Chemo-preventive effect of crocin against experimentally-induced hepatocarcinogenesis via regulation of apoptotic and Nrf2 signaling pathways

The results of the current study investigated the chemo-preventive effect of crocin against hepatocarcinogenesis in rats with particular focus on the evaluation of the modulatory impact of crocin on apoptotic and nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathways. Thioacetamide (TAA) (200 mg/kg, I.P.) was used for experimental induction of hepatocarcinogenesis in rats. Crocin administration significantly attenuated TAA-induced cancerous lesions with concomitant attenuation of impaired liver functions. This was associated with significant enhancement in hepatic Nrf2 and heme oxygenase-1 (HO-1) expression with parallel suppression in Keap-1 expression. Inline, crocin induced a significant improvement in hepatic oxidative status with enhanced antioxidant batteries. Crocin administration significantly suppressed the hepatic content of c-Jun N-terminal kinase (c-JNK) with significant upregulation in TNF-related apoptosis-inducing ligand (TRAIL) and caspase-8 protein expression as well as p53 gene expression; biomarkers of apoptosis. Moreover, hepatic expression of the apoptotic BAX significantly increased and the anti-apoptotic Bcl-2 significantly decreased in the liver specimen; biomarkers of intrinsic apoptosis. In conclusion; crocin attenuates experimentally induced hepato-carcinogenesis via modulation of oxidative/apoptotic signaling. Namely, crocin induced hepatic expression of Nrf2 with downstream modulation of endogenous HO-1 and Keap-1 signaling with modulation of various key players of apoptosis including; c-JNK, p53, TRAIL, caspase-8, BAX, and Bcl-2.     

Proteomic screening of molecular targets of crocin

Background

Traditional drug discovery approaches are mainly relied on the observed phenotypic changes following administration of a plant extract, drug candidate or natural product. Recently, target-based approaches are becoming more popular. The present study aimed to identify the cellular targets of crocin, the bioactive dietary carotenoid present in saffron, using an affinity-based method.

Methods

Heart, kidney and brain tissues of BALB/c mice were homogenized and extracted for the experiments. Target deconvolution was carried out by first passing cell lysate through an affinity column prepared by covalently attaching crocin to agarose beads. Isolated proteins were separated on a 2D gel, trypsinized in situ and identified by MALDI-TOF/TOF mass spectrometry. MASCOT search engine was used to analyze Mass Data.

Results

Part of proteome that physically interacts with crocin was found to consist of beta-actin-like protein 2, cytochrome b-c1 complex subunit 1, ATP synthase subunit beta, tubulin beta-3 chain, tubulin beta-6 chain, 14-3-3 protein beta/alpha, V-type proton ATPase catalytic subunitA, 60 kDa heat shock protein, creatine kinase b-type, peroxiredoxin-2, cytochrome b-c1 complex subunit 2, acetyl-coA acetyltransferase, cytochrome c1, proteasome subunit alpha type-6 and proteasome subunit alpha type-4.

Conclusion

The present findings revealed that crocin physically binds to a wide range of cellular proteins such as structural proteins, membrane transporters, and enzymes involved in ATP and redox homeostasis and signal transduction.     

A comparative study of the role of crocin and sitagliptin in attenuation of STZ-induced diabetes mellitus and the associated inflammatory and apoptotic changes in pancreatic β-islets

Type 1 diabetes mellitus (T1DM) describes a complex group of metabolic disorders associated with elevated blood glucose levels and increased risks of complications development. Exploring new drug therapies would reduce the increased diabetes-associated morbidity and mortality and will reduce the excessive health care costs. Crocin is the major active ingredient of saffron. In the current study, DM was induced by single intraperitoneal injection of streptozocin (50 mg/kg).DM progression was associated with a significant increase in blood glucose level with reduced insulin and increased glucagon secretion. Pancreatic malondialdehyde (MDA) content significantly escalated, while superoxide dismutase (SOD) activity, reduced glutathione (GSH) concentration, catalase activity, thioredoxin level and serum total antioxidant capacity significantly declined. This was associated with a significant increase in pancreatic caspase-3 contents and pancreatic infiltration with inflammatory cells in β-islets. Both sitagliptin and crocin significantly reduced blood glucose levels, enhanced pancreatic insulin expression and secretion and suppressed glucagon secretion with enhancement of anti-oxidant defenses and reduction of oxidative burden, with evident anti-inflammatory impacts. Interestingly, the effect of crocin on DM indices, inflammatory and apoptotic changes was comparable to that of sitagliptin; the standard oral hypoglycemic agent. Nevertheless, crocin had a superior effect compared to sitagliptin on blood sugar level, β-islets diameter and insulin immune-reactivity. In conclusion, crocin reduced blood glucose level mainly via reduction of oxidative burden, modulation of apoptotic pathway and attenuation of pancreatic inflammation.     

Evidence of neuroprotective effects of saffron and crocin in a Drosophila model of parkinsonism

Evidence suggests that saffron and its major bioactives exhibit significant neuromodulatory effects in various animal models. However, specific data related to their efficacy to attenuate oxidative stress and neurotoxicity in animal models of Parkinson’s disease (PD) are limited. Hence, we investigated the neuroprotective efficacy of saffron methanolic extract (SME) and its active constituent, crocin (CR) employing a Drosophila model of parkinsonism. We focussed on attenuation of Rotenone (ROT)-induced locomotor phenotype, oxidative stress, mitochondrial dysfunction and neurotoxicity in this model. SME and CR-enrichment significantly reduced ROT (500 μM) induced mortality, rescued the locomotor phenotype and diminished the enhanced levels of oxidative stress markers in head/body regions of flies. The reduced levels of reduced glutathione (GSH) and total thiols (TSH) resulting from ROT exposure were significantly restored with concomitant enhancement of the antioxidant enzymes activities. Further, ROT-induced mitochondrial dysfunctions (MTT reduction, activities of SDH and NADH-Cyt C reductase (complexes I–III) enzymes) were markedly attenuated by SME/CR enrichment. While ROT elevated the activity of acetylcholinesterase (AChE) in head/body regions, both the treatments caused marked diminution of AChE activity and restored the dopamine levels suggesting their effectiveness to mitigate cholinergic function. Interestingly, SME/CR enrichment significantly delayed the onset of locomotor deficits and extended life span of flies among ROT (50 μM)-stressed flies. In a satellite study, flies provided with SME/CR prophylaxis exhibited marked resistance to an acute Paraquat (PQ) challenge as evidenced by the lower incidence of lethality and improved locomotor phenotype. Taken together, the neuroprotective effects of saffron and crocin in the fly model may be largely attributable to its antioxidant action. Based on our findings, we propose that saffron may be exploited as a supplementary therapeutic agent in PD and other oxidative stress mediated neurodegenerative conditions.     

Crocin inhibits RANKL-induced osteoclast formation and bone resorption by suppressing NF-κB signaling pathway activation

Crocin is a dietary compound with antioxidant and anti-inflammatory properties, but its effects on bone resorption have not been well characterized. Here we address this issue by examining the direct effects of crocin on osteoclast cells in vitro. Osteoclastogenesis was induced by RANKL (receptor activator of NF-κB ligand) in mouse bone marrow-derived macrophages in the absence or presence of crocin at various concentrations. Further, the bone resorption activity of mature osteoclast treated with crocin was assessed by pit assay. Without altering cell viability, crocin was shown to inhibit the differentiation and function of osteoclast cells in a dose-dependent manner. Mechanistically, RANKL-induced NF-κB and NFATc1 activation, the critical signaling pathways for osteoclast differentiation and function, were both repressed by crocin in bone marrow-derived macrophages. Thus, crocin suppresses osteoclastogenesis through direct inhibition of intracellular molecular pathways, which may contribute to future development of anti-bone resorption treatment.     

提高坐珠达西质量标准的研究

目的 提高坐珠达西品种的质量标准。方法 采用薄层色谱法对熊胆、牛黄、木香、丁香等主要药味进行了定性鉴别以及马钱子的液相鉴别,以高效液相色谱法对其主药西红花中西红花苷I进行含量测定。结果 定性鉴别分离度好,专属性强;西红花苷I在0.031 62～0.284 58 μg范围内呈良好的线形关系,r=0.999 7(n=7),平均回收率为 97.7%,RSD 为 1.55%(n=6)。结论 本法可靠,准确,专属性强,可有效控制坐珠达西的质量。     

藏红花素诱导人脑胶质瘤细胞U87凋亡的作用机制研究

目的探讨藏红花素促进人脑胶质瘤细胞U87凋亡的作用及其机制。方法培养U87细胞系,随机分为二甲基亚砜对照组（对照组）和藏红花素治疗组（藏红花素组）。采用四甲基偶氮唑盐（MTT）法检测不同剂量藏红花素（低浓度组、中浓度组、高浓度组）对人胶质瘤细胞U87增殖的影响;流式细胞仪观察细胞凋亡情况。Western—blot检测ERK1／2,p-ERK1／2的表达变化。结果与对照组比较,藏红花素组U87细胞的生存率明显降低;随着藏红花素浓度增加,U87细胞的凋亡率显著上升（18．92％→51．68％→70．12％）,组间差异有统计学意义（x2=14．102,P〈0．05）;Western—blot结果显示,与对照组比较,藏红花素组的p-ERK1／2表达降低,而高浓度藏红花素组p-ERK1／2表达降低最显著,不同组间差异有统计学意义（P〈0．05）。结论藏红花素可能呈剂量依赖性的通过抑制。p-ERK1/2但讲TIR7缃晌桶广学垤雷尊的精肿瘤作用     

Protective effect of crocin on diazinon induced vascular toxicity in subchronic exposure in rat aorta ex-vivo

Abstract

Context: Diazinon (DZN) is a widely used organophosphate insecticide. Although mechanism of DZN cardiovascular toxicity is primarily mediated through inhibition of acetylcholinesterase, however, DZN causes remarkable atropine-insensitive hypotension in rats. It has been proved that oxidative stress is an important mechanism of DZN toxicity especially in chronic exposure. Crocin, an active ingredient of saffron, has been found to antagonize the hypotensive effects of DZN in rats, but do not reverse acetylcholinesterase inhibition. Objective: In this study the effects of DZN on contractile and relaxant responses in rat aorta as well as ex-vivo antioxidant actions of crocin have been investigated. Materials and methods: Rats were divided into 7 groups: corn oil (control), DZN (15?mg/kg/day, gavage), crocin (12.5, 25 and 50?mg/kg/day, i.p.) plus DZN, vitamin E (200?IU/kg, i.p., three days a week) plus DZN and crocin (50?mg/kg/day, i.p.) groups. Treatments were continued for 4 weeks. Contractile and relaxant responses were evaluated on the isolated aorta. Results: Our results showed that DZN not only decreased the contractile responses to KCl and Phenylephrine (PE) (p?<?0.001), but also attenuated the relaxant response to acetylcholine (ACh) (p?<?0.01). Crocin and vitamin E attenuated lipid peroxidation, improved the reduction of contractile responses by KCl and PE and restored the decrease in ACh relaxation in rat aorta. Conclusion: DZN induced vascular toxicity which may be due to oxidative stress and not to a cholinergic mechanism. Crocin improved toxic effects of DZN via reducing lipid peroxidation and restoring altered contractile and relaxant responses in rat aorta.     

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H₂O₂ (200 μM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 μM. Furthermore, in the H₂O₂-treated group, agarose gel electrophoresis displayed a “DNA ladder”. Whereas in the 10 μM crocin-pretreated group, cells remained intact and no “DNA ladder” was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 μM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H₂O₂, which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).     

Antiproliferative effects of crocin in HepG2 cells by telomerase inhibition and hTERT down-regulation

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin (IC50 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 μg protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the Tm telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.