gigantol and Cataract

Gigantol is a phenolic component of precious Chinese medicine Dendrobii Caulis, which has many pharmacological activities such as prevent tumor and diabetic cataract. This paper aimed to investigate the molecular mechanism of gigantol in transmembrane transport in human lens epithelial cells(HLECs). Immortalized HLECs were cultured in vitro and inoculated in the laser scanning confocal microscopy(LSCM) medium at 5 000 cells/mL. The fluorescence distribution and intensity of gigantol marked by fluorescence in HLECs were observed by LSCM, and the absorption and distribution of gigantol were expressed as fluorescence intensity. The transmembrane transport process of gigantol in HLECs were monitored. The effects of time, temperature, concentration, transport inhibitors, and different cell lines on the transmembrane absorption and transport of gigantol were compared. HLECs were inoculated on climbing plates of 6-well culture plates, and the ultrastructure of HLECs was detected by atomic force microscopy(AFM) during the transmembrane absorption of non-fluorescent labeled gigantol. The results showed that the transmembrane absorption of gigantol was in time and concentration-dependent manners, which was also able to specifically target HLECs. Energy and carrier transport inhibitors reduced gigantol absorption by HLECs. During transmembrane process of gigantol, the membrane surface of HLECs became rougher and presented different degrees of pits, indicating that the transmembrane transport of gigantol was achieved by active absorption of energy and carrier-mediated endocytosis.

Topics: Apoptosis; Bibenzyls; Cataract; Cells, Cultured; Epithelial Cells; Humans; Lens, Crystalline

We previously reported that gigantol extracted from Caulis Dendrobii has significant therapeutic benefits for the treatment of galactosemic cataracts through its ability to inhibit aldose reductase (AR) activity. In this study, we identified the binding sites and structurally characterized the interaction between gigantol and AR, to understand the mechanism (s) of the effects of gigantol on cataracts. Gigantol was found to be protective against diabetic cataracts (DC) in rats induced by streptozotocin. Molecular docking predicted the binding sites between AR and gigantol to be residues Trp111, His110, Tyr48 and Trp20. Mutation of each of these residues led to a significant reduction in AR activity. Cold-spray ionization mass spectrometry measurements showed that the binding of gigantol to AR is  oncentration-dependent and that the maximum stoichiometric ratio of non-covalent bonding is 1:24.4. pH and temperature did not influence the interaction. Taken together, we provide further mechanistic evidence of the beneficial effects of gigantol on DC.

Topics: Aldehyde Reductase; Animals; Bibenzyls; Binding Sites; Catalysis; Cataract; Female; Guaiacol; Lens, Crystalline; Male; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutation; Protein Binding; Rats; Rats, Sprague-Dawley; Recombinant Proteins

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.. To characterize the interaction between gigantol extracted from D. chrysotoxum Lindl and the AR gene, and determine gigantol's efficacy against cataractogenesis.. 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.. 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.

Topics: Aldehyde Reductase; Bibenzyls; Cataract; Cells, Cultured; Dendrobium; Diabetes Complications; Epithelial Cells; Gene Expression Regulation, Enzymologic; Glucose; Guaiacol; Humans; Lens, Crystalline; Reverse Transcriptase Polymerase Chain Reaction; Spectrum Analysis, Raman

Gigantol and syringic acid (SA) have been shown to synergistically prevent formation of diabetic cataract (DC). However, the exact mechanism of this effect is unknown. Here, we investigate the effect of these compounds on the activity of aldose reductase (AR) and cataract formation.. We examined the synergistic anti-cataract efficacy of gigantol and SA in the high glucose- and streptozotocin -induced DC rat model; synergism was evaluated using Jin's formula. We investigated possible mechanisms of action by measuring AR expression and activity and levels of sorbitol using enzyme kinetics, Western blot, and RT-PCR. Finally, we examined binding interaction between AR and both compounds using a combination of site-directed mutagenesis, recombinant expression of wild-type and mutant proteins, and enzyme kinetics.. Combination treatment of gigantol and SA synergistically protected both HLECs(human lens epithelial cells) grown in vitro and DC formation in STZ-induced rats in vivo. Synergism was attributed to inhibition of AR activity, downregulation of AR expression via impaired transcription, and decreased sorbitol levels. Enzyme kinetics studies showed that the activity of an AR Asn160Ala mutant protein was significantly decreased compared to wild-type AR, confirming that Asn160 is a key residue for interaction between AR and both compounds.. Combined administration of gigantol and SA synergize to enhance anti-cataract efficacy. The synergistic effect is mainly attributed to disruption of the polyol pathway and inhibition of AR activity.

Topics: Aldehyde Reductase; Animals; Bibenzyls; Cataract; Cell Line; Cell Survival; Diabetes Complications; Disease Models, Animal; Drug Synergism; Female; Gallic Acid; Guaiacol; Humans; Male; Rats; Rats, Wistar

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.. 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.. 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.. 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.. 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.

Topics: Animals; Antioxidants; Bibenzyls; Cataract; Dendrobium; Drugs, Chinese Herbal; Galactosemias; Guaiacol; Lens, Crystalline; Medicine, Chinese Traditional; Molecular Docking Simulation; Osmotic Pressure; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction

As a continuation of our efforts directed towards the development of natural anti-diabetic cataract agents, gigantol was isolated from Herba dendrobii and was found to inhibit both aldose reductase (AR) and inducible nitric oxide synthase (iNOS) activity, which play a significant role in the development and progression of diabetic cataracts. To improve its bioefficacy and facilitate use as a therapeutic agent, gigantol (compound 14f) and a series of novel analogs were designed and synthesized. Analogs were formulated to have different substituents on the phenyl ring (compounds 4, 5, 8, 14a-e), substitute the phenyl ring with a larger steric hindrance ring (compounds 10, 17c) or modify the carbon chain (compounds 17a, 17b, 21, 23, 25). All of the analogs were tested for their effect on AR and iNOS activities and on D-galactose-induced apoptosis in cultured human lens epithelial cells. Compounds 5, 10, 14a, 14b, 14d, 14e, 14f, 17b, 17c, 23, and 25 inhibited AR activity, with IC50 values ranging from 5.02 to 288.8 μM. Compounds 5, 10, 14b, and 14f inhibited iNOS activity with IC50 ranging from 432.6 to 1188.7 μM. Compounds 5, 8, 10, 14b, 14f, and 17c protected the cells from D-galactose induced apoptosis with viability ranging from 55.2 to 76.26%. Of gigantol and its analogs, compound 10 showed the greatest bioefficacy and is warranted to be developed as a therapeutic agent for diabetic cataracts.

Topics: Aldehyde Reductase; Apoptosis; Bibenzyls; Cataract; Cell Survival; Cells, Cultured; Diabetes Complications; Enzyme Inhibitors; Epithelial Cells; Guaiacol; Humans; Hypoglycemic Agents; Lens, Crystalline; Molecular Structure; Nitric Oxide Synthase Type II; Structure-Activity Relationship

To study the anti-cataract effect of gigantol combined with syringic acid and their action mechanism.. H202-induced lens oxidative injury in vitro rat model was establish to observe the impact of gigantol combined with syringic acid on lens transparency under a dissecting microscope. D-galactose-induced cataract rat model was established to observe the impact of gigantol combined with syringic acid on lens transparency under a slit-lamp. UV spectrophotometry was adopted to detect the inhibitory activity of gigantol combined with syringic acid against AR. Molecular docking method was used to detect binding sites, binding types and pharmacophores of gigantol combined with syringic acid in prohibiting aldose reductase.. Both in vitro and in vivo experiments showed a good anti-sugar cataract activity in the combination of gigantol and syringic acid and a better collaborative effect than single component-gigantol and syringic acid and positive control drug Catalin. Molecular docking and dynamic simulation showed their collaborative AR-inhibiting amino acid residue was Asn160 and the major acting force was Van der Waals' force, which formed common pharmacophores.. Gigantol combined with syringic acid shows good anti-cataract, their action mechanism is reflected in their good collaborative inhibitory effect on AR.

Topics: Aldehyde Reductase; Animals; Bibenzyls; Cataract; Drug Synergism; Female; Gallic Acid; Guaiacol; Humans; In Vitro Techniques; Lens, Crystalline; Male; Rats; Rats, Wistar