Use of Transcriptome Sequencing to Analyze the Effects of Different Doses of an Astragalus-Rhubarb-Saffron Mixture in Mice with Diabetic Kidney Disease.

Purpose: To investigate the therapeutic effect and underlying mechanism of a traditional Chinese medicine (TCM) mixture consisting of Astragalus, rhubarb, and saffron in a mouse model of diabetic kidney disease (DKD). Methods: Forty-eight db/db mice received no TCM (DKD model), low-dose TCM, medium-dose TCM, or high-dose TCM, and an additional 12 db/m mice received no TCM (normal control). Intragastric TCM or saline (controls) was administered daily for 24 weeks. Blood glucose, body weight, serum creatinine (SCr), blood urea nitrogen (BUN), blood lipids, and urinary microalbumin were measured every four weeks, and the urinary albumin excretion rate (UAER) was calculated. After 24 weeks, kidney tissues were collected for transcriptome sequencing, and the main functions of these genes were determined via functional enrichment analysis. Results: Compared with the DKD model group, the medium-dose and high-dose TCM groups had significantly decreased levels of SCr, BUN, total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and UAER (all p<0.05). We identified 42 genes that potentially functioned in this therapeutic response, and the greatest effect on gene expression was in the high-dose TCM group. We also performed functional enrichment analysis to explore the potential mechanisms of action of these different genes. Conclusion: A high-dose of the Astragalus-rhubarb-saffron TCM provided the best prevention of DKD. Analysis of the kidney transcriptome suggested that this TCM mixture may prevent DKD by altering immune responses and oxygen delivery by hemoglobin.

A novel method for simultaneously measuring boronophenylalanine uptake in brain tumor cells and number of cells using inductively coupled plasma atomic emission spectroscopy.

Boron neutron capture therapy (BNCT) combines neutron irradiation with boron compounds that are selectively uptaken by tumor cells. Boronophenylalanine (BPA) is a boron compound used to treat malignant brain tumors. The determination of boron concentration in cells is of great relevance to the field of BNCT. This study was designed to develop a novel method for simultaneously measuring the uptake of BPA by U87 and U251 cells (two brain tumor cell lines) and number of cells using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results revealed a linear correlation between phosphorus intensity and the numbers of U87 and U251 cells, with correlation coefficients (R2) of 0.9995 and 0.9994, respectively. High accuracy and reliability of phosphorus concentration standard curve were also found. Using this new method, we found that BPA had no significant effect on phosphorus concentration in either U87 or U251 cells. However, BPA increased the boron concentration in U87 and U251 cells in a concentration-dependent manner, with the boron concentration in U87 cells being higher than that in U251 cells. In both U87 and U251 cells, boron was mainly distributed in the cytoplasm and nucleus, accounting for 85% and 13% of the total boron uptake by U87 cells and 86% and 11% of the total boron uptake by U251 cells, respectively. In the U87 and U251 cell-derived xenograft (CDX) animal model, tumor exhibited higher boron concentration values than blood, heart, liver, lung, and brain, with a tumor/blood ratio of 2.87 for U87 cells and 3.11 for U251 cells, respectively. These results suggest that the phosphorus concentration in U87 and U251 cells can represent the number of cells and BPA is easily uptaken by tumor cells as well as in tumor tissue.

The yin-yang of immunity: Immune dysregulation in myelodysplastic syndrome with different risk stratification.

Myelodysplastic syndrome (MDS) is a heterogeneous group of myeloid clonal diseases with diverse clinical courses, and immune dysregulation plays an important role in the pathogenesis of MDS. However, immune dysregulation is complex and heterogeneous in the development of MDS. Lower-risk MDS (LR-MDS) is mainly characterized by immune hyperfunction and increased apoptosis, and the immunosuppressive therapy shows a good response. Instead, higher-risk MDS (HR-MDS) is characterized by immune suppression and immune escape, and the immune activation therapy may improve the survival of HR-MDS. Furthermore, the immune dysregulation of some MDS changes dynamically which is characterized by the coexistence and mutual transformation of immune hyperfunction and immune suppression. Taken together, the authors think that the immune dysregulation in MDS with different risk stratification can be summarized by an advanced philosophical thought "Yin-Yang theory" in ancient China, meaning that the opposing forces may actually be interdependent and interconvertible. Clarifying the mechanism of immune dysregulation in MDS with different risk stratification can provide the new basis for diagnosis and clinical treatment. This review focuses on the manifestations and roles of immune dysregulation in the different risk MDS, and summarizes the latest progress of immunotherapy in MDS.

Inhibition of miRNA-1-Mediated Inflammation and Autophagy by Astragaloside IV Improves Lipopolysaccharide-Induced Cardiac Dysfunction in Rats.

Introduction: Astragaloside IV (AS-IV) is one of the main active components isolated from the traditional Chinese medicinal herb, Astragalus membranaceus. The present study was designed to investigate whether the regulation of microRNA-1 (miR-1)-mediated inflammation and autophagy contributes to the protective effect of AS-IV against cardiac dysfunction in rats treated with lipopolysaccharides (LPS). Methods: Animal model of cardiac dysfunction in rats or cellular model of injured H9c2 heart cell line was established by using LPS. Echocardiography, electron microscopy, enzyme-linked immunosorbent assay, immunofluorescence, quantitative RT-PCR, and Western blotting were used to determine the cardiac function and expression of inflammation- and autophagy-related proteins at both the mRNA and protein levels. Results: LPS caused cardiac dysfunction in rats or injury in H9c2 cells and induced inflammation and autophagy. Compared with LPS treatment, AS-IV treatment attenuated cardiac dysfunction or cell injury, accompanied by inhibition of inflammation and autophagy. However, the miR-1 mimics partly abolished the effects of AS-IV. In addition, the effect of the miR-1 inhibitor was similar to that of AS-IV in the LPS model. Further analyses showed that AS-IV treatment decreased the mRNA expression of miR-1 in the heart tissue of rats and H9c2 cells treated with LPS. Conclusion: These results suggest that AS-IV attenuated cardiac dysfunction caused by LPS by inhibiting miR-1-mediated inflammation and autophagy, thereby providing a novel mechanism for the protection against cardiac diseases.

Gene-Edited Interleukin CAR-T Cells Therapy in the Treatment of Malignancies: Present and Future.

In recent years, chimeric antigen receptor T cells (CAR-T cells) have been faced with the problems of weak proliferation and poor persistence in the treatment of some malignancies. Researchers have been trying to perfect the function of CAR-T by genetically modifying its structure. In addition to the participation of T cell receptor (TCR) and costimulatory signals, immune cytokines also exert a decisive role in the activation and proliferation of T cells. Therefore, genetic engineering strategies were used to generate cytokines to enhance tumor killing function of CAR-T cells. When CAR-T cells are in contact with target tumor tissue, the proliferation ability and persistence of T cells can be improved by structurally or inductively releasing immunoregulatory molecules to the tumor region. There are a large number of CAR-T cells studies on gene-edited cytokines, and the most common cytokines involved are interleukins (IL-7, IL-12, IL-15, IL-18, IL-21, IL-23). Methods for the construction of gene-edited interleukin CAR-T cells include co-expression of single interleukin, two interleukin, interleukin combined with other cytokines, interleukin receptors, interleukin subunits, and fusion inverted cytokine receptors (ICR). Preclinical and clinical trials have yielded positive results, and many more are under way. By reading a large number of literatures, we summarized the functional characteristics of some members of the interleukin family related to tumor immunotherapy, and described the research status of gene-edited interleukin CAR-T cells in the treatment of malignant tumors. The objective is to explore the optimized strategy of gene edited interleukin-CAR-T cell function.

Retinoic acid modulates iron metabolism imbalance in anemia of inflammation induced by LPS via reversely regulating hepcidin and ferroportin expression.

The present study was designed to investigate the effect of retinoic acid (RA) on anemia of inflammation (AI) induced by lipopolysaccharide (LPS) and explore the potential mechanisms. BALB/c mice were randomly assigned into four groups: control group; LPS (10 mg/kg) group, LPS + RA (3 mg/kg) and LPS + RA (15 mg/kg) groups. Red blood cell count (RBC), hemoglobulin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin contentration (MCHC), erythropoietin (EPO) and iron content in both serum and liver tissue were measured. The AI model induced by LPS was successfully established represented by the decreases in RBC, Hb, HCT, MCV, MCHC and EPO for anemia indicators and by the increases in TNF-α, IL-18 and IL-1ß contents for inflammation indicators. However, supplementation of RA increased the levels of anemia indicators and decreased the content of inflammation indicators. In addition, RA increased the content of iron in serum, while decreased its content in liver tissue. Furthermore, RA down-regulated the protein expression of hepcidin, toll-like receptor 4 (TLR4) and p-p65 in liver tissue, while up-regulated that of ferroportin. RA modulates iron metabolism imbalance in AI induced by LPS via reversely regulating hepcidin and ferroportin expression, which might be mediated by TLT-4/NFκB signaling pathway.

Astragalus polysaccharide ameliorates H2O2-induced human umbilical vein endothelial cell injury.

Endothelial dysfunction caused by reactive oxygen species (ROS) has been implicated in numerous cardiovascular diseases. Astragalus polysaccharide (APS), an important bioactive component extracted from the Chinese herb Astragalus membranaceus, has been widely used for the treatment of cardiovascular disease. The present study aimed to investigate the effects of APS on hydrogen peroxide (H2O2)­induced human umbilical vein endothelial cell (HUVEC) injury. Following treatment with 400 µM H2O2 for 24 h, cell viability was decreased and apoptosis was increased. However, pretreatment with APS for 1 h significantly attenuated H2O2­induced injury in HUVECs. In addition, APS decreased intracellular ROS levels, increased the protein expression of endothelial nitric oxide synthase and copper­zinc superoxide dismutase, elevated intracellular cyclic guanosine monophosphate (an activity marker for nitric oxide) levels and restored the mitochondrial membrane potential, compared with cells treated with H2O2 only. In conclusion, the results of the present study suggested that APS may protect HUVECs from injury induced by H2O2 via increasing the cell antioxidant capacity and nitric oxide (NO) bioavailability, which may contribute to the improvement of the imbalance between ROS and NO levels.

Protective effects of Astragalus polysaccharides against endothelial dysfunction in hypertrophic rats induced by isoproterenol.

Astragalus polysaccharide (APS) is an important bioactive component extracted from Chinese herb Astragalus membranaceus. It has been widely used in treatment of cardiovascular diseases. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy. The present study was designed to evaluate the protective effect of APS on vascular endothelia in cardiac hypertrophy rats induced by isoproterenol (ISO). ISO (10mg×kg(-1)) was intraperitoneally injected once daily for 2weeks to induce cardiac hypertrophy. APS (400 and 800mg×kg(-1)) was intragastrically injected once daily along with ISO. The results showed that combination with APS significantly ameliorates the endothelial dysfunction while attenuates cardiac hypertrophy induced by ISO. We found that administration with APS could attenuate the increase in number of circulating endothelial cell (CEC). APS also decreases the superoxide anion generation and the protein expression of p65 and the levels of TNF-α and IL-6; while increases the cGMP levels, an activity marker for nitric oxide (NO) in aortas. In addition, APS improves the relaxation dysfunction in isolated aortic rings and increases the protein expression of IκBα and Cu/Zn-SOD in aortas. In conclusion, our results suggested that APS had a protective effect against endothelial dysfunction in hypertrophic rats induced by ISO. The underlining mechanisms may be contributed to the anti-inflammatory effects and the improvement of the imbalance between reactive oxygen species (ROS) and NO.

Astragaloside IV improves the isoproterenol-induced vascular dysfunction via attenuating eNOS uncoupling-mediated oxidative stress and inhibiting ROS-NF-κB pathways.

OBJECTIVE: Oxidative stress and inflammation are regarded as two important triggers of endothelial dysfunction and play pivotal role in progression of vascular damage associated with cardiac hypertrophy. Our previous studies demonstrated that astragaloside IV (AsIV) could protect against cardiac hypertrophy in rats induced by isoproterenol (Iso), but its effects on the aorta are not known. In present study, we aimed to assess the effects of AsIV on Isoinduced vascular dysfunction. METHODS: Sprague-Dawley (SD) rats were treated with Iso (10mg/kg/d) alone or in combination with AsIV (50mg/kg/d). RESULTS: Compared with Isotreated alone, AsIV significantly reduced the ratios of heart weight/body weight and left ventricular weight/body weight. AsIV ameliorated the increased vasoconstriction response to phenylephrine induced by Iso and suppressed superoxide anion generation in rat aorta, increased endothelial nitric oxide synthase (eNOS) dimer/monomer ratio and its critical cofactor tetrahydrobiopterin (BH4) content in aorta as well as the NO production in the serum, reduced the plasmatic peroxynitrite (ONOO-). Moreover, in contrast with Isotreatment alone, AsIV decreased the ratio of nuclear-to-cytosolic protein expression of the NF-κB p65 subunit while enhanced its inhibited protein expression of IκB-α, down-regulated mRNA expression of IL-1ß, IL-6 and TNF-α of the aorta. CONCLUSIONS: The present study suggested that AsIV protects against Isoinduced vascular dysfunction probably via attenuating eNOS uncoupling-mediated oxidative stress and inhibiting ROS-NF-κB pathways.

Calpain-1 Mediated Disorder of Pyrophosphate Metabolism Contributes to Vascular Calcification Induced by oxLDL.

We previously reported that oxidized low density lipoprotein (oxLDL) accelerated the calcification in aorta of rats and rat vascular smooth muscle cells (RVSMCs). However, the molecular mechanism underlying the acceleration remains poorly understood. The present study aimed to investigate the role of calpain-1, Ca2+-sensitive intracellular cysteine proteases, in the vascular calcification of rats treated with both high dose of vitamin D2 and high cholesterol diet. The results showed that calpain activity significantly increased in calcified aortic tissue of rats and RVSMCs treated with oxLDL. Specific calpain inhibitor I (CAI, 0.5mg/kg, intraperitoneal) inhibited the vascular calcification in rats with hypercholesterolemia accompanied by the increase in the level of extracellular inorganic pyrophosphate (PPi), the endogenous inhibitor of vascular calcification. In addition, CAI increased the content of adenosine triphosphate (ATP), decreased the activity, mRNA and protein expression of alkaline phosphatase (ALP) and reduced the production of superoxide anion in calcified aortic tissue. CAI also increased the activity of ATP synthase as well as protein expression of ATP5D, δ subunit of ATP synthase. In the in vitro study, suppression of calpain-1 using siRNA assay inhibited the calcium deposition, increased the levels of PPi and ATP, improved the activity of ATP synthase as well as protein expression of ATP5D in RVSMCs treated with oxLDL. Calpain-1 suppression also decreased the activity, mRNA and protein expression of ALP and reduced the mitochondrial ROS (Mito-ROS) production in RVSMCs. However, mito-TEMPO, the mitochondria-targeted ROS scavenger, reduced the calcium deposition, increased the PPi in culture medium, decreased the activity, mRNA and protein expression of ALP in RVSMCs treated with oxLDL. Taken together, the results suggested that calpain-1 activation plays critical role in vascular calcification caused by oxLDL, which might be mediated by PPi metabolism disorder. The results also implied that Mito-ROS might contribute to the PPi metabolism disorder through regulation of the activity and expression of ALP.

Astragalus polysaccharide attenuates isoproterenol-induced cardiac hypertrophy by regulating TNF-α/PGC-1α signaling mediated energy biosynthesis.

We previously reported that Astragalus polysaccharide (APS) extracted from Chinese medicine Astragalus membranaceus (Fisch.) Bge, attenuates hypertrophy of neonatal rat ventricular myocytes (NRVMs) induced by isoproterenol (Iso). The present study was designed to investigate the effects and the possible mechanism of APS on Iso-induced hypertrophy in rats and NRVMs with focus on tumor necrosis factor α (TNF-α)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) signaling mediated energy biosynthesis. 36-Week old rats were randomly divided into 3 groups: (1) Control, rats received vehicle; (2) Iso, rats received isoproterenol injections; (3) Iso+APS, rats received isoproterenol injections and APS. NRVMs were divided into similar groups as rats. The results showed that combination of APS with Iso significantly attenuated the pathological changes, reduced the ratios of heart weight/body weight (HW/BW) and left ventricular weight/BW (LVW/BW), improved the cardiac hemodynamics, down-regulated mRNA and protein expression of atrial natriuretic peptide (ANP), increased the ratios of ATP/ADP and ATP/AMP, and decreased the content of free fatty acid (FFA) in heart tissue of rats compared with Iso alone. In addition, pretreatment with APS significantly decreased the surface area and protein content, down-regulated mRNA and protein expression of ANP, increased the ratios of ATP/ADP and ATP/AMP, and decreased the content of FFA in NRVMs compared with Iso alone. Furthermore, APS increased the protein expressions of ATP5D, the σ subunit of ATP synthase, PGC-1α and pyruvate dehydrogenase kinase 4 (PDK4) in tissue and NRVMs respectively and inhibited the production of TNF-α in serum and culture medium compared with Iso alone. The results suggested that APS attenuates Iso-induced cardiac hypertrophy through regulating TNF-α/PGC-1α signaling mediated energy biosynthesis.

Astragaloside IV attenuates injury caused by myocardial ischemia/reperfusion in rats via regulation of toll-like receptor 4/nuclear factor-κB signaling pathway.

Myocardial ischemia/reperfusion (MI/R) injury, in which inflammatory response and cell apoptosis play a vital role, is frequently encountered in clinical practice. Astragaloside IV (AsIV), a small molecular saponin of Astragalus membranaceus, has been shown to confer protective effects against many cardiovascular diseases. The present study was aimed to investigate the antiinflammatory and antiapoptotic effects and the possible mechanism of AsIV on MI/R injury in rats. Rats were randomly divided into sham operation group, MI/R group and groups with combinations of MI/R and different doses of AsIV. The results showed that the expressions of myocardial toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) were significantly increased, and apoptosis of cardiomyocytes was induced in MI/R group compared with that in sham operation group. Administration of AsIV attenuated MI/R injury, downregulated the expressions of TLR4 and NF-κB and inhibited cell apoptosis as evidenced by decreased terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells, B-cell lymphoma-2 associated X protein and caspase-3 expressions and increased B-cell lymphoma-2 expression compared with that in MI/R group. In addition, AsIV treatment reduced levels of inflammatory cytokines induced by MI/R injury. In conclusion, our results demonstrated that AsIV downregulates TLR4/NF-κB signaling pathway and inhibits cell apoptosis, subsequently attenuating MI/R injury in rats.

Vitamin E conditionally inhibits atherosclerosis in ApoE knockout mice by anti-oxidation and regulation of vasculature gene expressions.

Lipid deposition in artery walls is implied in the pathogenesis of atherosclerosis and imbalance between uptake and efflux of cholesterol favors the deposition. We investigated the effect of vitamin E with the same dose and duration on the different stages of atherosclerosis in Apolipoprotein E knockout (ApoE KO) mice and explored the potential mechanisms. The results showed that the ApoE KO mouse spontaneously develops atherosclerosis in an age-dependent manner from 14 to 46 weeks on the regular chow. Vitamin E (100 mg/kg) supplementation to ApoE KO mice at 6, 14, and 22 weeks for 8 weeks significantly reduced the atherosclerotic lesion area by 41, 29 and 19% respectively compared to the age-matched control mice; however had no significant effect on the lesion when given at 30 and 38 weeks. In addition, vitamin E supplemented at the ages from 6 to 30 weeks decreased the contents of serum oxLDL and TBARS without affecting the TC and TAG contents in serum and liver. Furthermore, vitamin E supplemented at 6, 14 and 22 weeks down-regulated vasculature mRNA expressions of scavenger receptor CD36 and up-regulated mRNA expressions of PPARγ, LXRα and ABCA1 which are involved in reverse cholesterol transportation; however had no significant effects on these genes when given at 30 and 38 weeks. In conclusion, vitamin E with same dose and duration inhibits the early but not advanced atherosclerotic lesion in ApoE KO mice by anti-oxidation and regulation of mRNA expression of genes involved in cholesterol uptake and efflux, which favors the improvement of atherosclerosis.

Tanshinone II-A inhibits oxidized LDL-induced LOX-1 expression in macrophages by reducing intracellular superoxide radical generation and NF-κB activation.

Lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1), a novel scavenger receptor highly expressed in human and experimental atherosclerotic lesions, is responsible for the uptake of oxLDL in vascular cells. We demonstrated previously that Tanshinone II-A (Tan), a pharmacologically active compound extracted from the rhizome of the Chinese herb Salvia miltiorrhiza Bunge, inhibits atherogenesis in hypercholesterolemic rats, rabbits, and apolipoprotein-E deficient (ApoE⁻/⁻) mice. However, the precise mechanism by which Tan protects against atherogenesis remains to be elucidated. Therefore, we hypothesized that Tan can suppress the uptake of oxLDL by diminishing the expression of LOX-1 via suppression of NF-κB signaling pathway, thereby contributing to reduced macrophage foam cell formation. In cultured murine macrophages, oxLDL induced LOX-1 expression at the mRNA and protein levels, was abrogated by addition of Tan or pyrrolidinedithiocarbamic acid ammonium salt (PDTC), a widely used inhibitor of NF-κB, suggesting the involvement of NF-κB. Tan also reduced LOX-1 expression in atherosclerotic lesions of ApoE⁻/⁻ mice fed a high cholesterol diet. Mechanistically, Tan suppressed the nuclear translocation of NF-κB P65 subunit and phosphorylation of IκB-α induced by oxLDL. Electrophoretic mobility shift assay (EMSA) demonstrated that Tan inhibited the nuclear protein binding to NF-κB consensus sequence. Functionally, we observed that Tan inhibited DiI-oxLDL uptake by macrophages in a fashion similar to that produced by LOX-1 neutralizing antibody. Our current findings reveal a novel mechanism by which Tan protects against atherogenesis and shed new light on the potential therapeutic application of Tan to the treatment and prevention of atherosclerotic cardiovascular diseases.

Effects of Fufang Zhenzhu Tiaozhi Prescription (), A Chinese herbal preparation, on atherosclerosis in ApoE-/- mice and related mechanisms.

OBJECTIVE: The present study was designed to observe the effects and the possible mechanisms of Fufang Zhenzhu Tiaozhi Prescription (, FZT), a Chinese herbal preparation, on atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice. METHODS: ApoE-/- mice were randomized to groups orally administrated without or with FZT (4.5 and 9 g crude drug/kg body weight, respectively). Atherosclerotic plaques, lipids profifiles in serum, aortic cholesterol content, serum indices of oxidative stress were measured, and mRNA expressions of scavenger receptors CD36 and scavenger receptor-A (SR-A) in aorta were analyzed. RESULTS: FZT dose-dependently suppressed the atherosclerotic plaques and reduced the cholesterol contents in aorta (P<0.05, P<0.01). In addition, FZT decreased the levels of total cholesterol and triglyceride in serum (P<0.05, P<0.01), inhibited the production of oxidized low density lipoprotein and malonaldehyde, and increased the superoxide dismutase activity in serum (P<0.05 or P<0.01). Furthermore, FZT down-regulated the mRNA expressions of CD36 and SR-A in the aorta (P<0.05, P<0.01). CONCLUSIONS: FZT reduced the atherosclerotic formation in ApoE-/- mice. The mechanisms might be related to the decrease of serum lipid level, the improvement of oxidative stress and the down-regulated expressions of scavenger receptors CD36 and SR-A, consequently reducing the foam cell formation, the hallmark of early atherosclerosis.

Tanshinone II-A attenuates cardiac fibrosis and modulates collagen metabolism in rats with renovascular hypertension.

The adaptive changes that develop in the pressure-overloaded left ventricular myocardium include cardiac hypertrophy and interstitial fibrosis. The objectives of the present study were to evaluate the effects of Tanshinone II-A, a bioactive diterpene quinone isolated from Danshen, on cardiac fibrosis and collagen metabolism in rats with renovascular hypertension. Male Sprague-Dawley rats were subjected to two-kidney two-clip (2K2C) or sham operation (sham) and treated with Valsartan (Val, 26.7 mg/kg/d), Tanshinone II-A (Tsn, 70, 35 mg/kg/d) or vehicle. Six weeks later, systolic blood pressure (BP), LV weight, collagen abundance, cardiac function parameters, hydroxyproline content and mRNA levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were evaluated. Both high-dose (Tsn-H, 70 mg/kg/d) and low-dose (Tsn-L, 35 mg/kg/d) of Tsn failed to attenuate 2K2C-induced BP elevation but significantly attenuated the attendant interstitial fibrosis. Val suppressed elevations of BP and left ventricular systolic pressure (LVSP) in 2K2C rats. Val and Tsn-H exerted comparable suppressive effects on the gene expression of MMP-9 and TIMP-1, while Val decreased the MMP-2 mRNA level without affecting the transcript levels of TIMP-2. Both Val and Tsn-H attenuated cardiac dysfunction, while Tsn-L showed slight improvement. These data demonstrate for the first time, that Tsn prevented cardiac fibrosis and improved cardiac function in a rat model of renovascular hypertensive independent of hypotensive effect. Tsn conferred its beneficial effects on the collagen metabolism probably through its regulation of transcript levels of the MMPs/TIMPs balance.

Suppression of vascular cell adhesion molecule-1 expression by crocetin contributes to attenuation of atherosclerosis in hypercholesterolemic rabbits.

To elucidate the molecular mechanism by which antioxidants alleviate atherosclerosis, we investigated the effect of crocetin, a naturally occurred carotinoid with potent antioxidant power, on vascular cell adhesion molecule-1 (VCAM-1) expression in atherosclerotic rabbits. Twenty-four male New Zealand White rabbits were allocated to three groups fed on standard diet (control group), high lipid diet (HLD group) or high lipid diet supplemented with crocetin (crocetin group), respectively. After 8 weeks of treatment, rabbits in HLD group developed severe hypercholesterolemia and atherosclerosis in aortas, together with a significantly up-regulated expression of both protein and mRNA for VCAM-1. In contrast, supplementation with crocetin resulted in markedly ameliorated atherosclerosis, coupled with a significantly decreased VCAM-1 expression, though plasma lipids level remained comparable to that of HLD group. Regression analysis revealed a positive correlation between VCAM-1 expression and the extent of atherosclerosis (P < 0.01). In addition, immunohistochemical analysis showed an increased activation of nuclear factor kappa B (NF-kappaB), a redox sensitive transcription factor essential for VCAM-1 expression, in aortas from rabbits fed on high lipid diet, which was evidently suppressed by crocetin supplementation. These findings suggest that the antiatherosclerotic effect of crocetin might be attributed, at least in part, to the suppressed expression of VCAM-1, which might result from reduced NF-kappaB activation. This study provides a further insight into the molecular mechanism by which antioxidants attenuate atherosclerosis and suggests a potential target for the treatment of atherosclerosis with antioxidants.

Effect of crocin on intracellular calcium concentration in cultured bovine aortic smooth muscle cells.

AIM: To study the effect of crocin on intracellular calcium concentration ([Ca2+]i) in cultured bovine aortic smooth muscle cells (BASMCs). METHODS: Cells were loaded with fluorescence probe Fluo-3/AM and [Ca2+]i was measured by laser scanning confocal microscope (LSCM). RESULTS: In the presence or absence of extracellular Ca2+, crocin (1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol x L(-1)) concentration-dependently inhibited the [Ca2+]i elevation induced by 1 x 10(-2) mol x L(-1) H2O2 (for the former, the inhibition rates were 34.1%, 57.1% and 74.3%, while for the latter were 26.2%, 32.1%, 50.0%). In the absence of extracellular Ca2+, crocin (1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol x L(-1)) could inhibit the [Ca2+]i elevation induced by 70 mmol x L(-1) CHCl3, the inhibition rates were 27.8%, 27.8% and 50.0% respectively. CONCLUSION: Crocin could inhibit the extracellular Ca2+ influx and release of intracellular Ca2+ stores in endoplasmic reticulum.