anti-obesity effect of hispidulin was predicted to act on estrogen, prolactin, Rap1, and PI3K-Akt signaling pathways by targeting AKT1, SRC, EGFR, and GSK3B. Preceding research have reported that these signaling pathways are related to obesity or adipocyte metabolism [570]. Also, p-synephrine was predicted to exert its antiobesity effect by way of calcium and cAMP signaling pathways by targeting adrenergic receptors, ADRB1, ADRB2, and ADRB3. In unique, a number of studies has supplied proof regarding the connection amongst 3-adrenergic receptors (ADRB3) and obesity [613]. Moreover, current research have shown that the calcium signaling pathway specifically plays a essential role in minimizing obesity by enhancing power consumption and promoting adipocyte differentiation and metabolism [647]. Determined by the results of previous studies, the network pharmacology analysis inside the present study predicted a feasible feasible mechanism of action of hispidulin and p-synephrine against obesity. In addition, the results in the mixture network evaluation from the two compounds showed fully distinct targets and pathways, which suggests that mixture treatment with hispidulin and p-synephrine could possibly exhibit additive and synergistic effects by means of diverse mechanisms of action. Among the commercially offered diet regime drugs, Qsymia(phentermine/topiramate) and Contrave(naltrexone/bupropion) will be the combinations of two drugs with diverse mechanisms of action [10,68]. These drugs show a stronger appetite suppressant effect than single drugs via the additive and synergistic effects in the combined components with distinct mechanisms of action. Determined by this evidence, the combination remedy of hispidulin and p-synephrine has a potential to show stronger effects against obesity than when employed alone. As a result, further experiments were performed to confirm the results of your network pharmacology evaluation and further evaluate the efficacy of hispidulin and p-synephrine in single and combination therapies. Each compounds have already been reported to be powerful against adipogenesis in 3T3-L1 cells. A earlier study showed that hispidulin at 40 exhibited a maximal inh