[Distribution of memory B cell subsets in peripheral blood of children with frequently relapsing nephrotic syndrome]. / é¢‘å¤å‘è‚¾ç— ç»¼åˆå¾æ‚£å„¿å¤–å‘¨è¡€è®°å¿†B细胞亚群分布变化.

OBJECTIVES: To investigate the change in the distribution of memory B cell subsets in children with frequently relapsing nephrotic syndrome (FRNS) during the course of the disease. METHODS: A total of 35 children with primary nephrotic syndrome (PNS) who attended the Department of Pediatrics of the Affiliated Hospital of Xuzhou Medical University from October 2020 to October 2021 were enrolled as subjects in this prospective study. According to the response to glucocorticoid (GC) therapy and frequency of recurrence, the children were divided into two groups: FRNS (n=20) and non-FRNS (NFRNS; n=15). Fifteen children who underwent physical examination were enrolled as the control group. The change in memory B cells after GC therapy was compared between groups, and its correlation with clinical indicators was analyzed. RESULTS: Before treatment, the FRNS and NFRNS groups had significantly increased percentages of total B cells, total memory B cells, IgD+ memory B cells, and IgE+ memory B cells compared with the control group, and the FRNS group had significantly greater increases than the NFRNS group (P<0.05); the FRNS group had a significantly lower percentage of class-switched memory B cells than the NFRNS and control groups (P<0.05). After treatment, the FRNS and NFRNS groups had significant reductions in the percentages of total B cells, total memory B cells, IgM+IgD+ memory B cells, IgM+ memory B cells, IgE+ memory B cells, IgD+ memory B cells, and IgG+ memory B cells (P<0.05) and a significant increase in the percentage of class-switched memory B cells (P<0.05). The FRNS group had a significantly higher urinary protein quantification than the NFRNS and control groups (P<0.05) and a significantly lower level of albumin than the control group (P<0.05). In the FRNS group, urinary protein quantification was negatively correlated with the percentage of class-switched memory B cells and was positively correlated with the percentage of IgE+ memory B cells (P<0.05). CONCLUSIONS: Abnormal distribution of memory B cell subsets may be observed in children with FRNS, and the percentages of IgE+ memory B cells and class-switched memory B cells can be used as positive and negative correlation factors for predicting recurrence after GC therapy in these children.

FOXC1 up-regulates the expression of toll-like receptors in myocardial ischaemia.

Myocardial ischaemia (MI) remains a major cause of death and disability worldwide. Accumulating evidence suggests a significant role for innate immunity, in which the family of toll-like receptors (TLRs) acts as an essential player. We previously reported and reviewed the changes of Tlr expression in models of MI. However, the underlying mechanisms regulating Tlr expression in MI remain unclear. The present study first screened transcription factors (TFs) that potentially regulate Tlr gene transcription based on in silico analyses followed by experimental verification, using both in vivo and in vitro models. Forkhead box C1 (FOXC1) was identified as a putative TF, which was highly responsive to MI. Next, by focusing on two representative TLR subtypes, an intracellular subtype TLR3 and a cell-surface subtype TLR4, the regulation of FOXC1 on Tlr expression was investigated. The overexpression or knockdown of FoxC1 was observed to up- or down-regulate Tlr3/4 mRNA and protein levels, respectively. A dual-luciferase assay showed that FOXC1 trans-activated Tlr3/4 promoter, and a ChIP assay showed direct binding of FOXC1 to Tlr3/4 promoter. Last, a functional study of FOXC1 was performed, which revealed the pro-inflammatory effects of FOXC1 and its destructive effects on infarct size and heart function in a mouse model of MI. The present study for the first time identified FOXC1 as a novel regulator of Tlr expression and described its function in MI.

Shenfu Injection attenuates rat myocardial hypertrophy by up-regulating miR-19a-3p expression.

Shenfu Injection (SFI) is a classical Chinese medicine used to treat heart failure. Our previous study demonstrated that miRNAs underwent changes in rats with myocardial hypertrophy induced by abdominal aortic constriction. Interestingly, there was a significant change in miR-19a-3p, whose target gene is known to be associated with MEF2 signaling. However, whether and how SFI regulates miR-19a-3p in the treatment of myocardial hypertrophy has not been investigated. The purpose of the present study was to investigate the regulatory effect of SFI on miR-19a-3p in MEF2 signaling in the rat hypertrophic myocardium. We found that the miR-19a-3p expression level was significantly decreased in the hypertrophic myocardium, and MEF2A was the target gene of miR-19a-3p. The protein expressions of MEF2A, ß-MHC, BNP and TRPC1 were significantly increased in hypertrophic cardiomyocytes. MiR-19a-3p was up-regulated after SFI treatment, and the protein expressions of these genes were significantly decreased. In addition, miR-19a-3p over-expression in hypertrophic cardiomyocytes could decrease MEF2A mRNA and protein expressions, and anti-miR-19a-3p showed the opposite result. Our study provided substantial evidence that miR-19a-3p played a functional role in MEF2 signaling in myocardial hypertrophy. SFI attenuated cardiomyocyte hypertrophy probably through up-regulating or maintaining the miR-19a-3p levels and regulating the MEF2 signaling pathway.

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction.

Toll-like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3-II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP-GFP-LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene-knockdown experiments showed that the TRIF-dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up-regulated TLR3 expression and increased TLR3-Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3-KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3-KO-derived protection, as wild-type and TLR3-KO hearts were comparable in inflammatory activity. It is concluded that up-regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.

Flavonoids from the roots of Artocarpus heterophyllus.

Four new flavonoids, artoheteroids A-D (1-4), together with six known ones (5-10), were isolated from the roots of Artocarpus heterophyllus. Their structures were elucidated by spectroscopic methods, including 1D and 2D NMR, UV, IR, CD, and HR-ESI-MS. All isolated compounds were screened for their inhibitory abilities against cathepsin K (CatK). Among them, compounds 1-2, 4-6, and 10 were found to have suppression capabilities against CatK with IC50 values ranging from 1.4 to 93.9µM.

MiR-126 Suppresses the Glucose-Stimulated Proliferation via IRS-2 in INS-1 ß Cells.

BACKGROUND: Increasing evidence suggests that miR-126 participates in the glucose homeostasis through its target molecules. Although bioinformatics analysis predicts that miR-126 can bind with the insulin receptor substrate-2(IRS-2) mRNA at the "seed sequence", but there are still no definitely reports to support it. In this study, we provided evidences that IRS-2 was one of the target genes of miR-126. And miR-126 has a proliferation inhibiting effects in INS-1 ß cells, mainly through the suppression of IRS-2. METHODS: The 3'-UTR of IRS-2 regulated by miR-126 was analyzed by the luciferase assay and western blot. Furthermore, proliferation of INS-1 ß cells stimulated by glucose was tested, and the association between IRS-2 and miR-126 were analyzed. RESULTS: We found that mutation of only three of the 6 "seed sequences" can eliminate the inhibition effect of miR-126. In INS-1 ß cells, administration of miR-126 suppresses the proliferation, together with the unbalanced down-regulation of IRS-2 and IRS-1. Over-expression of IRS-2 can reverse the proliferation effect of miR-126, while not of IRS-1. These results suggested that miR-126 inhibited the ß-cell proliferation via the inhibition of IRS-2 instead of IRS-1.Additionally, we also found that high glucose and insulin could stimulate the rapid production of endogenous miR-126 within 6 hours, together with the short term suppression of IRS-1 and IRS-2 expression, and intensify the unbalanced expression of IRS-1 and IRS-2. CONCLUSIONS: IRS-2 was one of the targets of miR-126. MiR-126 inhibited the ß-cell proliferation through IRS-2 instead of IRS-1. MiR-126 may take part in the glucose homeostasis both through its target IRS-2 and IRS-1. The unbalance between IRS-1 and IRS-2 caused by miR-126 may play an important role in type 2 diabetes.

Centrally acting drug moxonidine decreases reactive oxygen species via inactivation of the phosphoinositide-3 kinase signaling in the rostral ventrolateral medulla in hypertensive rats.

OBJECTIVE: Centrally acting antihypertensive action of moxonidine is a result of activation of Imidazoline-1 receptor (I1R) in the rostral ventrolateral medulla (RVLM). Hypertension shows an increase in reactive oxygen species (ROS) in the RVLM. The present objective was to determine the phosphoinositide-3 kinase (PI3K) signaling pathway involved in the effect of moxonidine on ROS generation in the RVLM of spontaneously hypertensive rat (SHR). METHODS: Wistar-Kyoto rats and SHR received intracisternal infusion (2 weeks) of tested agents which were subjected to subsequent experiments. In-situ ROS in the RVLM was evaluated by the oxidative fluorescence dye. Western blot and PCR analysis were performed to detect the expression levels of PI3K signaling pathway. Lentivirus was injected bilaterally into the RVLM for silencing PI3K signaling. RESULTS: ROS production in the RVLM was dose-dependently reduced in SHRs treated with infusion of moxonidine (20 nmol/day), which was prevented by the I1R antagonist efaroxan but not by the α2-adrenoceptor antagonist yohimbine. Moxonidine pretreatment significantly blunted cardiovascular sensitivity to injection of tempol (5 nmol) or angiotensin II (10 pmol) into the RVLM in SHR. Expression levels of PI3K/Akt, nuclear factor kappa-B (NFκB), NADPHase (NOX4), and angiotensin type I receptor (AT1R) in the RVLM were markedly decreased in SHR treated with moxonidine. Infection of lentivirus containing PI3K shRNA in the RVLM effectively prevented effects of moxonidine on cardiovascular activity and expression levels of Akt, NFκB, NOX4, and AT1R. CONCLUSION: The centrally antihypertensive drug moxonidine decreases ROS production in the RVLM through inactivation of the PI3K/Akt signaling pathway in hypertension.

Up-regulated TLR4 in cardiomyocytes exacerbates heart failure after long-term myocardial infarction.

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll-like receptors (TLRs) that respond to pathogen- and damage-associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4-mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (Bmax ) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4-blocking antibodies. We conclude that the expression, ligand-binding capacity and pro-inflammatory function of cardiomyocyte TLR4 are up-regulated after long-term MI, which promote inflammation and exacerbate heart failure.

[Chemical Constitutes from Root of Artocarpus styracifolius].

OBJECTIVE: To study the chemical constituents from root of Artocarpus styracifolius. METHODS: Tne constituents were isolated from the root of Artocarpus styracifolius by column chromatography over silica gel, RP-18 silica gel, MCI GEL CHP-20P, macroporous resin HP-20, Sephadex LH-20, Toyopearl HW-40C and by preparative HPLC. Their structures were elucidated by analysis of physical and chemical properties and spectral data. RESULTS: Nine compounds were isolated and their structures were identified as p-hydroxy benzoic acid (1), syringic acid (2), 2,4-dihydroxy benzaldehyde (3), (+)-lyoniresinol (4), 5,5'-dimethoxysecoisolariciresinol (5), (+)- syringaresinol (6), scopoletin (7), xylarolide (8) and trans-oxyresveratrol (9). CONCLUSION: Compounds 2, 5, 6 and 8 are isolated from Moraceae for the first time. Compounds 1, 4 and 7 are firstly characterized in the genus Artocarpus, compounds 3 and 9 are characterized in Artocarpus styracifolius for the first time.

ZBTB20 regulates nociception and pain sensation by modulating TRP channel expression in nociceptive sensory neurons.

In mammals, pain sensation is initiated by the detection of noxious stimuli through specialized transduction ion channels and receptors in nociceptive sensory neurons. Transient receptor potential (TRP) channels are the key sensory transducers that confer nociceptors distinct sensory modalities. However, the regulatory mechanisms about their expression are poorly defined. Here we show that the zinc-finger protein ZBTB20 regulates TRP channels expression in nociceptors. ZBTB20 is highly expressed in nociceptive sensory neurons of dorsal root ganglia. Disruption of ZBTB20 in nociceptors led to a marked decrease in the expression levels of TRPV1, TRPA1 and TRPM8 and the response of calcium flux and whole-cell currents evoked by their respective specific agonists. Phenotypically, the mice lacking ZBTB20 specifically in nociceptors showed a defect in nociception and pain sensation in response to thermal, mechanical and inflammatory stimulation. Our findings point to ZBTB20 as a critical regulator of nociception and pain sensation by modulating TRP channels expression in nociceptors.

Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats.

The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in the brain has been suggested to be beneficial to hypertension. This study was designed to determine the effect of ACE2 gene transfer into the RVLM on tonically active glutamatergic input in spontaneously hypertensive rats (SHRs). Lentiviral particles containing enhanced green fluorescent protein (lenti-GFP) or ACE2 (lenti-ACE2) were injected bilaterally into the RVLM. Both protein expression and activity of ACE2 in the RVLM were increased in SHRs after overexpression of ACE2. A significant reduction in blood pressure and heart rate in SHRs was observed 6 wk after lenti-ACE2 injected into the RVLM. The concentration of glutamate in microdialysis fluid from the RVLM was significantly reduced by an average of 61% in SHRs with lenti-ACE2 compared with lenti-GFP. ACE2 overexpression significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity evoked by bilateral injection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nl) into the RVLM in SHRs. Therefore, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced tonically active glutamatergic input in SHRs, which may be an important mechanism underlying the beneficial effect of central ACE2 to hypertension.

Some new traveling wave exact solutions of the (2+1)-dimensional Boiti-Leon-Pempinelli equations.

We employ the complex method to obtain all meromorphic exact solutions of complex (2+1)-dimensional Boiti-Leon-Pempinelli equations (BLP system of equations). The idea introduced in this paper can be applied to other nonlinear evolution equations. Our results show that all rational and simply periodic traveling wave exact solutions of the equations (BLP) are solitary wave solutions, the complex method is simpler than other methods, and there exist some rational solutions ur,2 (z) and simply periodic solutions us,2-6(z) which are not only new but also not degenerated successively by the elliptic function solutions. We believe that this method should play an important role for finding exact solutions in the mathematical physics. For these new traveling wave solutions, we give some computer simulations to illustrate our main results.

[Salusins and its cardiovascular effects].

Salusins are newly discovered cardiovascular active peptides, including salusin-alpha and salusin-beta, which are peptides containing 28 and 20 amino acids respectively. Salusins are widely distributed in tissuse and organs of human and rat, and have a series of cardiovascular effects, including lowering blood pressure, slowing down the heart rate, inhibiting myocardial contraction, reducing cardiac ischemic injury, and promoting hypertrophy of cardiomyocytes and proliferation of vascular smooth muscle cells. It is noteworthy to mention that salusin-alpha and salusin-beta are polypeptides produced by the same precursor and play opposite roles in the development and progression of atherosclerosis.

Exercise training lowers the enhanced tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats.

AIMS: It is well known that low-intensity exercise training (ExT) is beneficial to cardiovascular dysfunction in hypertension. The tonically active glutamatergic input to the rostral ventrolateral medulla (RVLM), a key region for control of blood pressure and sympathetic tone, has been demonstrated to be increased in hypertensive rats. The aim of this study was to determine the effect of ExT on the increased glutamatergic input to the RVLM in spontaneously hypertensive rat (SHR). METHODS: Normotensive rats Wistar-Kyoto (WKY) and SHR were treadmill trained or remained sedentary (Sed) for 12 weeks and classed into four groups (WKY-Sed, WKY-ExT, SHR-Sed, and SHR-ExT). The release of glutamate in the RVLM and its contribution to cardiovascular activity were determined in WKY and SHR after treatment of ExT. RESULTS: Blood pressure and sympathetic tone were significantly reduced in SHR after treatment with ExT. Bilateral microinjection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nL) into the RVLM significantly decreased resting blood pressure, heart rate, and renal sympathetic nerve activity in SHR-Sed but not in WKY groups (WKY-Sed and WKY-ExT). However, the degree of reduction in these cardiovascular parameters evoked by KYN was significantly blunted in SHR-ExT compared with SHR-Sed group. The concentration of glutamate and the protein expression of vesicular glutamate transporter 2 in the RVLM were significantly increased in SHR-Sed compared with WKY-Sed, whereas they were reduced after treatment with ExT. CONCLUSION: Our findings suggest that ExT attenuates the enhancement in the tonically acting glutamatergic input to the RVLM of hypertensive rats, thereby reducing the sympathetic hyperactivity and blood pressure.

Extracellular HSP60 induces inflammation through activating and up-regulating TLRs in cardiomyocytes.

AIMS: The molecular events leading from cardiomyocyte ischaemia to inflammatory cytokine production are not well understood. We previously found that heat shock protein 60 (HSP60) appeared in extracellular space after cardiomyocyte ischaemia. This study examined the activation and regulation of toll-like receptors (TLRs) by HSP60 in cardiomyocytes. METHODS AND RESULTS: Cytokine production and TLRs regulation mediated by TLRs signalling were examined in response to exogenous HSP60 (exHSP60) and endogenous HSP60 (enHSP60) released extracellularly under ischaemia. The results showed that exHSP60 induced inflammatory cytokine production in adult rat cardiomyocytes and H9c2 cells (a standard cardiac cell line derived from embryonic cells), through a pathway dependent on TLR4, myeloid differentiation factor 88 (MyD88), p38, and nuclear factor-κB (NF-κB). Further study revealed up-regulated expression of both TLR2 and TLR4 by exHSP60, which was dependent on the activation of TLR4, MyD88, c-Jun NH2-terminal kinase (JNK), and NF-κB, but not on p38. In myocytes exposed to ischaemia, enHSP60 was released into the media, and triggered cytokine production and TLR2/4 overexpression, through the same pathways as exHSP60. In rats subjected to LAD ligation, the released enHSP60 contributed to cytokine production and TLR2/4 overexpression in the ischaemic myocardium. CONCLUSION: Extracellular HSP60 induces cytokine production via TLR4-MyD88-p38/NF-κB pathway, and up-regulates TLR2/4 expression via TLR4-MyD88-JNK/NF-κB pathway. Both pathways contribute to myocardial inflammation induced by ischaemia.

Effects of Salusin-ß on action potential and ionic currents in ventricular myocytes of rats.

AIM: Salusin-ß is a regulatory peptide that exerts negative inotropic effect on ventricular muscle, but its electrophysiological effects on ventricular myocytes are still unknown. METHODS: Action potential and channel currents such as sodium current (I(N) (a) ), transient outward potassium current (I(to) ), steady-state potassium current (I(sus) ), sodium-calcium exchange current (I(N) (aCa) ) and inward rectifier potassium current (I(K) (1) ) were measured in ventricular myocytes isolated from 12 to 16 weeks rats by whole-cell voltage-clamp techniques. RESULTS: Salusin-ß dose-dependently shortened the duration of action potential in rat ventricular myocytes. Furthermore, salusin-ß significantly inhibited I(N) (aCa) and increased I(to) , but did not affect I(N) (a) , I(sus) and I(K) (1) . CONCLUSION: These results suggest that the effect of salusin-ß on action potential may be partly attributed to a decrease in inward currents and an increase in outward currents.

The inhibitory effect of miRNA-1 on ET-1 gene expression.

There is a close correlation between endothelin-1 (ET-1) and microRNA-1 (miRNA-1) expression in the heart, but whether ET-1 expression is regulated by miRNA-1 warrants further research. Our results revealed multiple clues suggesting that miRNA-1 may participate in inhibiting ET-1 gene expression. The inhibitory effect of miRNA-1 on recombinant luciferase reporter gene was mediated by the target sequence at the 127th nucleotide on ET-1 3'UTR. We further confirmed that miRNA-1 could inhibit endogenous ET-1 gene expression at the post-transcriptional level. Our study provides a new perspective on the regulatory mechanism of ET-1 gene.

Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes.

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O(2)), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.

Paradoxical effects of streptozotocin-induced diabetes on endothelial dysfunction in stroke-prone spontaneously hypertensive rats.

Although both diabetes and hypertension are risk factors for cardiovascular disease, the role of hyperglycaemia per se in endothelial dysfunction is controversial. This study was designed to examine whether hyperglycaemia, or streptozotocin-induced diabetes, could aggravate endothelial dysfunction in stroke-prone spontaneously hypertensive rats (SHRSP). Hyperglycaemia was induced by streptozotocin in 2-month-old SHRSP and age-matched normotensive Wistar-Kyoto (WKY) rats. The aorta was isolated 8 weeks after induction of hyperglycaemia to record its function and to examine its morphology with transmission electron microscopy. Endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive haem oxygenase (HO-1/HO-2) levels were determined with Western blotting. Aortic endothelial function and production of reactive oxygen species and nitric oxide were assayed after incubation in vitro in hyperglycaemic, hyperosmolar solution. Streptozotocin-induced diabetes of 8 weeks duration did not result in endothelial dysfunction in normotensive WKY rats. In contrast, hyperglycaemic WKY rats showed significantly enhanced endothelium-dependent vasodilatation, which was abrogated by simultaneous blocking of NOS and HO. The enhanced vasodilatation was associated with elevation of vascular eNOS and HO-1. Significant endothelial dysfunction and massive macrophage-monocyte infiltration were found in SHRSP aorta (the ratio of the number of macrophages to endothelial cells in the intima, expressed as a percentage, was 20.9 ± 2.8% in SHRSP versus 1.9 ± 0.5% in WKY rats, P < 0.01), which was attenuated significantly in hyperglycaemic SHRSP (11.3 ± 1.6%, P < 0.01 versus SHRSP). Acute hyperglycaemia (10 min) aggravated endothelial dysfunction in SHRSP, with a marked increase in intracellular reactive oxygen species and NO production. Sustained in vitro incubation in hyperglycaemic/hyperosmolar conditions (addition of an extra 50 mmol L(-1) of glucose or mannitol to the usual buffer, to produce a final osmolarity of 350 mosmol L(-1)) for 5 h enhanced endothelium-dependent vasodilatation, with elevated vessel NO production and upregulation of eNOS/HO-1 proteins. Sustained hyperglycaemia does not aggravate endothelial dysfunction and macrophage infiltration in SHRSP. Hyperglycaemia/hyperosmolarity-induced upregulation of eNOS and HO-1 may play a role in this paradoxical adaptation of endothelial function.

Sildenafil improves diabetic vascular activity through suppressing endothelin receptor A, iNOS and NADPH oxidase which is comparable with the endothelin receptor antagonist CPU0213 in STZ-injected rats.

OBJECTIVES: Abnormal vascular activity in diabetes is related not only to impaired nitric oxide bioavailability but also to inflammatory cytokines, endothelin A receptor (ET(A) ) activation and NADPH oxidase in the vasculature. The potential role of sildenafil in improving vascular function was investigated. Its action was likely blocking upregulated ET(A) and NADPH oxidase, and was compared with the endothelin receptor antagonist CPU0213. METHODS: Diabetes was induced by single-dose administration of streptozotocin (65 mg/kg, i.p.) to rats and the vascular activity of the thoracic aorta was measured. KEY FINDINGS: An increase in contractile tone to phenylephrine and a decrease in relaxant tone to acetylcholine was found in the thoracic aorta. Oxidative stress was evident by increased malondialdehyde and reduced glutathione peroxidase levels in serum and upregulation of ET(A), MMP-9 (matrix metalloproteinase-9), inducible nitric oxide synthase and NADPH oxidase p67(phox) were found in the vascular wall. The vascular abnormalities and abnormal biomarkers were attenuated significantly by either sildenafil or CPU0213 along with an improvement of nitric oxide bioavailability and vascular activity. CONCLUSIONS: Improvement of diabetic vascular abnormal activity by sildenafil results from its suppression of activation of ET(A) and NADPH oxidase in the vasculature, and these actions are comparable with those of the endothelin receptor antagonist CPU0213.