The Natural Alkaloid (-)-N-Hydroxyapiosporamide Suppresses Colorectal Tumor Progression as an NF-κB Pathway Inhibitor by Targeting the TAK1-TRAF6 Complex.

Colorectal cancer (CRC) is an exceptionally deadly disease, whereas effective therapeutic drugs for CRC have declined over the past few decades. Natural products have become a reliable source of anticancer drugs. Previously we isolated an alkaloid named (-)-N-hydroxyapiosporamide (NHAP), which exerts potent antitumor effects, but its effect and mechanism in CRC remain unclear. This study aimed to reveal the antitumor target of NHAP and identify NHAP as a promising lead compound for CRC. Various biochemical methods and animal models were used to investigate the antitumor effect and molecular mechanism for NHAP. These results showed that NHAP exhibited potent cytotoxicity, induced both apoptosis and autophagic cell death of CRC cells, and inhibited the NF-κB signaling pathway by blocking the interaction of the TAK1-TRAF6 complex. NHAP also markedly inhibited CRC tumor growth in vivo without obvious toxicities and possessed good pharmacokinetic characteristics. These findings identify, for the first time, that NHAP is an NF-κB inhibitor with potent antitumor activity in vitro and in vivo. This study clarifies the antitumor target of NHAP against CRC, which will contribute to the future development of NHAP as a novel therapeutic lead compound for CRC.

[Real-Time Monitoring Method for Pedicle Screw Fixation Surgery Based on Near-Infrared Spectrum and Computer Tomography Images].

Spine is one of the most important organs in the human body. One of the most commonly used method for the treatment of spinal diseases is the internal fixation and accurate placement of pedicle screw, which is a key factor of spinal surgery. However, due to the large differences as to the appearance of pedicles, it is hard to place the pedicle screw precisely, which will cause complication. Therefore, to find a new real-time intra-operative monitoring method with navigation is the direction of clinical application research. In this paper, a new method was firstly proposed. This method combined computer tomography (CT) values and near-infrared spectroscopy (NIRs) measurement data to guide the PS placement, and the relationship between NIRs parameters and CT values along the PS trajectory in vertebrae was investigated. First, we took pig vertebrae as samples and different puncture paths were planned. Second, a near-infrared monitoring device was utilized in experiments of fresh pig vertebrae to acquire the best NIRs monitoring pattern factors. Finally, the correlation function between NIRs data and CT values pattern factors was obtained. The results showed that CT values have a linear relationship with NIRs monitoring pattern factors, which provide references for real-time monitoring method in pedicle screw fixation surgery. This model can be applied in monitoring the pedicle screw implantation and alarming. The proposed method will be potential in improving the accuracy of PS placement and reduce the risk caused by the misplacement of pedicle screw.

[Effects of crocetin on VCAM-1 expression in human umbilical vein endothelial cells and monocyte-endothelial cell adhesion].

Crocetin, a naturally occurring carotenoid, possesses antioxidant and antiatherosclerotic properties, of which the underlying mechanism remains unclear. In the present study, we examined the effects of crocetin (0.1, 1, 10 µmol·L(-1)) on angiotensin II (Ang II, 0.1 µmol·L(-1)) induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and monocyte-endothelial cell adhesion. The effects of crocetin on the activation of nuclear factor kappa B (NF-κB) and intracellular reactive oxygen species (ROS) were also observed. The results demonstrated that crocetin notably suppressed Ang II induced NF-κB activation (P<0.01) and VCAM-1 expression (P<0.05, P<0.01) in HUVECs, accompanied by a markedly reduced monocyte-endothelial cell adhesion (P<0.05, P<0.01). In addition, preincubation with crocetin resulted in a significant enhancement of cellular antioxidant capacity (P<0.05, P<0.01), while Ang II induced intracellular ROS decreased markedly (P<0.05, P<0.01). These results indicated that crocetin was capable of suppressing Ang II induced VCAM-1 expression and monocyte-endothelial cell adhesion by suppression of NF-κB activation, which might be derived from the enhancement of antioxidant capacity and subsequent reduction of intracellular ROS.

[Applications and progress of Fourier transform infrared spectroscopic microimaging in bone disease research].

Fourier transform infrared spectroscopy (FTIRS) and microimaging technique have been integrated together to evolve into Fourier transform infrared spectroscopic imaging (FTIRI) system. This system can provide not only the morphological information of the sample by visible image and FTIR image, but also the abundant information on the spectral, component and structure of specimen by FTIRS, especially of the heterogeneous solid mixture. The richer and more visualized information obtained by FTIRI greatly raised the research efficiency and usability of the spectral technique in biomedicine, pharmacology, forensic medicine, material science and chemistry, etc. The present paper depicts FTIRI development process, system structure, imaging principle and mode selection; and then introduces that FTIRI opened a new area of investigation for biomedicine, namely, research on bone disease by FTIRI. Then the paper illustrates the related research findings and progress in FTIRI use for osteopetrosis, osteogenesis imperfecta, osteoporosis and osteomalacia, as well as a couple of limitations. The prospective study for FTIRI in biomedical research field is also addressed.

[Preliminary study of wireless biofeedback therapy for treatment of bruxism].

OBJECTIVE: To access the effect of wireless biofeedback therapy on bruxism. METHODS: Ten voluntary bruxers (seven female and three male, mean age 26.1 years) were invited to participate in this clinical research. An electric resistance strain gauge was embedded in the position of canine of a maxillary splint for monitoring the abnormal clenching or grinding movement of teeth during sleep. The relevant details of bruxism events, including value of relative force, occurring time and duration were recorded and analyzed by the receiver device and monitoring program respectively. Meanwhile, for the purpose of nerve system and muscle relaxation, a watch-style device around the patient's wrist will vibrate to alert the patient of teeth grinding or clenching if the value of biting force and duration exceed the threshold. Total average episodes of bruxism and duration was observed during eight hours sleep, and was analyzed with one-way analysis of variance in SPSS 19.0 by the end of 6th week and three months following biofeedback therapy. RESULTS: The average episodes of bruxism has declined dramatically from (9.8 ± 2.2) times to (3.0 ± 1.2) times during one night (P < 0.05), and the average duration of bruxism events was reduced from (20.7 ± 12.2) s to (10.0 ± 3.4) s (P < 0.05) after six weeks biofeedback therapy. By the end of three months, the average episodes declined to (2.9 ± 1.2) times (P < 0.05), and the average duration decline to (9.2 ± 2.9) s (P < 0.05) with contrast to preliminary night. CONCLUSIONS: The pressure-based wireless biofeedback device is able to monitoring clenching and grinding of bruxism. The results suggest that biofeedback therapy may be an effective, novel and convenient measure for treatment of bruxism according to several months therapy.

[Study on brain tissue characteristics of rat model of Parkinson's disease based on functionality near-infrared spectroscopy (fNIRs) technology].

Functionality near infrared spectroscopy (fNIRs) technology was utilized in the present paper to explore functional properties of brain tissue of rat model of Parkinson's disease(PD). Imaging data of rat model were detected by small animal MRI and CT; and characteristic parameters of striatum of rat brain were detected by fNIRs system. Experimental results show that, between PD and normal rat, there is no obvious change in morphological structure, but significant differences existed in reduced scattering coefficient (mu's) and cerebral blood volume (CBV) of rat striatum; there exists correlation between parameters (mu's, CB3V) obtained by fNIRs and parameters (cerebral blood flow (CBF), CBV) obtained by CT perfusion (CTP). These results indicate that fNIRs can be used as important reference for PD research.

Protein kinase C pathway is involved in the inhibition by crocetin of vascular smooth muscle cells proliferation.

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study showed that crocetin inhibits angiotensin II (Ang II)-induced proliferation of vascular smooth muscle cells (VSMCs). The present study investigated the involvement of the protein kinase C (PKC) pathway in the growth-inhibitory action of crocetin in VSMCs. The findings showed that PKC activity in the membrane fraction of VSMCs increased following stimulation with Ang II, which was suppressed significantly by pretreating the cells with crocetin. Inhibition of PKC activity by crocetin appeared to be associated with growth inhibition in VSMCs, because chelerythrine chloride, a specific PKC inhibitor, likewise decreased cell proliferation. PKC-a, a conventional PKC isoform, was detected in bovine aorta VSMCs by RT-PCR and western blotting analysis. Crocetin inhibited Ang II-induced membrane translocation of PKC-a, and the inhibition of crocetin on PKC activity in membrane fraction coincided with its suppression on membrane translocation of PKC-a. In addition, Ang II-induced mRNA expressions of c-fos, c-jun and c-myc were also decreased by crocetin. Taken together, the data suggest that the inhibition by crocetin of PKC activity, at least in part due to inactivation of PKC-a, and the subsequent suppression of proto-oncogene expressions might mediate its inhibitory effect on VSMCs proliferation.

Crocetin inhibits cell cycle G1/S transition through suppressing cyclin D1 and elevating p27kip1 in vascular smooth muscle cells.

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study shows that crocetin inhibits angiotensin II (Ang II)-induced vascular smooth muscle cells (VSMCs) proliferation. To further explore the mechanism by which crocetin inhibits VSMCs proliferation, in the present study we examined the effect of crocetin on cell cycle progression and cell cycle regulatory proteins. Flow cytometry analysis showed that Ang II elicited significant increase in the percentage of VSMCs in the S phase, with a concomitant decline in the percentage of VSMCs in the G(0)/G(1) phase. However, on pretreatment of VSMCs with crocetin, the percentage of VSMCs in the S phase decreased, while that in the G(0)/G(1) phase increased significantly. In addition, Ang II-induced increase of cell proliferation index was also decreased by crocetin. Western blotting analysis indicated that crocetin markedly inhibited the protein expression of cyclin D1 but not cyclin E. Crocetin also increased the level of cyclin-dependent kinase inhibitor (CDKI) p27(kip1) but not CDKI p21(waf1/cip1). In conclusion, our present results suggest that the inhibition of cell cycle G(1)/S transition in VSMCs by crocetin can be attributed, at least in part, to its suppression of cyclin D1 and elevation of CDKI p27(kip1).

[Research on traumatic brain real-time monitoring by near-infrared technology].

In order to monitoring the development of traumatic brain edema in vivo, a specifically designed optical parameters of tissue testing system with a mini-invasion bifurcated optical fiber probe and a fiber spectrometer was used to monitor the reduced scattering coefficient (mu's) of t he rat traumatic brain while the counterpart parameter, i. e. brain water content (BWC), was also measured. Acute rat regional brain trauma was applied according to Feeney's apparatus. The changes of brain edema were monitored by near-infrared spectroscopy (NIRS) technology and by measuring the water content of the brain. Experiment result showed that distinct brain edema in injured areas was found at 6 hours later after trauma, which reached a summit of severity at 24-72 hours later after trauma, then gradually declined. After using the dehydrant, the brain edema situation became better, and then, the edema occurred again whilet he medicamentosus effect of dehydrant was gradually lost. It can be showed that mu's had similar change profile with BWC and the two parameters were well linearly relative to each other. mu's is a good indicator for monitoring traumatic brain edema and t he medicamentosus effect of dehydrant. As a result near-infrared spectroscopy is a new feasible method of monitoring the development of traumatic brain edema in vivo.

Crocetin attenuates norepinephrine-induced cytotoxicity in primary cultured rat cardiac myocytes by antioxidant in vitro.

This study aims to investigate the protective role of crocetin, a natural antioxidant, against cytotoxicity produced by exposure to norepinephrine (NE) in primary cultured rat cardiac myocytes. Reactive oxygen species (ROS) and Ca(2+) in cells were evaluated by fluorescence microplate reader using 6-carboxy-2',7'-dichlorofluorescein and fluoro-3-acetoxymethyl ester, respectively. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances. The activities of superoxide dismutase (SOD) and contents of glutathione (GSH) were detected by xanthine/xanthime oxidase-mediated ferricytochrome c reduction assay, and recycling effection of glutathione disulfide with GSH reductase and NADPH, respectively. The apoptotic cells were assayed by fluorescein diacetate (FDA)-ethidium bromide (EB) two-staining method. Intracellular accumulation of ROS, Ca(2+), and products of lipid peroxidation resulting from NE were significantly reduced by crocetin. Preincubation of primary cultured rat cardiac myocytes with crocetin remarkably prevented the decrease in SOD activity and quantities of GSH induced by NE. The percentage of NE-induced apoptosis in the cells was decreased by FDA-EB two-staining assay after pretreated with crocetin. The results showed that crocetin may ameliorate NE-induced injury in cardiac myocytes by enhanced SOD activity and increased quantities of GSH, decreased lipid peroxidation and Ca(2+) in cells, and apoptosis death ratio that may represent the cellular mechanisms for its cardioprotective role.

[Study on assessment of effects of mannitol on treatment of TBE based on measurement of mini-invasive optical parameter (micro'(s))].

Near infrared spectroscopy (NIRs) technology was utilized for assessing effects in treating traumatic brain edema (TBE). Firstly, models for rats with traumatic brain edema were copied according to Feeney's apparatus. Then rats were given mannitol with different dosages (large and little) according to their groups. Simultaneously rat's brain tissues were monitored in vivo and real-time by NIRs mini-invasive detector developed by the authors' laboratory. And the water content of the brain tissues was measured by the wet and dry weight method at 1, 6, 24, 72 and 120 h after the injury and the treatment. Then, effects in treating TBE with different dosages were assessed by analyzing reduced scattering coefficient (micro'(s)) data measured by NIRs and brain water content (BWC) before and after injecting dehydration. Finally, the authors found that reduced scattering coefficient (micro'(s)) of rat's local cortex is a good indicator of assessing effects of treatment of TBE and that may be a preferable approach to assessing effects in vivo and real-time in treatment of brain injury.

[Estimation of tissue's blood oxygen parameters from visible absorption spectrum of tissues by artificial neural network].

Total hemoglobin concentration (THC) and hemoglobin oxygen saturation (SO2) are essential parameters to doctors who wonder patients' hematogenous conditions and oxygen supplies and consumptions. Instruments presently used for measuring these parameters have big size of detecting probes that limit their applications to inner bodies. An optical probe involving two fibers with source-detector separations of one hundred micrometers was developed in the present study for purpose of minimally invasive inner detecting, which uses steady-state, broadband (300-1 000 nm) light source. The source light is delivered to targets through one fiber and the reflected light from the targets is collected and transferred to a spectrometer through the other fiber. Reflectance spectrum is obtained from the spectrometer. The method of reading THC and SO2 from the reflectance spectrum was developed using liquid-tissue phantoms containing intralipid and blood. Firstly, reflex spectrum of intralipid was recorded before mixtures of intralipid and blood with different THC were made as tissue phantoms. Then the fiber optical spectrometer was used to obtain reflex spectra as the phantoms' SO2 changed; simultaneously their corresponding THC and SO2 were recorded as the scale values by an oximeter. Differences of reflex spectra in 520-590 nm between intralipid and tissue models were proved reliably. Secondly, after data collections of absorption spectra and scale values were finished, two artificial neural networks (ANN) were build to model the relationship between scale values and absorption spectra. After being trained, the ANNs could output THC and SO2 correctly when an absorption spectrum was input. The ANNs produced errors of less than 4 micromol x L(-1) for THC and 5% for SO2. In vivo and minimally invasive measurements of THC and SO2 of brain tissues in different depth were finished on 30 rats by this specific system with the ANNs. The probe was inserted stereotactically to a depth of 6 mm with measurements obtained every 0.2 mm. SO2 of gray mater and white mater of rats was respectively obtained as 0.60-0.70 and 0.45-0.55. The highest THC, 110 micromol x L(-1) was measured around rat cortex. THC of brain tissue in other depth is 70-90 micromol x L(-1). These values agree well with reported data. This simple, inexpensive method deserves further study to establish its efficacy for THC and SO2 measurements of inner body.

[Effects of triptolide on the production of interferon-gamma in human peripheral blood mononuclear cell and phosphorylation of signal transducer and activator of transcription-1 and production of interleukin-8].

OBJECTIVE: To investigate the effects of triptolide on the production of interferon-gamma (IFN-gamma) in human peripheral blood mononuclear cell (PBMC) and interleukin-8 (IL-8) in HaCaT keratinocytes and phosphorylation of signal transducer and activator of transcription-1 (STAT1) of IFN-gamma signal transduction pathways in HaCaT cells. METHODS: Human PBMC was induced by phytohaemagglutinin (PHA-L) and HaCaT cells were stimulated by recombinant human IFN-gamma (rhIFN-gamma). The productions of IFN-gamma and IL-8 in cells were detected by ELISA. The expression of STAT1 and its phosphorylation were analyzed by Western blot. RESULTS: Triptolide inhibited the production of IFN-gamma in human PBMC induced by PHA-L in a dose-dependent manner (P < 0.05, P < 0.01, P < 0.001) and the 50% inhibitory concentration (IC50) value was 5.96 x 10(-11) mol/L. IL-8 production in HaCaT cells induced by rhIFN-gamma in vitro was also inhibited by triptolide (P < 0.001) and the IC50 value was about 1.15 x 10(-13) mol/L. The expressions of phosphorylated STAT1 in HaCaT cells stimulated by rhIFN-gamma was inhibited by triptolide (P < 0.01) and the IC50 value was about 9.45 x 10(-11) mol/L. CONCLUSION: Triptolide can inhibit the production of IFN-gamma in human PBMC and downregulate IL-8 level in HaCaT keratinocytes induced by rhIFN-gamma. Triptolide can inhibit the phosphorylations of STAT1 of IFN-gamma signal pathway in HaCaT keratinocytes stimulated by IFN-gamma.

Influence of Crocetin on experimental atherosclerosis in hyperlipidamic-diet quails.

Antioxidants have been expected to have potential as antiatherogenic agents. Crocetin is a natural carotenoid antioxidant isolated from Gardenia jasminoids Ellis. Therefore, in the present study, we investigated the inhibitory effect of Crocetin on experimental atherosclerosis in quails. The atherosclerosis model was established by feeding hyperlipidamic diet to quail and Crocetin (25, 50, 100 mg/kg/day) was administered by oral gavage. At the 9th week, serum lipids, malondialdehyde and nitric oxide were measured, and Hematoxylin-Eosin (H&E) stains was used to investigate the histopathological changes of aorta. Results showed that Crocetin could reduce the levels of serum total cholesterol, triglyceride, low density lipoprotein cholesterol and inhibit the formation of aortic plaque. Crocetin could also reduce malondialdehyde and inhibit the descending of nitric oxide in serum. The results suggested that Crocetin could inhibit the formation of atherosclerosis in quails, which might be related to the hypolipidemic effects along with the antioxidative properties of Crocetin.

Involvement of Ca2+ in the inhibition by crocetin of angiotensin II-induced ERK1/2 activation in vascular smooth muscle cells.

Crocetin, a carotenoid compound, was isolated from Gardenia jasminoids Ellis. Our recent study shows that crocetin inhibits angiotensin II-induced extracellular signal-regulated kinases 1/2 (ERK1/2) activation and subsequent proliferation in vascular smooth muscle cells (VSMCs). To further explore the mechanism involved, in the present study, we investigated the effect of Ca(2+) in the activation of ERK1/2 and whether Ca(2+) is involved in the suppression by crocetin of angiotensin II-induced ERK1/2 activation. Our findings showed that crocetin pretreatment partially attenuated both the intracellular Ca(2+) mobilization and the extracellular Ca(2+) influx induced by angiotensin II. Moreover, angiotensin II-induced ERK1/2 activation was completely abolished by acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator, and partially inhibited by EGTA, an extracellular Ca(2+) chelator, or verapamil, an L-type Ca(2+) channel blocker. These findings suggest that Ca(2+) may play an important role in angiotensin II-induced ERK1/2 activation in VSMCs, and Ca(2+)-dependent pathway may be involved in the inhibitory effect by crocetin of angiotensin II-induced ERK1/2 activation.

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 H(2)O(2) (200 microM) 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 microM. Furthermore, in the H(2)O(2)-treated group, agarose gel electrophoresis displayed a "DNA ladder". Whereas in the 10 microM 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 muM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H(2)O(2), which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).

Effects of crocetin on antioxidant enzymatic activities in cardiac hypertrophy induced by norepinephrine in rats.

Crocetin, a carotenoid isolated from the Chinese herbal medicine Crocus sativus L. (Saffron), has been shown to have cardiovascular protective effects. The present study investigated the protective action of the antioxidant crocetin against cardiac hypertrophy induced by norepinephrine (NE). This was evaluated by assaying for pathological histological changes with an optical microscope and cell image analysis system. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances (TBARS). Myocardial superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and myocardial catalase (CAT) activities were assayed to evaluate the antioxidant capacity. After long term treatment with NE, antioxidant enzymatic activities were significantly decreased, while products of lipid peroxidation increased. Crocetin markedly reduced the content of lipid peroxidation (LPO), increased the GSH-Px and SOD activity in cardiac hypertrophy, and significantly improved the myocardial pathological histological changes induced by NE. These results suggest that the cardioprotective effects of crocetin are related to modulation of endogenous antioxidant enzymatic activities. Comparing crocetin with captopril, our results indicated that antioxidant activity is an important factor in the therapy of cardiac hypertrophy, but as an antioxidant only, its effects may be limited.

ERK1/2 pathway is involved in the inhibitory effect of crocetin on angiotensin II-induced vascular smooth muscle cell proliferation.

Angiotensin II (Ang II) induces vascular smooth muscle cells (VSMCs) proliferation, which plays an important role in the development and progression of atherosclerosis. Ang II-induced cellular events have been implicated, in part, in the activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. In the present study, we investigated the effect of crocetin on the Ang II-induced VSMCs proliferation and ERK1/2 activation. 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and [3H]thymidine incorporation assay showed that the Ang II-induced VSMCs proliferation was inhibited significantly by crocetin. In-gel kinase assay indicated that Ang II elicited rapid and significant increase of ERK1/2 activity in VSMCs, which was suppressed by crocetin markedly. Western blotting analysis and cell-based enzyme-linked immunosorbent assay (ELISA) demonstrated that crocetin significantly inhibited the phosphorylation and activation of ERK1/2 induced by Ang II. Using the indirect immunofluorescent technique, we also found that crocetin inhibited nuclear translocation of activated ERK1/2 induced by Ang II. These findings suggest that the suppression by crocetin of the Ang II-induced VSMCs proliferation can be attributed, at least in part, to its inhibitory effect on ERK1/2 pathway.

Crocetin inhibits leukocyte adherence to vascular endothelial cells induced by AGEs.

Advance glycation end products (AGEs) have been postulated to play an important role in diabetic complications such as atherosclerosis disease. Adhesion and migration of leukocyte to endothelial cells (EC) is one of the early key steps in the pathogenesis. Crocetin is an important ingredient of diet in India and also used in various systems of indigenous medicine. In this study, we investigated effect of crocetin on leukocyte adherence to bovine endothelial cells (BEC) induced by AGEs in vitro and the possible mechanisms involved. BEC were pre-incubated with crocetin (0.01, 0.1, and 1 microM) for 12 h and exposed to AGEs (100 microg/ml). Cells proliferation was determined by MTT; leukocyte-endothelial cell adhesion was assayed by myeloperoxidase methods; intercellular adhesion molecular-1 (ICAM-1) protein expression was studied by immunocytochemistry and mitochondrial membrane potential (MMP) was analyzed by the retention of rhodamine 123 (RH123); furthermore, levels of anion (O(2)(-)), malonicdialdehyde (MDA) in super cells culture and superoxide dismutase (SOD) in cells were also detected, respectively. Results demonstrated that crocetin could inhibit AGE-induced BEC growth suppression and significantly reduce adhesion rate of leukocyte to BEC (P < 0.01 or P < 0.05); ICAM-1 protein was also suppressed (P < 0.05). Furthermore, crocetin could increase activity of SOD (P < 0.05), decrease levels of MDA and O(2)(-) (P < 0.01). In addition, down-regulated MMP was also increased by crocetin (P < 0.01 or P < 0.05). These data revealed crocetin could prevent the adhesion of leukocyte to BEC and down-regulation the expression of ICAM-1, and the possible mechanisms might be related to its antioxidant activity, which is through up-regulation of the activity of antioxidant enzymes and protection for mitochondrion.

Effect of crocin on experimental atherosclerosis in quails and its mechanisms.

In the present study, we examined the prophylaxis effect of crocin on experimental atherosclerosis and its possible mechanisms. The atherosclerosis formation was induced by hyperlipidamic diet in quails. At the 9th week, serum lipid, MDA and NO were measured, and HE staining was used to investigate the histopathological changes of aorta. Bovine aortic endothelial cells (EC) were obtained from the thoracic aorta of newborn calves. After incubation of the cells with Ox-LDL (50 mg x L(-1)) for 24 h, the activities of LDH, NO in culture media and activity of NOS in endothelial cells were measured, flow cytometer was used to determine the rate of endothelial cells apoptosis. Peritoneal macrophages were obtained from thioglycolate-injected mice. Cholesterol and free cholesterol in cells were assayed after incubation of the cells with Ox-LDL. Bovine aortic smooth muscle cells (SMC) were obtained from the thoracic aorta of newborn calf. Proliferation was induced by 100 microg x L(-1) Ox-LDL and antiproliferative effect of crocin on SMCs were observed. SMCs cycle phases were measured by flow cytometry. SMCs were loaded with Fluo-3/AM and [Ca2+]i was measured by Laser Scanning Confocal Microscope (LSCM). Crocin could reduce the level of serum TC, TG, LDL-C and inhibit the formation of aortic plaque. Crocin could reduce MDA and inhibit the descending of NO in serum. Compared with control, Ox-LDL group could increase the activity of LDH and decrease activity of NO in culture media and activity of NOS in endothelial cells, preincubated with crocin, the effects of Ox-LDL were inhibited. Crocin could decrease the EC apoptosis induced by Ox-LDL. Crocin concentration-dependently inhibited the TC and CE elevation induced by Ox-LDL in macrophages. Crocin could inhibit the proliferation of SMCs induced by Ox-LDL. In the presence or absence of extracellular Ca2+, crocin concentration-dependently inhibited the [Ca2+]i elevation induced by 120 mg x L(-1)Ox-LDL, In the absence of extracellular Ca2+, crocin could inhibit the [Ca2+]i elevation induced by CHCl3 in a concentration-dependent manner. The results indicated that crocin could inhibit the formation of atherosclerosis in quails. Crocin had protective effects on endothelial cells. Crocin could decrease CE in macrophages and uptake of Ox-LDL, inhibiting the formation of foam cell, which would promote the initiation and progression of atherosclerosis. Crocin could inhibit the [Ca2+]i elevation in smooth muscle cell, Ca2+ is an important second messenger that regulates a variety of cellular processes, including smooth muscle cell proliferation and gene expression . Crocin exerted antiatherosclerotic effects through decreasing the level of Ox-LDL that plays an important role in the initiation and progression of atherosclerosis.