Dali Luo

Low Molecular Weight Fucoidan Alleviates Cardiac Dysfunction in Diabetic Goto-Kakizaki Rats by Reducing Oxidative Stress and Cardiomyocyte Apoptosis

Diabetic cardiomyopathy (DCM) is characterized by cardiac dysfunction and cardiomyocyte apoptosis. Oxidative stress is suggested to be the major contributor to the development of DCM. This study was intended to evaluate the protective effect of low molecular weight fucoidan (LMWF) against cardiac dysfunction in diabetic rats. Type 2 diabetic goto-kakizaki rats were untreated or treated with LMWF (50 and 100 mg/kg/day) for three months. The establishment of DCM model and the effects of LMWF on cardiac function were evaluated by echocardiography and isolated heart perfusion. Ventricle staining with H-E or Sirius Red was performed to investigate the structural changes in myocardium. Functional evaluation demonstrated that LMWF has a beneficial effect on DCM by enhancing myocardial contractility and mitigating cardiac fibrosis. Additionally, LMWF exerted significant inhibitory effects on the reactive oxygen species production and myocyte apoptosis in diabetic hearts. The depressed activity of superoxide dismutase in diabetic heart was also improved by intervention with LMWF. Moreover, LMWF robustly inhibited the enhanced expression of protein kinase C β, an important contributor to oxidative stress, in diabetic heart and high glucose-treated cardiomyocytes. In conclusion, LMWF possesses a protective effect against DCM through ameliorations of PKCβ-mediated oxidative stress and subsequent cardiomyocyte apoptosis in diabetes.

Potential regulatory role of calsequestrin in platelet Ca 2+ homeostasis and its association with platelet hyperactivity in diabetes mellitus

Summary.  Background: Altered Ca2+ homeostasis contributes significantly to platelet hyperactivity in diabetes mellitus. Calsequestrin (CSQ), as a Ca2+ buffer protein in the sarcoplasmic reticulum, also regulates the Ca2+ release process in muscles. We hypothesized that CSQ may be expressed in platelets, but is altered and involved in diabetic platelet Ca2+ abnormalities and hyperaggregability. Methods: CSQ expression in platelets from streptozotocin‐induced type 1 diabetes rats, type 2 diabetes volunteers and Goto‐Kakizaki rats were analyzed by western blotting and RT‐qPCR. Platelet Ca2+ and aggregation were evaluated with Fura2 and an aggregometer, respectively. Results: Platelets from diabetic patients and rats exhibited increased resting Ca2+ levels, and hyperactive Ca2+ and aggregation responses to agonists. This enhanced basal Ca2+ was largely dependent on intracellular Ca2+ and insensitive to inositol 1,4,5‐trisphosphate receptor (IP3R) antagonism. Additionally, the expression of the skeletal CSQ isotype (CSQ‐1) was detected in both rat and human platelets, but its levels were significantly lowered in diabetic platelets as compared with normal platelets. Impairment of CSQ by trifluoperazine caused concentration‐dependent Ca2+ release in normal platelets and HEK293 cells. Knocking down CSQ‐1 in HEK293 cells resulted in increased leakage of Ca2+, which was also insensitive to IP3R inhibition, and exaggerated Ca2+ release following carbachol treatment. Conclusions: Downregulation of CSQ‐1 in diabetic platelets and impairment of CSQ‐1 in normal cells leads to disturbed Ca2+ release, demonstrating a potential role for CSQ‐1 in the regulation of the platelet Ca2+ release process and a possible causal contribution to diabetic platelet hyperactivity.

α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes

AbstractAim:It is unclear why α1D-adrenergic receptors (α1D-ARs) play a critical role in the mediation of peripheral vascular resistance and blood pressure in situ but function inefficiently when studied in vitro. The present study examined the causes for these inconsistencies in native α1-adrenergic functional performance between the vascular smooth muscle and myocytes.Methods:The α1-adrenergic mediated contraction, Ca2+ signaling and the subcellular receptor distribution were evaluated using the Fluo-4, BODIPY-FL prazosin and subtype-specific antibodies.Results:Rat aortic rings and freshly dissociated myocytes displayed contractile and increased intracellular Ca2+ responses to stimulation with phenylephrine (PE, 10 μmol), respectively. However, the PE-induced responses disappeared completely in cultured aortic myocytes, whereas PE-enhanced Ca2+ transients were seen in cultured rat cardiac myocytes. Further studies indicated that α1D-ARs, the major receptor subtype responsible for the α1-adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the α1A-AR subcellular localization in the cultured cardiomyocytes. In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for α1D-ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of α1D-ARs at the cell surface and a partial restoration of the Ca2+ signal response to PE in approximately 30% of the cultured cells.Conclusion:Reduction in α1D-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to α1-adrenergic receptor activation.

Low molecular weight fucoidan ameliorates streptozotocin-induced hyper-responsiveness of aortic smooth muscles in type 1 diabetes rats☆

ETHNOPHARMACOLOGICAL RELEVANCE Low molecular weight fucoidan (LMWF) was prepared from Laminaria japonica Areschoug, a popular seafood and medicinal plant consumed in Asia. Chinese have long been using it as a traditional medicine for curing hypertension and edema. AIM OF THE STUDY This study was intent to investigate the possible beneficial effect of LMWF on hyper-responsiveness of aortic smooth muscles instreptozotocin (STZ)-induced type 1 diabetic rats. MATERIALS AND METHODS Sprague-Dawley rats were made diabetic by injection of STZ, followed by the administration of LMWF (50 or 100mg/kg/day) or probucol (100mg/kg/day) for 12 weeks. Body weight, blood glucose level, basal blood pressure, serum lipid profiles, oxidative stress, prostanoids production, and vasoconstriction response of endothelium-denuded aorta rings to phenylephrine were measured by Real time-PCR, Western blots, ELISA assay, and force myograph, respectively. RESULTS LMWF (100mg/kg/day)-treated group showed robust improvements on STZ-induced body weight-loss, hypertension, and hyperlipidaemia as indicated by decreased serum level of total cholesterol, triglyceride, and low density lipoprotein cholesterol; while probucol, a lipid-modifying drug with antioxidant properties, displayed mild effects. In addition, LMWF appreciably ameliorated STZ-elicited hyper-responsiveness and oxidative stress in aortic smooth muscles as indicated by decreased superoxide level, increased glutathione content and higher superoxide dismutase activity. Furthermore, administration with LMWF dramatically prevented cyclooxygenase-2 stimulation and restored the up-regulation of thromboxane synthase and down-regulation of 6-keto-PGF1α (a stable metabolic product of prostaglandin I2) in the STZ-administered rats. CONCLUSION This study demonstrates for the first time that LMWF can protect against hyperlipidaemia, hypertension, and hyper-responsiveness of aortic smooth muscles in type 1 diabetic rat via, at least in part, amelioration of oxidative stress and restoration of prostanoids levels in aortic smooth muscles. Therefore, LMWF can be a potential adjuvant treatment against cardiovascular complications in type 1 diabetes.

Altered platelet calsequestrin abundance, Na+/Ca2 + exchange and Ca2 + signaling responses with the progression of diabetes mellitus

INTRODUCTION Downregulation of calsequestrin (CSQ), a major Ca(2+) storage protein, may contribute significantly to the hyperactivity of internal Ca(2+) ([Ca(2+)]i) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca(2+) signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca(2+) influx in diabetic platelets. MATERIALS AND METHODS Type 1 diabetes was induced by streptozotocin in rats. Platelet [Ca(2+)]i and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting. RESULTS During the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca(2+)]i and ADP-induced Ca(2+) release were progressively altered in diabetic platelets, while the elevated Ca(2+) influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca(2+) channel blocker, almost completely abolished ADP-induced Ca(2+) influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na(+)/Ca(2+) exchange induced much slower Ca(2+) extrusion and more Ca(2+) influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca(2+)-ATPase inhibition, ionomycin caused greater Ca(2+) mobilization and Ca(2+) influx in diabetic platelets than in normal platelets. CONCLUSIONS These data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca(2+) homeostasis, insufficient Na(+)/Ca(2+) exchange also contributes, at least in part, to the hyperactive Ca(2+) response to stimulation in diabetic platelets.

Low molecular weight fucoidan ameliorates the inflammation and glomerular filtration function of diabetic nephropathy

Diabetic nephropathy (DN) is a type of serious microangiopathy that is caused by diabetes mellitus (DM). It is the most common cause of chronic renal failure and end-stage renal disease, and it severely affects patients’ quality of life. This work aims to study the effect and mechanism of low molecular weight fucoidan (LMWF) on streptozotocin (STZ)-induced DN. The experimental results showed that LMWF prevented weight loss in DN rats, significantly reduced the levels of biochemical indexes in blood and urine samples, and also lowered hyaluronic acid (HA) levels and advanced glycosylation end product-specific receptor (AGER) levels in DN rats. LMWF maintained the structural integrity of glomerular basement membrane (GBM) and glomerulus, improved the glomerular filtration function, protected glycosaminoglycan from abnormal degrading, prevented advanced glycosylation end product (AGE) from being generated and accumulating, and also alleviated inflammatory response in DN rats. LMWF could obviously ameliorate and slow the development and progression of DN in rats.

Low molecular weight fucoidan modulates P-selectin and alleviates diabetic nephropathy

Diabetic nephropathy (DN) is a serious microvascular complication that can lead to chronic and end-stage renal failure. It is understood that inflammation is associated with the onset and process of DN. Low molecular weight fucoidan (LMWF) isolated from Saccharina japonica has anti-inflammatory properties. Therefore, this study aimed to explore the mechanism of LMWF in DN model induced by streptozotocin. The biochemical indices levels showed LMWF reduced the DN diagnostic indices to protect renal function. The HE stained sections exhibited LMWF protected normal morphological structures and reduced inflammatory cell infiltration in the kidneys of DN rats. Furthermore, the levels of P-selectin and selectin-dependent inflammatory cytokines resulting from LMWF were obviously decreased at both the transcriptional and protein levels. Thus, our results found that LMWF protected the renal function in DN rats and alleviated inflammation through the modulation of P-selectin and inflammatory cytokines. LMWF may have therapeutic potential against DN.

Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling

BackgroundInositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.ResultsIn neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.ConclusionsThese results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation.

Background/Aims: Stromal interacting molecule-1 (STIM1) aggregation and redistribution to plasma membrane to interact with Orai1 constitute the core mechanism of store-operated Ca2+ entry (SOCE). Previous study has revealed that calsequestrin-1 (CSQ1) regulates SOCE in HEK293 cells through interacting with STIM1 and inhibiting STIM1/Orai1 interaction. Here, we further investigate how CSQ1/STIM1 interaction affects SOCE. Methods: Using confocal microscopy, STIM1 aggregation and co-localizations with CSQ1 or Orai1 upon Ca2+ store depletion by thapsigargin were measured and quantified by Imaris software in HeLa cells transfected with different CSQ1 mutants. The interactions of CSQ1/STIM1 and STIM1/Orai1, and internal Ca2+ changes were detected by co-immunoprecipitation and Fura2, respectively. Results: Wt-CSQ1 overexpression significantly reduced STIM1 clustering in the perimembrane and cytosolic regions, whereas over-expression of a C-terminal amino acid 362-396 deletion mutant (C35) did not. Consistently, a significant depression of SOCE, increased CSQ1 monomerization and CSQ1/STIM1 interaction, and a reduced STIM1/Orai1 association were observed in wt-CSQ1 but not in C35-transfected cells. Additionally, mutant lacking C-terminal AA 388-396 deletion exerted weaker potency in inhibiting STIM1 aggregation and association with Orai1 than wt-CSQ1. Conclusions: Our results demonstrate that CSQ1 monomers suppress SOCE by interacting with STIM1 and attenuating STIM1 aggregation via its C-terminal amino acid 362-396.

Low molecular weight fucoidan attenuates liver injury via SIRT1/AMPK/PGC1α axis in db/db mice

Non-alcoholic fatty-liver disease (NAFLD), caused by elevated hepatic lipids, inflammation and oxidative stress, is the most common liver disease globally. Low molecular weight fucoidan (LMWF), a sulfated polysaccharide extracted from brown seaweeds, has shown strong anti-inflammatory and antioxidant activities, which has not been explored in diabetes-induced NAFLD. Therefore, the present study sought to determine whether LMWF protects obese diabetic db/db mice against NAFLD. Results showed LMWF administration decreased plasma level of alanine aminotransferase, aspartate aminotransferase, total cholesterol, and triglyceride, as well as alleviated hepatic accumulation of triglyceride and total cholesterol in db/db mice. LMWF also ameliorated hepatic oxidative stress by suppressing superoxide production and lipid peroxidation, and increasing catalase and superoxide dismutase activity in the liver of db/db mice. Furthermore, LMWF down-regulated several pro-inflammatory cytokines and transcription factor, and up-regulated the anti-inflammatory adiponectin. These changes were accompanied by the activation of hepatic SIRT1/AMPK/PGC1α signaling with LMWF treatment. In addition, blocking SIRT1 or AMPK by inhibitor notably abolished LMWF-elicited protection against palmitic acid-induced oxidative stress and inflammation in hepatocytes. These results suggest LMWF prevents NAFLD in db/db mice by activation of SIRT1/AMPK/PGC1α signaling pathway, which prevents lipotoxicity-related oxidative stress and inflammation. Therefore, LMWF provides a potential supplementary treatment for obesity/diabetes-induced NAFLD.