Yuanyuan Zheng

Attenuation of streptozotocin-induced diabetic retinopathy with low molecular weight fucoidan via inhibition of vascular endothelial growth factor.

Diabetic retinopathy (DR) is a hyperglycemia-induced ischemic disorder characterized by microvascular dysfunction and neovascularization. It is a leading cause of blindness in many countries, yet efficient drugs are limited now for prevention and treatment of DR. Low molecular weight fucoidan (LMWF), extract from brown algae, has been shown to possess multiple biological activities like anti-inflammation, anti-oxidation and anti-aggregation, which all could be beneficial for attenuating ischemia-induced tissue damages. Here, by comparing with calcium dobesilate, the potent antioxidant compound currently used for the treatment of DR, we investigated the protective effect of LMWF against DR in streptozotocin-induced diabetic mice and high glucose-promoted vascular endothelial growth factor (VEGF) production and cell proliferation in microvascular endothelial cells. One week after diabetes induction, the mice were administered with LMWF (50, 100 or 200xa0mg/kg/day) or calcium dobesilate (200xa0mg/kg/day) for four months, then the retinal pathological changes and neovascularization were detected by hematoxylin-eosin staining and fluorescein dextran angiography, respectively. Immunofluorescence staining, ELISA and RT-PCR were used to examine the expression levels of hypoxia-inducible factor-1α (HIF-1α) and VEGF in retina and endothelial cells. Here, we found that LMWF resembled calcium dobesilate, in alleviating retinal pathological change and hindering neovascularization due to diabetes inxa0vivo. The relative levels of VEGF expression and HIF-1α induction were also less in retinas of LMWF- or calcium dobesilate-treated diabetic mice than those in retinas of control mice. Furthermore, high glucose-induced VEGF overexpression and cell proliferation in primary cultured vascular endothelial cells were also inhibited by LMWF in a dose-dependent manner. Therefore, this study demonstrated that LMWF alleviates diabetic retinal neovascularization and damage likely through lowering HIF-1α and VEGF expressions, providing a potential candidate drug for prevention and treatment of diabetic retinopathy.

Retrograde regulation of STIM1-Orai1 interaction and store-operated Ca2+ entry by calsequestrin.

Interaction between the endoplasmic reticulum (ER)-located stromal interaction molecue1 (STIM1) and the plasma membrane-located Ca2+ channel subunit, Orai1, underlies store-operated Ca2+ entry (SOCE). Calsequestrin1 (CSQ1), a sarcoplasmic reticulum Ca2+ buffering protein, inhibits SOCE, but the mechanism of action is unknown. We identified an interaction between CSQ1 and STIM1 in HEK293 cells. An increase in monomeric CSQ1 induced by depleted Ca2+ stores, or trifluoperazine (TFP), a blocker of CSQ folding and aggregation, enhanced the CSQ1-STIM1 interaction. In cells with Ca2+ stores depleted, TFP further increased CSQ1 monomerization and CSQ1-STIM1 interaction, but reduced the association of STIM1 with Orai1 and SOCE. Over-expression of CSQ1 or a C-terminal (amino acid 388–396) deletion mutant significantly promoted the association of CSQ1 with STIM1, but suppressed both STIM1-Orai1 interaction and SOCE, while over-expression of the C-terminal (amino acid 362–396) deletion mutant had no effect. The physical interaction between low polymeric forms of CSQ1 and STIM1 likely acts by interfering with STIM1 oligimerization and inhibits STIM1-Orai1 interaction, providing a brake to SOCE under physiological conditions. This novel regulatory mechanism for SOCE may also contribute to the pathological Ca2+ overload in calsequestrin deficient diseases, such as malignant hyperthermia and ventricular tachycardia.

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

ETHNOPHARMACOLOGICAL RELEVANCEnLow 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.nnnAIM OF THE STUDYnThis 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.nnnMATERIALS AND METHODSnSprague-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.nnnRESULTSnLMWF (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.nnnCONCLUSIONnThis 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

INTRODUCTIONnDownregulation 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.nnnMATERIALS AND METHODSnType 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.nnnRESULTSnDuring 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.nnnCONCLUSIONSnThese 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.

Protective effect of low dose gadolinium chloride against isoproterenol-induced myocardial injury in rat.

Acute myocardial injury remains a leading cause of morbidity and mortality worldwide, and large amount of released arachidonic acid (AA) is found to be related to cardiomyocyte apoptosis and necrosis. Previous study suggested that GdCl3 completely abolished AA-induced Ca2+ response. Thus, this study aims to investigate possible cardioprotection effect of GdCl3 on isoproterenol (ISO)-induced myocardial injury and its underlying mechanism(s). Rats that were randomly allocated to five groups: control, GdCl3, ISO, ISOxa0+xa0GdCl3, and ISOxa0+xa0verapamil. Serum levels of AA and cardiac markers, infarct area, and cell apoptosis in heart were measured by ELISA assay, TTC and TUNEL staining, respectively. Chemical interaction between AA and GdCl3 was evaluated by mass and UV spectrometry. The expressions and translocations of death receptor related molecules into lipid rafts were detected in neonatal rat ventricular myocytes by Western blots. Compared with ISO-administered rats, GdCl3 significantly ameliorated the myocardium injury, demonstrated by restoring serum cardiac troponin I, lactate dehydrogenase, creatine kinase MB and AA to near normal levels, and decreasing infarct area and cell apoptosis. In addition, an activation of AA-Fas pathway was found in ISO-induced myocardial injury, which was abrogated by GdCl3. Furthermore, AA induced cell apoptosis through clustering and activating death receptor related molecules TNFR1, Fas and FADD in lipid rafts, a process significantly prevented by the pretreatment with GdCl3. Finally, GdCl3 at the molar ratio of 1/3 (GdCl3/AA) was mostly effective in abolishing AA-induced Ca2+ response and cell apoptosis, because an obvious change in the chemical identity of AA was obtained by GdCl3 according to this molar ratio. In conclusion, this study demonstrates for the first time that GdCl3 protects myocardium against ISO-induced cell apoptosis through, at least partly, serving as a scavenger of AA, therefore abolishing its downstream activation of the death receptor regulated apoptosis pathway.

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.

Low molecular weight fucoidan ameliorates hindlimb ischemic injury in type 2 diabetic rats

ETHNOPHARMACOLOGICAL RELEVANCEnLow molecular weight fucoidan (LMWF), extracted from Laminaria japonica Areschoug, is a traditional Chinese medicine, commonly used to alleviate edema, particularly for feet with numbness and pain.nnnAIM OF THE STUDYnDiabetic mellitus (DM) patients are at high risk of developing peripheral arterial disease (PAD). Individuals with DM and PAD co-morbidity have a much higher risk of critical limb ischemia. LMWF showed several beneficial effects, such as anti-inflammation, anti-thrombosis, and enhancing revascularization. Therefore, we hypothesized that LMWF might be beneficial to diabetes-induced PAD, and investigated the therapeutic potential of LMWF on diabetic PAD rats.nnnMATERIALS AND METHODSnType 2 diabetic Goto-Kakizaki (GK) rats were made PAD by injection of sodium laurate into femoral artery. LMWF (20, 40 or 80mg/kg/day) or cilostazol (100mg/kg/day) were given to diabetic PAD rats for 4 weeks, respectively. The effects of LMWF on foot ulceration and claudication, plantar blood flow, collateral vessel formation, endothelium morphology, gastrocnemius injury, platelet aggregation, vessel vasodilation, and the expressions of inflammation factors, VEGF, eNOS, and nitric oxide were measured.nnnRESULTSnWe found that LMWF markedly ameliorated foot ulceration and claudication, and improved the plantar perfusion by reversing hyperreactive platelet aggregation, ameliorating endothelium-dependent vasodilation and revascularization on diabetic PAD rats. In addition, upregulation of several inflammatory factors, such as ICAM-1 and IL-1β in the gastrocnemius muscles of ischemic hindlimb were suppressed by LMWF administration. And eNOS phosphorylation at Ser1177 and NO production were significantly enhanced in LMWF-treated diabetic PAD rats.nnnCONCLUSIONSnTaken together, our findings demonstrated that LMWF exhibits therapeutic effect on hindlimb ischemia in type 2 diabetic rats likely through ameliorating endothelium eNOS dysfunction and enhancing revascularization, thus, providing a potential supplementary non-invasive treatment for diabetes-induced PAD.

Pretreatment with low-dose gadolinium chloride attenuates myocardial ischemia/reperfusion injury in rats

Aim:We have shown that low-dose gadolinium chloride (GdCl3) abolishes arachidonic acid (AA)-induced increase of cytoplasmic Ca2+, which is known to play a crucial role in myocardial ischemia/reperfusion (I/R) injury. The present study sought to determine whether low-dose GdCl3 pretreatment protected rat myocardium against I/R injury in vitro and in vivo.Methods:Cultured neonatal rat ventricular myocytes (NRVMs) were treated with GdCl3 or nifedipine, followed by exposure to anoxia/reoxygenation (A/R). Cell apoptosis was detected; the levels of related signaling molecules were assessed. SD rats were intravenously injected with GdCl3 or nifedipine. Thirty min after the administration the rats were subjected to LAD coronary artery ligation followed by reperfusion. Infarction size, the release of serum myocardial injury markers and AA were measured; cell apoptosis and related molecules were assessed.Results:In A/R-treated NRVMs, pretreatment with GdCl3 (2.5, 5, 10 μmol/L) dose-dependently inhibited caspase-3 activation, death receptor-related molecules DR5/Fas/FADD/caspase-8 expression, cytochrome c release, AA release and sustained cytoplasmic Ca2+ increases induced by exogenous AA. In I/R-treated rats, pre-administration of GdCl3 (10 mg/kg) significantly reduced the infarct size, and the serum levels of CK-MB, cardiac troponin-I, LDH and AA. Pre-administration of GdCl3 also significantly decreased the number of apoptotic cells, caspase-3 activity, death receptor-related molecules (DR5/Fas/FADD) expression and cytochrome c release in heart tissues. The positive control drug nifedipine produced comparable cardioprotective effects in vitro and in vivo.Conclusion:Pretreatment with low-dose GdCl3 significantly attenuates I/R-induced myocardial apoptosis in rats by suppressing activation of both death receptor and mitochondria-mediated pathways.

Low molecular-weight fucoidan protects against hindlimb ischemic injury in type 2 diabetic mice through enhancing endothelial nitric oxide synthase phosphorylation: LMWF protects against diabetic foot

Diabetes mellitus (DM) complications are associated with ischemic injury. Angiogenesis is a therapeutic strategy for diabetic foot. The aim of this study was to investigate the possible angiogenic effect of low molecular weight fucoidan (LMWF) in diabetic peripheral arterial disease (PAD).

CaMKII Potentiates Store-Operated Ca 2+ Entry Through Enhancing STIM1 Aggregation and Interaction with Orai1

Background/Aims: Upon Ca2+ store depletion, stromal interaction molecule 1 (STIM1) oligomerizes, redistributes near plasmalemma to interact with Ca2+ selective channel-forming subunit (Orai1) and initiates store-operated Ca2+ entry (SOCE). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a regulator of SOCE, but how CaMKII regulates SOCE remains obscure. Methods: Using Fura2, confocal microscopy, co-immunoprecipitation, specific blocker and overexpression/knockdown approaches, we evaluated STIM1 aggregation and its interaction with Orai1, and SOCE upon Ca2+ store depletion in thapsigargin (TG) treated HEK293 and HeLa cells. Results: Overexpression of CaMKIIδ enhanced TG-induced STIM1 co-localization and interaction with Orai1 as well as SOCE. In contrast, CaMKIIδ knockdown and a specific inhibitor of CaMKII suppressed them. In addition, overexpression or knockdown of CaMKIIδ in TG treated cells exhibited increased or reduced STIM1 clustering and plasmalemma redistribution, respectively. Conclusion: CaMKII up-regulates SOCE by increasing STIM1 aggregation and interaction with Orai1. This study provides an additional insight into SOCE regulation and a potential mechanism for CaMKII involvement in some pathological situations through crosstalk with SOCE.