Chebulagic acid and Chebulinic acid inhibit TGF-ß1 induced fibrotic changes in the chorio-retinal endothelial cells by inhibiting ERK phosphorylation.

PURPOSE: Diabetic retinopathy (DR) is characterized by pro-inflammatory, pro-angiogenic and pro-fibrotic environment during the various stages of the disease progression. Basement membrane changes in the retina and formation of fibrovascular membrane are characteristically seen in DR. In the present study the effect of Alcoholic (AlE) extracts of Triphala an ayurvedic herbal formulation and its chief compounds, Chebulagic (CA), Chebulinic (CI) and Gallic acid (GA) were evaluated for TGFß1-induced anti-fibrotic activity in choroid-retinal endothelial cells (RF/6A). METHOD: RF/6A cells were treated with TGFß1 alone or co-treated with AlE, CA, CI or GA. The mRNA and protein expression of fibrotic markers (αSMA, CTGF) were assessed by qPCR and western blot/ELISA. Functional changes were assessed using proliferation assay and migration assay. To deduce the mechanism of action, downstream signaling was assessed by western blot analysis along with in silico docking studies. RESULT: AlE (50 µg/ml) CA and CI at 10 µM reduced the expression of pro-fibrotic genes (αSMA and CTGF) induced by TGFß1, by inhibiting ERK phosphorylation. GA did not inhibit TGFß1 mediated changes in RF/6A cells. In silico experiments shows that CA and CI has favourable binding energy to bind with TGFß receptor and inhibit the downstream signaling, while GA did not. CONCLUSION: Hence this study identifies Triphala and its chief compounds CA and CI as potential adjuvants in the management of DR.

Chebulagic acid Chebulinic acid and Gallic acid, the active principles of Triphala, inhibit TNFα induced pro-angiogenic and pro-inflammatory activities in retinal capillary endothelial cells by inhibiting p38, ERK and NFkB phosphorylation.

Tumor necrosis factor-α (TNFα) a pleiotropic cytokine induces pro-inflammatory and pro-angiogenic changes in conditions such as diabetic retinopathy (DR) and neovascular age related macular degeneration (NV-AMD). Hence, inhibition of TNFα mediated changes can benefit the management of DR and NV-AMD. Triphala, an ayurvedic herbal preparation is known to have immunomodulatry functions. In this study we evaluated the alcoholic extract of triphala (AlE) and its compounds Chebulagic acid (CA), Chebulinic acid (CI) and Gallic acid (GA) for their anti-TNFα activity. TNFα induced pro-inflammatory and pro-angiogenic changes in the retinal-choroid microvascular endothelial cells (RF/6A). Treatment with CA/CI/GA and the whole Triphala extract showed characteristic inhibition of MMP-9, cell proliferation/migration and tube formation as well the expression of IL-6, IL-8 and MCP-1 without affecting cell viability. This was mediated by inhibition of p38, ERK and NFκB phosphorylation. Ex vivo angiogenesis assay using chick chorioallantoic membrane (CAM) model also showed that TNFα-induced angiogenesis and it was inhibited by AlE and its active principles. Further, in silico studies revealed that CA, CI and GA are capable of binding the TNFα-receptor-1 to mediate anti-TNFα activity. This study explains the immunomodulatory function of Triphala, evaluated in the context of retinal and choroid vasculopathies in vitro and ex vivo; which showed that CA, CI and GA can be a potential pharmacological agents in the management of DR and NV-AMD.

Flavonoids from Litsea japonica Inhibit AGEs Formation and Rat Lense Aldose Reductase In Vitro and Vessel Dilation in Zebrafish.

In our ongoing efforts to identify effective naturally sourced agents for the treating of diabetic complications, two new (1 and 2) and 11 known phenolic compounds (3-13) were isolated from an 80 % ethanol extract of Litsea japonica leaves. The structures of the new compounds were established by spectroscopic and chemical studies. These isolates (1-13) were subjected to an in vitro bioassay evaluating their inhibitory activity on advanced glycation end products formation and rat lens aldose reductase activity. Of the compounds evaluated, the flavonoids (3, 4, 6-8, 11, and 12) markedly inhibited advanced glycation end products formation, with IC50 values of 7.4-72.0 µM, compared with the positive control, aminoguanidine (IC50 = 975.9 µM). In the rat lens aldose reductase assay, consistent with the inhibition of advanced glycation end products formation, the flavonoids (3, 4, 6-8, 11, and 12) exhibited considerable inhibition of rat lens aldose reductase activity, with IC50 values of 1.1-12.5 µM. In addition, the effects of kaempferol (4) and tiliroside (7) on the dilation of hyaloid-retinal vessels induced by high glucose in larval zebrafish were investigated. Only kaempferol significantly reduced the diameters of high glucose-induced hyaloid-retinal vessels, by 52.2 % at 10 µM, compared with those in the high glucose-treated control group.

Inhibition of cyclooxygenase-2 expression by zinc-chelator in retinal ischemia.

The zinc ion (Zn2+) is abundant in neurons. However, excessive Zn2+ can induce neuronal cell death. This study examined the role of Zn2+ in transient retinal ischemia in adult male rats. The rats were sacrificed 4-24 h after retinal ischemia by high intra-ocular pressure, and the retinas were prepared for microscopic examination of retinal cell degeneration, and fluorescence microscopy using zinquin ethyl ester as the zinc ion-specific probe. Moreover, COX-2 expression was observed by Western blotting. In control retinas, there was a low Zn2+ concentration in the inner plexiform layer (IPL), a high Zn2+ concentration in the outer plexiform layer (OPL), and no detectable Zn2+ in either the ganglion cell layer (GCL) or the inner nuclear layer (INL). In contrast, in the retinas exposed to ischemia without the administration of the zinc ion chelators (Ca2+-EDTA and TPEN), Zn2+ deposits were found in the IPL and INL beginning 4 h after ischemia and degeneration of neurons was found in the GCL and INL. Less Zn2+ accumulation in the IPL and INL and less neuronal degeneration in the GCL and INL were found in the retinas treated with Ca2+-EDTA or TPEN before ischemia. Furthermore, the COX-2 protein levels increased 4-8 h after retinal ischemia, and chelation of zinc ion inhibited this effect. These results suggest that the accumulation of Zn2+ following an ischemic insult can cause retinal degeneration and induce abnormal COX-2 expression.

Oxygen-induced retinopathy in the rat. Vitamins C and E as potential therapies.

Oxygen-induced retinopathy (OIR) was produced by subjecting newborn albino rats to a 60% oxygen atmosphere for 14 days before they were killed and retinal analysis was done. The extent of OIR was measured by estimating the severity of retinal vasoobliteration in ink-perfused flat-mounted retinas. This was done with the aid of a digitizing camera and an image-analysis system designed to create binary images of the retinal blood vessels. Retinal levels of several antioxidant molecules also were measured. Alpha-tocopherol and ascorbic acid were reduced in oxygen-exposed rats by 34% and 20%, respectively, compared with room air-raised control animals. Retinal glutathione reductase, S-transferase, and peroxidase showed no differences between oxygen-treated and -untreated rats. Attempts to increase the newborn rats' retinal ascorbic acid by administering daily subcutaneous injections (5 g/kg body weight) to the mother rats were unsuccessful. However, the level of retinal alpha-tocopherol of newborn rats could be altered by dietary manipulation of the mothers. The mothers were fed diets containing either 1 g alpha-tocopherol acetate/kg food or none, starting 21-25 days before the birth of their litters and lasting throughout the exposure period. This treatment resulted in three- to fourfold differences in the retinal alpha-tocopherol levels of the pups. The combination of dietary and oxygen treatments also resulted in significant differences in retinal glutathione peroxidase activity, with the vitamin E-deprived, oxygen-exposed group having highest levels. Newborn rats both supplemented with and deprived of alpha-tocopherol had less vasoobliteration than did those nursed by mothers fed rat chow.

Attenuation of phosphoinositidase activity and phosphatidylinositol bisphosphate level of bovine retinal capillary pericytes in high glucose.

Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined. When glucose in growth medium was increased from 5- to 15- or 30 mM, PIase activity was attenuated to 82% or 55%, respectively. In contrast, when glucose (5-, 15-, 30 mM) was added to an enzyme extract from cells grown in the standard growth medium (5 mM glucose, 0.04 mM myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose. When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase. Phosphatidylinositol bisphosphate (PIP2). Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol. The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium. However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation. These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions. Further explanation of the role of the altered hydrolysis of PIP2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.