A modified method for isolating sinoatrial node myocytes from adult mice.

Sinoatrial node (SAN) is the pacemaker of the heart in charge of initiating spontaneous electronical activity and controlling heart rate. Myocytes from SAN can generate spontaneous rhythmic action potentials, which propagate through the myocardium, thereby triggering cardiac myocyte contraction. Acutely, the method for isolating sinoatrial node myocytes (SAMs) is critical in studying the protein expression and function of myocytes in SAN. Currently, the SAMs were isolated by transferring SAN tissue directly into the digestion solution, but it is difficult to judge the degree of digestion, and the system was unstable. Here, we present a modified protocol for the isolation of SAMs in mice, based on the collagenase II and protease perfusion of the heart using a Langendorff apparatus and subsequent dissociation of SAMs. The appearance and droplet flow rate of the heart could be significantly changed during enzymatic digestion via perfusion, which allowed us to easily judge the degree of digestion and avoid incomplete or excessive digestion. The SAMs with stable yield and viability achieved from our optimized approach would facilitate the follow-up experiments.

Study on the characteristics of genetic diversity of different populations of Guizhou endemic plant Rhododendron pudingense based on microsatellite markers.

BACKGROUND: Rhododendron pudingense, firstly discovered in Puding county of Guizhou province in 2020, have adapted to living in rocky fissure habitat, which has important ornamental and economic values. However, the genetic diversity and population structure of this species have been rarely described, which seriously affects the collection and protection of wild germplasm resources. RESULTS: In the present study, 13 pairs of primers for polymorphic microsatellite were used to investigate the genetic diversity of 65 R. pudingense accessions from six different geographic populations. A total of 254 alleles (Na) were obtained with an average of 19.5 alleles per locus. The average values of polymorphic information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He) were 0.8826, 0.4501, and 0.8993, respectively, These results indicate that the microsatellite primers adopted demonstrate good polymorphism, and the R. pudingense exhibits a high level of genetic diversity at the species level. The average genetic differentiation coefficient (Fst) was 0.1325, suggested that moderate divergence occurred in R. pudingense populations. The average values of genetic differentiation coefficient and gene flow among populations were 0.1165 and 3.1281, respectively. The analysis of molecular variance (AMOVA) indicated that most of the population differences (88%) were attributed to within-population variation. The PCoA results are consistent with the findings of the UPGMA clustering analysis, supporting the conclusion that the six populations of R. pudingense can be clearly grouped into two separate clusters. Based on Mantel analysis, we speculate that the PD population may have migrated from WM-1 and WM-2. Therefore, it is advised to protect the natural habitat of R. pudingense in situ as much as possible, in order to maximize the preservation of its genetic diversity. CONCLUSIONS: This is the first comprehensive analysis of genetic diversity and population structure of R. pudingense in Guizhou province. The research results revealed the high genetic diversity and moderate population diferentiation in this horticulture plant. This study provide a theoretical basis for the conservation of wild resources of the R. pudingense and lay the foundation for the breeding or cultivation of this new species.

Corrigendum to "Connexin 43 dephosphorylation at serine 282 induces spontaneous arrhythmia and increases susceptibility to ischemia/reperfusion injury" [Heliyon 9 (5), May 2023 Article e15879].

[This corrects the article DOI: 10.1016/j.heliyon.2023.e15879.].

Downregulated calmodulin expression contributes to endothelial cell impairment in diabetes.

Endothelial dysfunction, a central hallmark of cardiovascular pathogenesis in diabetes mellitus, is characterized by impaired endothelial nitric oxide synthase (eNOS) and NO bioavailability. However, the underlying mechanisms remain unclear. Here in this study, we aimed to identify the role of calmodulin (CaM) in diabetic eNOS dysfunction. Human umbilical vein endothelial cells and murine endothelial progenitor cells (EPCs) treated with high glucose (HG) exhibited downregulated CaM mRNA/protein and vascular endothelial growth factor (VEGF) expression with impeded eNOS phosphorylation and cell migration/tube formation. These perturbations were reduplicated in CALM1-knockdown cells but prevented in CALM1-overexpressing cells. EPCs from type 2 diabetes animals behaved similarly to HG-treated normal EPCs, which could be rescued by CALM1-gene transduction. Consistently, diabetic animals displayed impaired eNOS phosphorylation, endothelium-dependent dilation, and CaM expression in the aorta, as well as deficient physical interaction of CaM and eNOS in the gastrocnemius. Local CALM1 gene delivery into a diabetic mouse ischemic hindlimb improved the blunted limb blood perfusion and gastrocnemius angiogenesis, and foot injuries. Diabetic patients showed insufficient foot microvascular autoregulation, eNOS phosphorylation, and NO production with downregulated CaM expression in the arterial endothelium, and abnormal CALM1 transcription in genome-wide sequencing analysis. Therefore, our findings demonstrated that downregulated CaM expression is responsible for endothelium dysfunction and angiogenesis impairment in diabetes, and provided a novel mechanism and target to protect against diabetic endothelial injury.

Connexin 43 dephosphorylation at serine 282 induces spontaneous arrhythmia and increases susceptibility to ischemia/reperfusion injury.

Background: Connexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to affect myocardium function and structure. Previously we found that deficiency in Cx43 S282 phosphorylation could impair intercellular communication and contribute to cardiomyocyte apoptosis by activating p38 mitogen-activated protein kinase (p38 MAPK)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway, which is involved in myocardium injury in ischemia/reperfusion (I/R) heart. In addition, mutant at Cx43 S282 substituted with alanine heterozygous mice (S282A+/-) exhibited different degrees of ventricular arrhythmias and only some underwent myocardium apoptosis. In this study, we aimed to investigate the role of Cx43 pS282 in different cardiac pathological phenotypes. Methods: We examined cardiac function, structure, and relevant protein expression in S282A+/- mice (aged 2, 10 and 30 weeks) by electrocardiograph, echocardiography, histological staining, and co-immunoprecipitation followed by Western blot. Intraperitoneal isoprenaline injection and I/R surgery were applied in S282A+/- mice as external stimulus. 2,3,5-triphenyltetrazolium chloride staining was used for myocardium infarction evaluation. Results: Adult S282A+/- mice (aged 10 and 30 weeks) still exhibited spontaneous arrhythmia. Unlike neonatal stage (aged around 2 weeks), no apoptosis-related manifestations and the activation of p38 MAPK-Fas-FADD apoptotic pathway were observed in adult S282A+/- hearts. S282A+/- neonatal mice with cardiomyocytes apoptosis exhibited more than 60% dephosphorylation at Cx43 S282 than WT mice, while less than 40% S282 dephosphorylation were found in adult S282A+/- mice. In addition, although S282A+/- mice displayed normal cardiac function, they were highly susceptible to isoproterenol-induced ECG alternans and prone to cardiac injury and deaths upon I/R attack. Conclusions: These results reinforce that Cx43 S282 dephosphorylation acts as a susceptibility factor in regulating cardiomyocyte survival and cardiac electrical homeostasis in basal conditions and contributes to myocardium injury in the setting of I/R. Cx43 S282 phosphorylation was competent to induce spontaneous arrhythmias, cardiomyocyte apoptosis and deaths based on the degree of S282 dephosphorylation.

Connexin 43 hyper-phosphorylation at serine 282 triggers apoptosis in rat cardiomyocytes via activation of mitochondrial apoptotic pathway.

Cx43 is the major connexin in ventricular gap junctions, and plays a pivotal role in control of electrical and metabolic communication among adjacent cardiomyocytes. We previously found that Cx43 dephosphorylation at serine 282 (pS282) caused cardiomyocyte apoptosis, which is involved in cardiac ischemia/reperfusion injury. In this study we investigated whether Cx43-S282 hyper-phosphorylation could protect cardiomyocytes against apoptosis. Adenovirus carrying rat full length Cx43 gene (Cx43-wt) or a mutant gene at S282 substituted with aspartic acid (S282D) were transfected into neonatal rat ventricular myocytes (NRVMs) or injected into rat ventricular wall. Rat abdominal aorta constriction model (AAC) was used to assess Cx43-S282 phosphorylation status. We showed that Cx43 phosphorylation at S282 was increased over 2-times compared to Cx43-wt cells at 24 h after transfection, while pS262 and pS368 were unaltered. S282D-transfected cells displayed enhanced gap junctional communication, and increased basal intracellular Ca2+ concentration and spontaneous Ca2+ transients compared to Cx43-wt cells. However, spontaneous apoptosis appeared in NRVMs transfected with S282D for 34 h. Rat ventricular myocardium transfected with S282D in vivo also exhibited apoptotic responses, including increased Bax/Bcl-xL ratio, cytochrome c release as well as caspase-3 and caspase-9 activities, while factor-associated suicide (Fas)/Fas-associated death domain expression and caspase-8 activity remained unaltered. In addition, AAC-induced hypertrophic ventricles had apoptotic injury with Cx43-S282 hyper-phosphorylation compared with Sham ventricles. In conclusion, Cx43 hyper-phosphorylation at S282, as dephosphorylation, also triggers cardiomyocyte apoptosis, but through activation of mitochondrial apoptosis pathway, providing a fine-tuned Cx43-S282 phosphorylation range required for the maintenance of cardiomyocyte function and survival.

Design, synthesis, and biological evaluation of novel triazoloquinazolinone derivatives as SHP2 protein inhibitors.

A novel series of triazoloquinazolinone derivatives were designed, synthesised, and evaluated for their in vitro biological activities against the SHP2 protein. Moreover, some compounds were evaluated against A375 cells. The results revealed that target compounds possessed moderate to excellent inhibitory activity against SHP2 protein, whereas compounds 12f, 12l, 12j, 17e, and 17f have strong antiproliferative activity on A375 cells. The compound 12l showed remarkable cytotoxicity against A375 cells and a strong inhibitory effect against SHP2 protein when compared with SHP244. The structure-activity relationships (SARs) indicated that electron-donating groups (EDGs) on phenyl rings are beneficial for improving the antitumor activity; compounds with a hydroxyl substituent at the 2-position of phenyl ring exhibited superior activities than compounds with a substituent at the 4-position. In addition, compound 12l displayed improved physicochemical properties as well as metabolic stability compared to SHP244. Our efforts identified 12l as a promising SHP2 protein inhibitor, warranting its further investigation.

Estrogen-dependent KCa1.1 modulation is essential for retaining neuroexcitation of female-specific subpopulation of myelinated Ah-type baroreceptor neurons in rats.

Female-specific subpopulation of myelinated Ah-type baroreceptor neurons (BRNs) in nodose ganglia is the neuroanatomical base of sexual-dimorphic autonomic control of blood pressure regulation, and KCa1.1 is a key player in modulating the neuroexcitation in nodose ganglia. In this study we investigated the exact mechanisms underlying KCa1.1-mediated neuroexcitation of myelinated Ah-type BRNs in the presence or absence of estrogen. BRNs were isolated from adult ovary intact (OVI) or ovariectomized (OVX) female rats, and identified electrophysiologically and fluorescently. Action potential (AP) and potassium currents were recorded using whole-cell recording. Consistently, myelinated Ah-type BRNs displayed a characteristic discharge pattern and significantly reduced excitability after OVX with narrowed AP duration and faster repolarization largely due to an upregulated iberiotoxin (IbTX)-sensitive component; the changes in AP waveform and repetitive discharge of Ah-types from OVX female rats were reversed by G1 (a selective agonist for estrogen membrane receptor GPR30, 100 nM) and/or IbTX (100 nM). In addition, the effect of G1 on repetitive discharge could be completely blocked by G15 (a selective antagonist for estrogen membrane receptor GPR30, 3 µM). These data suggest that estrogen deficiency by removing ovaries upregulates KCa1.1 channel protein in Ah-type BRNs, and subsequently increases AP repolarization and blunts neuroexcitation through estrogen membrane receptor signaling. Intriguingly, this upregulated KCa1.1 predicted electrophysiologically was confirmed by increased mean fluorescent intensity that was abolished by estrogen treatment. These electrophysiological findings combined with immunostaining and pharmacological manipulations reveal the crucial role of KCa1.1 in modulation of neuroexcitation especially in female-specific subpopulation of myelinated Ah-type BRNs and extend our current understanding of sexual dimorphism of neurocontrol of BP regulation.

Functional Calsequestrin-1 Is Expressed in the Heart and Its Deficiency Is Causally Related to Malignant Hyperthermia-Like Arrhythmia.

BACKGROUND: Calsequestrins (Casqs), comprising the Casq1 and Casq2 isoforms, buffer Ca2+ and regulate its release in the sarcoplasmic reticulum of skeletal and cardiac muscle, respectively. Human inherited diseases associated with mutations in CASQ1 or CASQ2 include malignant hyperthermia/environmental heat stroke (MH/EHS) and catecholaminergic polymorphic ventricular tachycardia. However, patients with an MH/EHS event often experience arrhythmia for which the underlying mechanism remains unknown. METHODS: Working hearts from conventional (Casq1-KO) and cardiac-specific (Casq1-CKO) Casq1 knockout mice were monitored in vivo and ex vivo by ECG and electric mapping, respectively. MH was induced by 2% isoflurane and treated intraperitoneally with dantrolene. Time-lapse imaging was used to monitor intracellular Ca2+ activity in isolated mouse cardiomyocytes or neonatal rat ventricular myocytes with knockdown, overexpression, or truncation of the Casq1 gene. Conformational change in both Casqs was determined by cross-linking Western blot analysis. RESULTS: Like patients with MH/EHS, Casq1-KO and Casq1-CKO mice had faster basal heart rate and ventricular tachycardia on exposure to 2% isoflurane, which could be relieved by dantrolene. Basal sinus tachycardia and ventricular ectopic electric triggering also occurred in Casq1-KO hearts ex vivo. Accordingly, the ventricular cardiomyocytes from Casq1-CKO mice displayed dantrolene-sensitive increased Ca2+ waves and diastole premature Ca2+ transients/oscillations on isoflurane. Neonatal rat ventricular myocytes with Casq1-knockdown had enhanced spontaneous Ca2+ sparks/transients on isoflurane, whereas cells overexpressing Casq1 exhibited decreased Ca2+ sparks/transients that were absent in cells with truncation of 9 amino acids at the C terminus of Casq1. Structural evaluation showed that most of the Casq1 protein was present as a polymer and physically interacted with ryanodine receptor-2 in the ventricular sarcoplasmic reticulum. The Casq1 isoform was also expressed in human myocardium. Mechanistically, exposure to 2% isoflurane or heating at 41 °C induced Casq1 oligomerization in mouse ventricular and skeletal muscle tissues, leading to a reduced Casq1/ryanodine receptor-2 interaction and increased ryanodine receptor-2 activity in the ventricle. CONCLUSIONS: Casq1 is expressed in the heart, where it regulates sarcoplasmic reticulum Ca2+ release and heart rate. Casq1 deficiency independently causes MH/EHS-like ventricular arrhythmia by trigger-induced Casq1 oligomerization and a relief of its inhibitory effect on ryanodine receptor-2-mediated Ca2+ release, thus revealing a new inherited arrhythmia and a novel mechanism for MH/EHS arrhythmogenesis.

Connexin 43 dephosphorylation contributes to arrhythmias and cardiomyocyte apoptosis in ischemia/reperfusion hearts.

Connexin 43 (Cx43)-associated gap junctions form electrical and mechanical conduits between adjacent ventricular cardiomyocytes, ensuring coordinate electrical excitation and synchronic contraction for each heartbeat. Cx43 dephosphorylation is a characteristic of ischemia, arrhythmia, and a failing and aging myocardium, but the exact phosphosite(s) triggering myocardial apoptosis and electrical disturbance and its underlying mechanisms are unclear. We previously found that Cx43-serine 282 phosphorylation (pS282) can regulate cardiomyocyte survival and electrical stability. Here, we investigated the hypothesis that S282 dephosphorylation occurs in and contributes to ischemia/reperfusion (I/R)-induced cardiac injury. We found enhanced Cx43-pS262 and Cx43-pS368 but decreased Cx43-pS282 in rat hearts subjected to I/R (30 min/2 h). I/R rats had ventricular arrhythmias and myocardial apoptosis with activation of the p38 mitogen-activated protein kinase (p38)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway. Similarly, S282 dephosphorylation, abnormal Ca2+ transients, cell apoptosis and p38/Fas/FADD activation also occurred in neonatal rat ventricular myocytes exposed to anoxia/reoxygenation (12/6 h). To confirm the causative role of S282 dephosphorylation in cardiac injury, rat hearts were intramyocardially injected with a virus carrying the S282 mutant substituted with alanine (S282A), thus causing arrhythmias and reducing cardiac output and myocardial apoptosis with p38/Fas/FADD pathway activation. Moreover, Cx43-S282A+/- mice displayed arrhythmias and impaired cardiac output with global myocardial apoptosis. Our findings revealed that Cx43 dephosphorylation at S282 triggers arrhythmias and, at least partly, contributes to cardiomyocyte death upon I/R by activating the p38/Fas/FADD pathway, providing a novel molecular mechanism and potential target for protecting against cardiac I/R injury.

Connexin 43-serine 282 modulates serine 279 phosphorylation in cardiomyocytes.

Connexin 43 (Cx43) phosphorylation plays a pivotal role in cardiac electrical and contractile performance. In a previous study we have found that Cx43 phosphorylation at serine 282 (pS282) regulates cardiomyocyte survival. Considering that both sites are altered simultaneously in many studies, we designed this study to identify the status of S279 phosphorylation upon pS282 manipulation. In heterozygous mice with S282 gene substituted with alanine (S282A), we found ventricular arrhythmias with inhibition of Cx43 phosphorylation at both S282 and S279 in the hearts. In cultured neonatal rat ventricular myocytes (NRVMs), transfection of virus carrying S282A mutant also blocked Cx43 phosphorylation at both S279/282 and gap junction coupling, while expression of wild-type Cx43 or S279A did not. Further, NRVMs transfected with S282 phospho-mimicking mutant substituted with aspartate or treated with ATP exhibited promotions of Cx43 phosphorylation at S279/282 and intercellular communication. Therefore, this study demonstrated a regulatory role of Cx43-S282 on S279 phosphorylation in cardiomyocytes, and suggested an involvement of S279 in the Cx43-S282 mediated cardiomyocyte homeostasis.

Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis.

Gap junction protein connexin 43 (Cx43) plays an important role in regulating cardiomyocyte survival in addition to regulating electrical coordination. Cx43 dephosphorylation, found in severe cardiac pathologies, is thought to contribute to myocardial injury. However, the mechanisms underlying Cx43 mediation of cell survival and myocardial lesions remain unknown. Here, we found that transfecting an adenovirus carrying a mutant gene of Cx43-serine 282 substituted with alanine (S282A) into neonatal rat ventricular myocytes (NRVMs) induced cell apoptosis and Ca2+ transient desynchronization, whereas using gap junction inhibitor or knocking down Cx43 expression with Cx43-miRNA caused uncoupled Ca2+ signaling without cell death. Similarly, while Cx43-S282A+/+ failed in generation, Cx43-S282A+/- mice exhibited cardiomyocyte apoptosis and ventricular arrhythmias dependent on S282 dephosphorylation. Further, Cx43 dephosphorylation at S282 activated p38 mitogen-activated protein kinase (p38 MAPK), factor-associated suicide and the caspase-8 apoptotic pathway by physically interacting with p38 MAPK. These findings uncovered a specific Cx43 phosphorylation residue involved in regulating cardiomyocyte homeostasis. S282 phosphorylation deficiency acts as a trigger inducing cardiomyocyte apoptosis and cardiac arrhythmias, providing a potential mechanism for Cx43-mediated myocardial injury in severe cardiac diseases.

CaMKII Potentiates Store-Operated Ca2+ 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.

Orai1 downregulation impairs lymphocyte function in type 2 diabetes mellitus.

BACKGROUND/AIMS: It has been suggested that diabetes is associated with immune dysfunction, in which Ca2+ signaling malfunction in lymphocyte may contributes most. However, the pattern of the Ca2+ signal disorder and the mechanism(s) that explains the change are unclear. Here, in this study we aimed to investigate possible changes and mechanism(s) accounting for the internal Ca2+ signals in diabetic T lymphocyte upon stimulation. METHODS AND RESULTS: Using Fura-2-AM, we found a significant decrease in Ca2+ influx induced by thapsigargin (TG) and anti-CD3 antibody (OKT3) in T lymphocytes from blood of both diabetes patients and animals. Furthermore, a downregulated Orai1 protein expression, but not mRNA, was also observed in these cells using western blot and qRT-PCR, respectively. In addition, in high-glucose and agonist treated Jurkat T cells, Ca2+ entry and the release of interleukin-2 (IL-2) were also decreased. Orai1 expression reduced, while stromal interaction molecule 1 (STIM1) and other downstream proteins remained unchanged. CONCLUSION: This study demonstrates that the declined Orai1 expression, at least partly, contributes to the downregulated Ca2+ entry during lymphocyte excitation, providing an important mechanism for T lymphocyte malfunction in diabetes.

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

BACKGROUND: 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). METHODS: Diabetic db/db mice and age-matched C57BL/6 mice underwent femoral artery ligation followed by LMWF (30, 60, 80 mg/kg per day, p.o.) or cilostazol (30 mg/kg/day, p.o.) treatment for 6 weeks. Endothelium-dependent vasodilation and blood flow of the hindlimb were measured. Histological and western blot analyses of CD34, vascular endothelial growth factor (VEGF), eNOS, and inflammatory factors in the gastrocnemius were performed. The effects of LMWF were confirmed in human umbilical vein endothelial cells (HUVEC). RESULTS: Diabetic mice with ligation exhibited hindlimb ulceration, hydrosarca, and necrosis, increased expression of inflammatory factors, and decreased levels of VEGF and eNOS phosphorylation. Treatment with LMWF markedly ameliorated foot lesions, suppressed expression of inflammatory factors, and improved plantar perfusion by promoting endothelium-dependent vasodilation and revascularization in diabetic PAD mice. In high-glucose treated HUVEC, LMWF (40 µg/mL) reversed blunted endothelial cell proliferation, migration, and tube formation, and promoted eNOS phosphorylation and VEGF expression, whereas HUVEC pretreatment with 100 µmol/L NG -nitro-l-arginine methyl ester, an eNOS antagonist, markedly inhibited the effects of LMWF. CONCLUSION: This study demonstrates that LMWF alleviates hindlimb ischemic damage, at least in part by promoting eNOS phosphorylation, nitric oxide production, and VEGF expression, resulting in enhanced angiogenesis in the ischemic region.

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 RELEVANCE: Low 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. AIM OF THE STUDY: Diabetic 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. MATERIALS AND METHODS: Type 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. RESULTS: We 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-1ß 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. CONCLUSIONS: Taken 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.

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.

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.

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.