Evidence of impaired mitochondrial cellular bioenergetics in ocular fibroblasts derived from glaucoma patients.

Glaucoma is a progressive optic neuropathy characterized by the neurodegeneration of the retinal ganglion cells (RGCs) resulting in irreversible visual impairment and eventual blindness. RGCs are extremely susceptible to mitochondrial compromise due to their marked bioenergetic requirements and morphology. There is increasing interest in therapies targeting mitochondrial health as a method of preventing visual loss in managing glaucoma. The bioenergetic profile of Tenon's ocular fibroblasts from glaucoma patients and controls was investigated using the Seahorse XF24 analyser. Impaired mitochondrial cellular bioenergetics was detected in glaucomatous ocular fibroblasts including basal respiration, maximal respiration and spare capacity. Spare respiratory capacity levels reflect mitochondrial bio-energetic adaptability in response to pathophysiological stress. Basal oxidative stress was elevated in glaucomatous Tenon's ocular fibroblasts and hydrogen peroxide (H2O2) induced reactive oxygen species (ROS) simulated the glaucomatous condition in normal Tenon's ocular fibroblasts. This work supports the role of therapeutic interventions to target oxidative stress or provide mitochondrial energetic support in glaucoma.

Transcriptomics and Network Pharmacology Reveal the Protective Effect of Chaiqin Chengqi Decoction on Obesity-Related Alcohol-Induced Acute Pancreatitis via Oxidative Stress and PI3K/Akt Signaling Pathway.

Obesity-related acute pancreatitis (AP) is characterized by increasing prevalence worldwide and worse clinical outcomes compared to AP of other etiologies. Chaiqin chengqi decoction (CQCQD), a Chinese herbal formula, has long been used for the clinical management of AP but its therapeutic actions and the underlying mechanisms have not been fully elucidated. This study has investigated the pharmacological mechanisms of CQCQD in a novel mouse model of obesity-related alcohol-induced AP (OA-AP). The mouse OA-AP model was induced by a high-fat diet for 12 weeks and subsequently two intraperitoneal injections of ethanol, CQCQD was administered 2 h after the first injection of ethanol. The severity of OA-AP was assessed and correlated with changes in transcriptomic profiles and network pharmacology in the pancreatic and adipose tissues, and further docking analysis modeled the interactions between compounds of CQCQD and their key targets. The results showed that CQCQD significantly reduced pancreatic necrosis, alleviated systemic inflammation, and decreased the parameters associated with multi-organ dysfunction. Transcriptomics and network pharmacology analysis, as well as further experimental validation, have shown that CQCQD induced Nrf2/HO-1 antioxidant protein response and decreased Akt phosphorylation in the pancreatic and adipose tissues. In vitro, CQCQD protected freshly isolated pancreatic acinar cells from H2O2-elicited oxidative stress and necrotic cell death. The docking results of AKT1 and the active compounds related to AKT1 in CQCQD showed high binding affinity. In conclusion, CQCQD ameliorates the severity of OA-AP by activating of the antioxidant protein response and down-regulating of the PI3K/Akt signaling pathway in the pancreas and visceral adipose tissue.

Aqueous extraction from dachengqi formula granules reduces the severity of mouse acute pancreatitis via inhibition of pancreatic pro-inflammatory signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Dachengqi decoction (DCQD) belongs to a family of purgative herbal formulas widely used in China for the treatment of acute pancreatitis (AP). AP is a prevalent digestive disease currently without an effective pharmacological intervention. Formula granules have become the preferred method for delivery of herbal formulation in China given its benefit of potency retention, dosing precision and ease of use. The efficacy of DCQD formula granules (DFGs) in experimental AP models has not been investigated. AIM OF THE STUDY: To analyse and compare the differences in chemical composition of DFGs, with their aqueous extraction (AE) and chloroform extraction (CE) derivatives. To assess their efficacy on severity and targeted pancreatic pro-inflammatory signalling pathways in freshly isolated acinar cells and two models of experimental AP. MATERIAL AND METHODS: UPLC-Q-TOF-MS was used to analyse chemical components of DFGs and their extractions. Freshly isolated mouse pancreatic acinar cells were treated with taurolithocholic acid 3-sulphate disodium salt (TLCS, 500 µM) with or without DFGs, AE and CE. Apoptotic and necrotic cell death pathway activation was measured by caspase 3/7 (10 µl/mL) and propidium iodide (PI, 1 µM), respectively, using a fluorescent plate reader. Necrotic acinar cells were also counted by epifluorescence microscopy. Mice received either 7 intraperitoneal injections of caerulein (50 µg/kg) at hourly intervals or retrograde infusion of TLCS (3 mM, 50 µl) to induce AP (CER-AP and TLCS-AP, respectively). In CER-AP, mice received oral gavage of DFGs (2.1, 4.2 and 5.2 g/kg), AE (0.6, 1.2, and 2.4 g/kg) and CE (4, 9 and 17 mg/kg), or matched DFGs (1.8 g/kg) and AE (1 g/kg) for 3 times at 2-hourly intervals, or a single intraperitoneal injection of DCQD-related monomers rhein (20 mg/kg), narigeinine (25 mg/kg), and honokiol (5 mg/kg) begun at the 3rd injection of caerulein. In TLCS-AP, DFGs (4.2 g/kg) were given orally at 1, 3 and 5 h post-surgery. Disease severity and pancreatic pro-inflammatory markers were determined. RESULTS: The main effective anthraquinones and their glycosides, flavonoids and their glycosides, polyphenols and lignans were found in the DFGs. A higher proportion of polar components including glycosides attached to anthraquinones, phenols and flavonoids was found in AE. Conversely, lower polar components containing methoxy substituted flavonoids and anthraquinones were more abundant in CE. DFGs were given at 4.2 g/kg, a consistent reduction in the pancreatic histopathology score and severity indices was observed in both CER-AP and TLCS-AP. In vitro, AE significantly reduced both apoptotic and necrotic cell death pathway activation, while CE increased TLCS-induced acinar cell necrosis. In vivo, AE at dose of 1.2 g/kg consistently reduced pancreatic histopathological scores and myeloperoxidase in the CER-AP that were associated with suppressed expression of pro-inflammatory meditator mRNAs and proteins. CE increased lung myeloperoxidase and failed to protect against CER-AP in all dosages. AE was demonstrated to be more effective than DFGs in reducing pancreatic histopathological scores and myeloperoxidase. CONCLUSIONS: AE from DFGs alleviated the severity of mouse AP models via an inhibition of pancreatic pro-inflammatory signalling pathways. Efficacy of AE on experimental AP was more potent than its original DFGs and DCQD monomers.

Mitochondrial Targeting of Antioxidants Alters Pancreatic Acinar Cell Bioenergetics and Determines Cell Fate.

Mitochondrial dysfunction is a core feature of acute pancreatitis, a severe disease in which oxidative stress is elevated. Mitochondrial targeting of antioxidants is a potential therapeutic strategy for this and other diseases, although thus far mixed results have been reported. We investigated the effects of mitochondrial targeting with the antioxidant MitoQ on pancreatic acinar cell bioenergetics, adenosine triphosphate (ATP) production and cell fate, in comparison with the non-antioxidant control decyltriphenylphosphonium bromide (DecylTPP) and general antioxidant N-acetylcysteine (NAC). MitoQ (µM range) and NAC (mM range) caused sustained elevations of basal respiration and the inhibition of spare respiratory capacity, which was attributable to an antioxidant action since these effects were minimal with DecylTPP. Although MitoQ but not DecylTPP decreased cellular NADH levels, mitochondrial ATP turnover capacity and cellular ATP concentrations were markedly reduced by both MitoQ and DecylTPP, indicating a non-specific effect of mitochondrial targeting. All three compounds were associated with a compensatory elevation of glycolysis and concentration-dependent increases in acinar cell apoptosis and necrosis. These data suggest that reactive oxygen species (ROS) contribute a significant negative feedback control of basal cellular metabolism. Mitochondrial targeting using positively charged molecules that insert into the inner mitochondrial member appears to be deleterious in pancreatic acinar cells, as does an antioxidant strategy for the treatment of acute pancreatitis.

Protective effects of flavonoids from Coreopsis tinctoria Nutt. on experimental acute pancreatitis via Nrf-2/ARE-mediated antioxidant pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative stress is a prominent feature of clinical acute pancreatitis (AP). Coreopsis tinctoria has been used traditionally to treat pancreas disorders like diabetes mellitus in China and Portugal and its flavonoid-rich fraction contain the main phytochemicals that have antioxidant and anti-inflammatory activities. AIM OF THE STUDY: To investigate the effects of flavonoids isolated from C. tinctoria on experimental AP and explore the potential mechanism. MATERIALS AND METHODS: LC-MS based online technique was used to analyse and isolate targeted flavonoids from C. tinctoria. Freshly isolated mouse pancreatic acinar cells were treated with taurocholic acid sodium salt hydrate (NaT, 5 mM) with or without flavonoids. Fluorescence microscopy and a plate reader were used to determine necrotic cell death pathway activation (propidium iodide), reactive oxygen species (ROS) production (H2-DCFDA) and ATP depletion (luminescence) where appropriate. AP was induced by 7 repeated intraperitoneal caerulein injections (50 µg/kg) at hourly interval in mice or retrograde infusion of taurolithocholic acid 3-sulfate disodium salt (TLCS; 5 mM, 50 µL) into the pancreatic duct in mice or infusion of NaT (3.5%, 1 mL/kg) in rats. A flavonoid was intraperitoneally administered at 0, 4, and 8 h after the first caerulein injection or post-operation. Disease severity, oxidative stress and antioxidant markers were determined. RESULTS: Total flavonoids extract and flavonoids 1-6 (C1-C6) exhibited different capacities in reducing necrotic cell death pathway activation with 0.5 mM C1, (2 R,3 R)-taxifolin 7-O-ß-D-glucopyranoside, having the best effect. C1 also significantly reduced NaT-induced ROS production and ATP depletion. C1 at 12.5 mg/kg and 8.7 mg/kg (equivalent to 12.5 mg/kg for mice) significantly reduced histopathological, biochemical and immunological parameters in the caerulein-, TLCS- and NaT-induced AP models, respectively. C1 administration increased pancreatic nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-medicated haeme oxygenase-1 expression and elevated pancreatic antioxidant enzymes superoxide dismutase and glutathione peroxidase levels. CONCLUSIONS: Flavonoid C1 from C. tinctoria was protective in experimental AP and this effect may at least in part be attributed to its antioxidant effects by activation of Nrf2-mediated pathways. These results suggest the potential utilisation of C. tinctoria to treat AP.

Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP.

OBJECTIVE: Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. DESIGN: We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. RESULTS: MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. CONCLUSIONS: This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.

Effects of anethole and structural analogues on the contractility of rat isolated aorta: Involvement of voltage-dependent Ca2+-channels.

Anethole is a naturally occurring aromatic oxidant, present in a variety of medicinal plant extracts, which is commonly used by the food and beverage industry. Despite its widespread occurrence and commercial use, there is currently little information regarding effects of this compound on the vasculature. Therefore the actions of anethole on the contractility of rat isolated aorta were compared with those of eugenol, and their respective isomeric forms, estragole and isoeugenol. In aortic rings precontracted with phenylephrine (PE; 1 microM), anethole (10(-6) M-10(-4) M) induced contraction in preparations possessing an intact endothelium, but not in endothelium-denuded tissues. At higher concentrations (10(-3) M-10(-2) M), anethole-induced concentration-dependent and complete relaxation of all precontracted preparations, irrespective of whether the endothelium was intact or not, an action shared by eugenol, estragole and isoeugenol. The contractile and relaxant effects of anethole in PE-precontracted preparations were not altered by L-NAME (10 microM) or indomethacin (10 microM), indicating that neither nitric oxide nor prostaglandins were involved in these actions. The mixed profile of effects was not confined to PE-mediated contraction, since similar responses were obtained to anethole when tissues were precontracted with 25 mM KCl. Anethole and estragole (10(-6)-10(-4) M), but not eugenol or isoeugenol, increased the basal tonus of endothelium-denuded aortic rings, an action that was abolished by VDCC blockers nifedipine (1 microM) and diltiazem (1 microM), or by withdrawal of extracellular Ca(2+). Our data suggest complex effects of anethole on isolated blood vessels, inducing contraction at lower doses, mediated via opening of voltage-dependent Ca(2+)-channels, and relaxant effects at higher concentrations that are shared by structural analogues.

Antinociceptive activity of Calotropis procera latex in mice.

This work evaluated the antinociceptive effect of proteins from the Calotropis procera (Asclepiadaceae) latex using three different experimental models of nociception in mice. The latex protein fraction administered intraperitoneally in male mice at the doses of 12.5, 25 and 50 mg/kg showed the antinociceptive effect in a dose dependent manner compared to the respective controls in all assays. Inhibitions of the acetic acid-induced abdominal constrictions were observed at the doses of 12.5 (67.9%), 25 (85%) and 50 (99.5%) mg/kg compared to controls. Latex protein at the doses of 25 (39.8%; 42%) and 50 mg/kg (66.6%; 99.3%) reduced the nociception produced by formalin in the 1st and 2nd phases, respectively, and this effect was not reversed by pretreatment with naloxone (1 mg/kg). In the hot plate test, an increase of the reaction time was observed only at 60 min after the treatment with latex at the doses of 25 (79.5%) and 50 (76.9%) mg/kg, compared to controls and naloxone was ineffective to reverse the effect. It was concluded that the protein fraction derived from the whole latex of Calotropis procera possesses antinociceptive activity, which is independent of the opioid system.

Red marine alga Bryothamnion triquetrum lectin induces endothelium-dependent relaxation of the rat aorta via release of nitric oxide.

We have investigated the vascular relaxant effects of the lectin from a red marine alga Bryothamnion triquetrum (BTL), in particular, the endothelial-dependency and the participation of a specific glycoprotein-binding site. BTL (1-100 microg mL(-1)) was applied to rat isolated aortic rings, with or without endothelium, tonically precontracted with phenylephrine (0.1 microM). Endothelium-dependent relaxation was assessed in the presence of indometacin (10 microM), L-nitro arginine methyl ester (L-NAME, 100 microM) and tetraethylammonium (TEA, 500 microM). For the involvement of the glycoprotein-binding site, BTL was assayed in presence of mucin (300 microg mL(-1)) or N-acetyl D-glucosamine (GlcNAc; 300 microg mL(-1)), a specific and non-specific lectin-binding sugar, respectively. BTL fully and concentration dependently relaxed preparations that possessed an intact endothelium (IC50 (concn producing 50% contraction) = 12.1 +/- 1.6 microg mL(-1)), whereas no significant relaxation was observed in endothelial-denuded tissue. L-NAME, but not indometacin or TEA, completely inhibited the lectin relaxation, suggesting the involvement of nitric oxide (NO). The lectin in association with mucin, but not with GlcNAc, inhibited BTL-induced relaxation, implicating the involvement of the lectin binding site. Our data suggest that the relaxant effect of the red marine alga Bryothamnion triquetrumlectin on isolated aorta occurs via interaction with a specific lectin-binding site on the endothelium, resulting in a release of NO.

Relaxant effects of the essential oil of Ocimum gratissimum on isolated ileum of the guinea pig.

The effects of the essential oil of Ocimum gratissimum L. (Labiatae) (EOOG) on guinea pig ileum were studied. EOOG (0.1-1000 microg/ml) reversibly and concentration-dependently relaxed the basal tone of the ileum and reversed the tonic contractions induced by 60 mM KCl and 10 microM acetylcholine, with IC(50) values of 23.8+/-5.2, 18.6+/-4.0 and 70.0+/-4.6 microg/ml, respectively. Our results show that EOOG exerts relaxant effects on intestinal smooth muscle, consistent with the popular use of the plant to treat gastrointestinal disorders.