Target PDE3B Validation And Signaling Pathway Study Of Phillygenin For Weight Loss

The abnormal or excessive accumulation of bodily fat is referred to as obesity.The number of overweight and obese people worldwide has increase significantly in recent years,and obesity has emerged as one of the major social issues that people worldwide must work together to solve.The intricacy and diversity of the causes of obesity,which also often result in a number of diseases like cancer,diabetes,and cardiovascular disease,put a lot of strain on societal health.Due to their multi-target,strong biocompatibility,and few harmful side effects,natural chemicals have emerged as the primary testing ground for possible weight loss medications.Phillyrin,the primary compound in Forsythia(Forsythia suspensa(Thunb.)Vahl)and forsythia leaves,have been discovered to significantly prevent fat buildup at the cellular level in earlier laboratory research.According to the literature,phillyrin significantly reduces body weight in animals.In fact,phillyrin can be converted into phillygenin in the human gut,the research team’s early cell and zebrafish experiments have proved that phillygenin also has a significant weight loss effect.Moreover,phillygenin has a higher bioavailability,good stability,and a high drug possibility than phillyrin,however,up to now,the target and signal pathway of phillygenin for phillygenin for weight loss are not known.This study first proved that phillygenin causes weight loss at the cellular level;then,based on the early prediction by network pharmacology that the phillygenin weight loss target may be PDE3B,ELISA and molecular simulation were utilized to confirm the phillygenin weight loss target PDE3B at the molecular lever;thirdly,DARTS/Western-blot and ELISA were used to confirm the phillygenin weight loss target PDE3B at the cellular level;finally,the phillygenin weight loss signaling pathway was studied using Western-blot and RT-qPCR.The precise research subjects and findings are as follows:(1)Phillygenin impact on lipid buildup in HepG2 cells subjected to high glucose.CCK8 testifyed that phillygenin had no toxicity to HepG2 at concentrations of 1-200 μM.Fluorescence detection demonstrated that phillygenin had an inhibitory effect on high glucose-induced red lipid fluorescence in HepG2 cells,and the lipid content in cells was decreased by 88.54%,80.56%,77.31%,71.98%,68.37%,with dose-dependent relationship at 40 μM,80 μM 120 μM,160 μM,and 200 μM,respectively.The findings demonstrated that phillygenin significantly inhibited the lipid buildup in high glucose-induced HepG2 cells.(2)The molecular level verification weight loss target is PDE3B.ELISA and molecular docking were used to explore the relationship between phillygenin and PDE3B.The results showed that phillygenin could significantly increase the cAMP content,which had the same effect as milrinone,that is,phillygenin increased the cAMP content by inhibiting PDE3B.The results of molecular docking showed that the binding energy between phillygenin and PDE3B(-7.9 kcal/mol)was greater than that of milrinone(an inhibitor of PDE3,-6.6 kcal/mol),whereas milrinone and PDE3B did not make any hydrogen bonds,phillygenin and PDE3B formed three hydrogen bonds.Therefore,it is preliminary possible to determine that the weight loss target of phillygenin is PDE3B.(3)Cellular level confirmation of PDE3B as the phillygenin weight loss target.DARTS/Western-blot and ELISA(cAMP content detection,competitive inhibition)were used to confirm the target PDE3B for weight loss.The outcomes demonstrated that phillygenin was capable of efficiently preventing pronase to hydrolyze PDE3B protein,demonstrating that PDE3B was the intended target of phillygenin’s weight reduction.The cAMP content test revealed that phillygenin could greatly boost the cAMP content on its own,and the optimal cAMP content was found at a concentration of 120 μM.The competitive inhibition investigation revealed that phillygenin and milrinone competed since the cAMP content did not change much when milrinone and phillygenin were incubated together at the optimal concentration of 5.5 μM and 120 μM,respectively.The results of the study on competitive inhibition demonstrated that phillygenin and milrinone competed with one another for the same PDE3B activity center and had a weight-loss effect when milrinone(optimal concentration 5.5 μM)and phillygenin(optimal concentration 120 μM)were incubated together.Consequently,PDE3B can be shown to be a weight-loss target for phillygenin at the cellular level.(4)Research on the PDE3B-cAMP-PKA-HSL weight-loss signaling pathway in phillygenin-mediated HepG2 cells.PDE3B’s mRNA expression was measured using RTqPCR,and the results showed that phillygenin may drastically lower it.Western-blot analysis was used to evaluate the expression of PDE3B,PKA,and HSL proteins,as well as the levels of p-PKA and p-HSL in the high-lipid model of HepG2 produced by high glucose.According to the findings,phillygenin mediated the PDE3B-cAMP-PKA-HSL signaling pathway to promote lipolysis and help people lose weight.The concentration of phillygenin(40 μM,80 μM,120 μM,160 μM)increased,the expression of PDE3B protein decreased,the content of cAMP increased,and the levels of p-PKA and p-HSL increased.