Proteomic screening identifies the direct targets of chrysin anti-lipid depot in adipocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Overweight/obesity was mentioned by many countries as an obstacle to good health and long life, which increases risk of diseases and disorders. Previous studies suggested that the chronic low-grade inflammation present in the body was considered as the essential pathogenesis for obesity. Chrysin is extracted from traditional Chinese medicine Oroxylum indicum (Linn.) Kurz and plays a superior anti-obesity role. Chrysin could reduce the lipid depot by inhibiting the obesity-related inflammation in adipose tissue. However, the target protein for chrysin to exert its anti-obesity role are not verified. AIM OF STUDY: The present study aimed to screen and validate the target protein for chrysin to reduce the lipid depot in palmitic acid-induced 3T3-L1 adipocytes. MATERIALS AND METHODS: Obesity model was established employing 0.5 mmol/L palmitic acid-induced 3T3-L1 adipocytes through "Cocktails" method. Two-dimensional gel electrophoresis (2-DE) combined with liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the differentially expressed proteins for chrysin intervention by lipid formation in adipocytes. Gene silencing was utilized to decrease gene expression of the candidate proteins, then production of triglyceride in 3T3-L1 was detected by triglycerides assay to determine the target proteins. Ultraviolet (UV) absorption together with fluorescence spectra validated the direct target proteins of chrysin. They also computed the correlation constants of combination between chrysin and the target proteins. Molecular docking was further employed to identify the main binding amino acids between chrysin and the target protein. RESULTS: 2-DE combined with LC-MS screened four candidate proteins which were related to metabolism and inflammation. The production of triglycerides in 3T3-L1 was reduced after decreasing gene expression of Annexin A2 (ANXA2), 60 kDa heat shock protein (HSP-60) and succinyl-CoA:3-ketoacid coenzyme A transferase 1 (SCOT-S), respectively. UV spectrum showed that the absorbance spectra of ANXA2 from 260 to 300 nm shifted upwards along with the increase in chrysin concentration, meanwhile the absorbance spectra of HSP-60 from 200 to 220 nm and from 265 to 280 nm shifted slightly upwards along with the increase in chrysin concentrations. The results indicated the conjugated structures between chrysin and ANXA2 or HSP-60. Fluorescence quenching further suggested a spontaneous interaction between chrysin and ANXA2 or HSP-60. Finally, molecular docking identified the main binding amino acids between ANXA2 and chrysin were Ser22, Tyr24, Pro267, Val298, Asp299, and Lys302. CONCLUSIONS: Chrysin can reduce the amount of triglycerides by directly downregulating the inflammation-related target proteins ANXA2 and HSP-60, exerting an anti-obesity role.

Gigantol from Dendrobium chrysotoxum Lindl. binds and inhibits aldose reductase gene to exert its anti-cataract activity: An in vitro mechanistic study.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium. chrysotoxum Lindl is a commonly used species of medicinal Dendrobium which belongs to the family of Orchidaceae, locally known as "Shihu" or "Huangcao". D. chrysotoxum Lindl is widely known for medicinal values in traditional Chinese medicine as it possesses anti-inflammatory, anti-hyperglycemic induction, antitumor and antioxidant properties. STUDY AIM: To characterize the interaction between gigantol extracted from D. chrysotoxum Lindl and the AR gene, and determine gigantol's efficacy against cataractogenesis. MATERIALS AND METHODS: Human lens epithelial cells (HLECs) were induced by glucose as the model group. Reverse transcription polymerase chain reaction (RT-PCR) was used to assess AR gene expression. Then, the mode of interaction of gigantol with the AR gene was evaluated by UV-visible spectroscopy, atomic force microscope (AFM) and surface-enhanced Raman spectroscopy (SERS). The binding constant was determined by UV-visible. RESULTS: Gigantol depressed AR gene expression in HLECs. UV-visible spectra preliminarily indicated that interaction between the AR gene and gigantol may follow the groove mode, with a binding constant of 1.85×103L/mol. Atomic force microscope (AFM) data indicated that gigantol possibly bound to insert AR gene base pairs of the double helix. Surface-enhanced Raman spectroscopy (SERS) studies further supported these observations. CONCLUSION: Gigantol extracted from D. chrysotoxum Lindl not only has inhibitory effects on aldose reductase, but also inhibits AR gene expression. These findings provide a more comprehensive theoretical basis for the use of Dendrobium for the treatment of diabetic cataract.

Anti-osmotic and antioxidant activities of gigantol from Dendrobium aurantiacum var. denneanum against cataractogenesis in galactosemic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium aurantiacum var. denneanumis widespread in southern China, locally known as "Shihu", "Huangcao" or "Fengdou", has long been used in traditional Chinese medicine for antipyretic, immunomodulatory, anti-aging effects and eye benefiting. AIM OF THIS STUDY: To investigate the effects of gigantol extracted from the stem of D. aurantiacum var. denneanum on the formation of galactose-induced cataractogenesis and the potential mechanisms underlying these effects. MATERIALS AND METHODS: Cataract lens models were induced by d-galactose both in vitro and in vivo. The transparency of the rat lenses in vitro and in vivo was observed with an anatomical microscope and a slit lamp microscope. The differential protein and action targets of gigantol were determined and compared among the control group, model group, and gigantol group using two-dimensional electrophoresis and mass spectrometry (MS). Enzyme kinetics was used to show the ability of gigantol to respress aldose reductase (AR) and inducible nitric oxide synthase (iNOS). Quantitative real-time PCR (RT-qPCR). was used to detect repression of the expression of AR and iNOS genes. Molecular docking and dynamic simulation were used to predict the interaction points and combination patterns between gigantol, AR, and iNOS. RESULTS: Gigantol was found to prevent galactose-induced damage to the rat lenses both in vitro and in vivo, to delay lens turbidity, and to keep the lenses transparent. Differential proteomes, MS, and RT-qPCR showed AR and iNOS to be the target proteins of gigantol. Gigantol reduced the galactose-induced AR and iNOS mRNA expression by 51.2% and 60.9%, respectively. The IC50 of gigantol for inhibition of AR and iNOS activities were 65.67 µg/mL and 8.768 µg/mL, respectively. Gigantol-AR binding sites were Trp111, His110, Tyr48, and Trp20, and gigantol-iNOS binding sites were Ile195 and Gln257. The main forms of interaction were hydrophobic forces, hydrogen bonds, and van der Waals forces. CONCLUSION: Gigantol extracted from D. aurantiacum var. denneanum was found to inhibit galactose-induced formation of cataracts through repression of the gene expression and activity of AR and iNOS.