The occurrence of hypogonadotropic hypogonadism in Chinese men with type 2 diabetes.

CONTEXT: The previous studies showed that hypogonadotropic hypogonadism (HH) occurred commonly in men with type 2 diabetes. However, since all the cohorts tested were from American and European studies, the occurrence of HH/nongonadal illness (NGI) in Chinese populations is unclear. OBJECTIVE: The study aimed to explore the occurrence of HH/NGI in Chinese men with type 2 diabetes. Furthermore, the correlative factors and predictors of hypogonadism were investigated. DESIGN: We conducted a cross-sectional study of 637 Chinese men with type 2 diabetes aged 20-75 years in our clinic. The prevalence of HH/NGI was investigated by measuring serum total testosterone (TT), sex hormone-binding globulin (SHBG), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the enrolled subjects. Free testosterone (FT) was calculated by using SHBG and TT levels and hypogonadism was defined as TT lower than 10.4 nmol/L and calculated FT (cFT) lower than 0.225 nmol/L. The LH cut-off value for defining HH/NGI was 9.4 mIU/ml. RESULTS: The results suggested that 31.9% of male Chinese type 2 diabetes patients had hypogonadism and 26.5% of subjects in our cohort were determined as HH/NGI. The occurrence of hypogonadism was markedly correlated with body mass index (BMI). There was a significant association between TT, cFT and SHBG levels with BMI. TT levels are inversely correlated with BMI and homeostasis model assessment-estimated insulin resistance (HOMA-IR) while positively related with SHBG. The cFT levels were inversely correlated with age, LH, FSH, BMI and HOMA-IR. Multiple regression analysis suggested that SHBG, BMI and HOMA-IR were significant predictors of TT and cFT. CONCLUSION: Our present study offered the first evidence that the occurrence of HH/NGI in Chinese male type 2 diabetes was 26.5%. TT and cFT were significantly correlated with BMI, SHBG and HOMA-IR in Chinese men with type 2 diabetes.

Dihydroartemisinin suppresses renal fibrosis in mice by inhibiting DNA-methyltransferase 1 and increasing Klotho.

Renal fibrosis is an unavoidable end result of all forms of progressive chronic kidney diseases (CKD). Discovery of efficacious drugs against renal fibrosis is in crucial need. In a preliminary study we found that a derivative of artemisinin, dihydroartemisinin (DHA), exerted strong renoprotection, and reversed renal fibrosis in adenine-induced CKD mouse model. In this study we investigated the anti-fibrotic mechanisms of DHA, particularly its specific target in renal cells. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) or oral administration of adenine (80 mg · kg-1), the mice received DHA (30 mg · kg-1 · d-1, i.g.) for 14 or 21 days, respectively. We showed that DHA administration markedly attenuated the inflammation and fibrotic responses in the kidneys and significantly improved the renal function in both the renal fibrosis mouse models. In adenine-treated mice, DHA was more effective than 5-azacytidine against renal fibrosis. The anti-fibrotic effects of DHA were also observed in TGF-ß1-treated HK-2 cells. In order to determine the target protein of DHA, we conducted pull-down technology coupled with shotgun proteomics using a small-molecule probe based on the structure of DHA (biotin-DHA). As a results, DNA methyltransferase 1 (DNMT1) was identified as the anti-fibrotic target of DHA in 3 different types of renal cell lines (HK-2, HEK293 and 3T3). We demonstrated that DHA directly bound to Asn 1529 and Thr 1528 of DNMT1 with a Kd value of 8.18 µM. In primary mouse renal tubular cells, we showed that DHA (10 µM) promoted DNMT1 degradation via the ubiquitin-proteasome pathway. DHA-reduced DNMT1 expression effectively reversed Klotho promoter hypermethylation, which led to the reversal of Klotho protein loss in the kidney of UUO mice. This subsequently resulted in inhibition of the Wnt/ß-catenin and TGF-ß/Smad signaling pathways and consequently conferred renoprotection in the animals. Knockdown of Klotho abolished the renoprotective effect of DHA in UUO mice. Our study reveals a novel pharmacological activity for DHA, i.e., renoprotection. DHA exhibits this effect by targeting DNMT1 to reverse Klotho repression. This study provides an evidence for the possible clinical application of DHA in the treatment of renal fibrosis.

UPLC-MS/MS Method for the Determination of Hyperoside and Application to Pharmacokinetics Study in Rat After Different Administration Routes.

A rapid and sensitive UPLC-MS/MS method was developed and fully validated for the quantification of hyperoside in rat plasma after intragastric, intraperitoneal and intravenous administration. Geniposide was used as an internal standard, and simple liquid-liquid extraction by ethyl acetate was utilized for to extracting the analytes from the rat plasma samples. Chromatographic separation was carried out on an InfinityLab Poroshell 120EC-C18column (2.1 mm × 50 mm, 1.9-Micro, Agilent technologies, USA). The mobile phase consisted of methanol (A) and water (B) (containing 0.1% acetic acid) at a flow rate of 0.4 mL/min. A run time of 3 min for each sample made it possible to analyze more than 300 plasma samples per day. The validated linear ranges of hyperoside were 2-1000 ng/mL in rat plasma. The intra-day and inter-day precision were within 2.6-9.3%, and accuracy were ± 8.6%. And the results of recovery and matrix interference studies were well within the accepted variability limits. Finally, this method was fully validated and successfully applied to the pharmacokinetic studies of hyperoside via different administration routes in rats.

Chronopharmacology of simvastatin on hyperlipidaemia in high-fat diet-fed obese mice.

The chronopharmacology refers to the utilization of physiological circadian rhythms to optimize the administration time of drugs, thus increasing their efficacy and safety, or reducing adverse effects. Simvastatin is one of the most widely prescribed drugs for the treatment of hypercholesterolaemia, hyperlipidemia and coronary artery disease. There are conflicting statements regarding the timing of simvastatin administration, and convincing experimental evidence remains unavailable. Thus, we aimed to examine whether different administration times would influence the efficacy of simvastatin. High-fat diet-fed mice were treated with simvastatin at zeitgeber time 1 (ZT1) or ZT13, respectively, for nine weeks. Simvastatin showed robust anti-hypercholesterolaemia and anti-hyperlipidemia effects on these obese mice, regardless of administration time. However, simvastatin administrated at ZT13, compared to ZT1, was more functional for decreasing serum levels of total cholesterol, triglycerides, non-esterified free fatty acids and LDL cholesterol, as well as improving liver pathological characteristics. In terms of possible mechanisms, we found that simvastatin did not alter the expression of hepatic circadian clock gene in vivo, although it failed to change the period, phase and amplitude of oscillation patterns in Per2::Luc U2OS and Bmal1::Luc U2OS cells in vitro. In contrast, simvastatin regulated the expression of Hmgcr, Mdr1 and Slco2b1 in a circadian manner, which potentially contributed to the chronopharmacological function of the drug. Taken together, we provide solid evidence to suggest that different administration times affect the lipid-lowering effects of simvastatin.

[Jujing Gouju Granules promote reproductive function in rats with spermatozoa].

OBJECTIVE: To observe the intervention effect of Jujing Gouju Granules (JGG) on teratozoospermia (TZ) in rats and explore its action mechanism. METHODS: Thirty-two male SD rats were randomly divided into four groups of an equal number: normal control, TZ model control, high-dose JGG and low-dose JGG. The TZ model was established in the latter three groups of rats by intragastric administration of methyl methanesulfonate at 4 mg/100 g body weight/day for 7 consecutive days. After successful modeling, the animals in the high- and low-dose JGG groups were treated with JGG at 0.288 and 0.072g/100 g body weight/d, respectively, while the normal controls with the same dose of normal saline, all for 48 days. At two days after medication, all the rats were sacrificed and the right epididymides harvested for sperm counting, sperm motility analysis, observation the sperm morphology, and determination of contents of fructose, zinc, α-glucosidase and superoxide dismutase (SOD) in the epididymal suspension, the levels of IL-6, IL-8 and TNF-α in the seminal plasma, and that of reactive oxygen species (ROS) in the sperm. RESULTS: Both sperm concentration and motility were significantly increased and the percentage of morphologically abnormal sperm decreased in the JGG groups compared with the model controls, even more significantly in the high- than in the low-dose JGG group (P < 0.05). No statistically significant difference was found in the contents of fructose and zinc in the epididymal suspension among the four groups, but that of α-glucosidase was remarkably lower in the TZ model than in the normal controls (ï¼»50.31 ± 2.12ï¼½ vs ï¼»67.23 ± 3.54ï¼½ U/L, P < 0.05), but higher in the high- and low-dose JGG groups (ï¼»79.36 ± 2.35ï¼½ and ï¼»56.25 ± 3.44ï¼½ U/L) than in the model control group (P < 0.05). The level of ROS was markedly higher and that of SOD lower in the TZ model than in the normal controls, while the former was lower and the latter was higher in the JGG groups than in the TZ model controls (P < 0.05), even more significantly in the in the high- than in the low-dose JGG group (P < 0.05). Compared with the TZ model controls, the rats in the JGG groups showed dramatically decreased levels of IL-6, IL-8 and TNF-α in the seminal plasma, even more significantly in the high- than in the low-dose JGG group (P < 0.05). CONCLUSIONS: Jujing Gouju Granules can improve sperm morphology in teratozoospermia rats, possibly by regulating oxidative stress and inflammation-related factors.

Adenosine kinase inhibition protects against cisplatin-induced nephrotoxicity.

Numerous studies have demonstrated that several mechanisms, including oxidative stress, DNA damage, and inflammatory responses, are closely linked to cisplatin-induced nephrotoxicity. Adenosine, emerging as a key regulatory molecule, is mostly protective in the pathophysiology of inflammatory diseases. A previous study showed that some of the adenosine receptors led to renal protection against ischemia-reperfusion injury. However, these adenosine receptor agonists lack a useful therapeutic index due to cardiovascular side effects. We hypothesized that inhibition of adenosine kinase (ADK) might exacerbate extracellular adenosine levels to reduce cisplatin-induced renal injury. In the present study, pretreatment with the ADK inhibitor ABT-702 could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis, oxidative stress, and inflammation in the kidneys. Consistent with in vivo results, inhibition of ADK suppressed cisplatin-induced apoptosis, reactive oxygen species production, and inflammation in HK2 cells. Additionally, the protective effect of ADK inhibition was abolished by A1 or A2B adenosine receptor antagonist and enhanced by A2A or A3 adenosine receptor antagonist. Collectively, the results suggest that inhibition of ADK might increase extracellular adenosine levels, which inhibited cisplatin-induced oxidative stress and inflammation via A1 and A2B adenosine receptors, finally suppressing cisplatin-induced cell apoptosis. Pharmacological therapies based on ADK will be of potential use in therapy of cisplatin-induced nephrotoxicity.

Ablation of Myeloid ADK (Adenosine Kinase) Epigenetically Suppresses Atherosclerosis in ApoE-/- (Apolipoprotein E Deficient) Mice.

Objective- Monocyte-derived foam cells are one of the key players in the formation of atherosclerotic plaques. Adenosine receptors and extracellular adenosine have been demonstrated to modulate foam cell formation. ADK (adenosine kinase) is a major enzyme regulating intracellular adenosine levels, but its functional role in myeloid cells remains poorly understood. To enhance intracellular adenosine levels in myeloid cells, ADK was selectively deleted in novel transgenic mice using Cre-LoxP technology, and foam cell formation and the development of atherosclerotic lesions were determined. Approach and Results- ADK was upregulated in macrophages on ox-LDL (oxidized low-density lipoprotein) treatment in vitro and was highly expressed in foam cells in atherosclerotic plaques. Atherosclerotic mice deficient in ADK in myeloid cells were generated by breeding floxed ADK (ADKF/F) mice with LysM-Cre (myeloid-specific Cre recombinase expressing) mice and ApoE-/- (apolipoprotein E deficient) mice. Mice absent ADK in myeloid cells exhibited much smaller atherosclerotic plaques compared with controls. In vitro assays showed that ADK deletion or inhibition resulted in increased intracellular adenosine and reduced DNA methylation of the ABCG1 (ATP-binding cassette transporter G1) gene. Loss of methylation was associated with ABCG1 upregulation, enhanced cholesterol efflux, and eventually decreased foam cell formation. Conclusions- Augmentation of intracellular adenosine levels through ADK knockout in myeloid cells protects ApoE-/- mice against atherosclerosis by reducing foam cell formation via the epigenetic regulation of cholesterol trafficking. ADK inhibition is a promising approach for the treatment of atherosclerotic diseases.

Polymorphisms of IKZF3 Gene and Autoimmune Thyroid Diseases: Associated with Graves' Disease but Not with Hashimoto's Thyroiditis.

BACKGROUND/AIMS: The IKZF3 gene encodes a zinc-finger protein that plays an important role in the proliferation and differentiation of B lymphocytes. Autoimmune thyroid diseases (AITDs), mainly include Graves' disease (GD) and Hashimoto's thyroiditis (HT), are probably caused by the aberrant proliferation of B cells. The objective of this study was to explore the association between IKZF3 polymorphisms and AITDs. METHODS: We examined 915 AITD patients (604 GD and 311 HT) and 814 healthy controls. IKZF3 variants (rs2941522, rs907091, rs1453559, rs12150079 and rs2872507) were tested by PCR-ligase detection reaction. RESULTS: It was manifested that that the minor alleles of the five loci increased susceptibility to GD (p<0.05 for rs2941522, and p<0.01 for rs907091, rs1453559, rs12150079 and rs2872507) but in HT patients, these loci showed no significant difference compared with controls. Similarly, the genotype distributions of GD patients manifested obvious differences in all these loci compared with the control group, whereas no statistical differences were observed between HT patients and controls. Furthermore, bioinformatics tools were used to analyze rs1453559, rs12150079 and rs907091. These variants were believed to be the transcription regulator. CONCLUSION: It is the first time we reported the association between the IKZF3 polymorphisms and GD, indicating that IKZF3 gene tends to bean important risk factor for the development of GD.

Hypothalamic DNA methylation in rats with dihydrotestosterone-induced polycystic ovary syndrome: effects of low-frequency electro-acupuncture.

NEW FINDINGS: What is the central question of this study? What is the role of hypothalamic DNA methylation in the development of polycystic ovary syndrome (PCOS) and the response to electro-acupuncture treatment. What is the main finding and its importance? Global DNA methylation and expression of DNA methyltransferases (DNMTs) were increased in PCOS-like rats, and electro-acupuncture (EA) decreased global DNA methylation and DNMT3b expression. Pyrosequencing showed that the DNA methylation of some PCOS candidate genes was changed in the PCOS and PCOS+EA groups, suggesting that hypothalamic DNA methylation plays an important role in the development of PCOS and in mediating the effects of electro-acupuncture treatment. ABSTRACT: Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disease of unknown aetiology. Recently, epigenetic studies focusing on DNA methylation in PCOS have received much attention, but the mechanisms are still unclear. In the present study, we used the 5α-dihydrotestosterone-induced PCOS-like rat model and treated the rats with electro-acupuncture (EA). Rats were randomly divided into four groups - controls, diet-induced obesity, PCOS and PCOS+EA. We examined the reproductive, metabolic and behavioural phenotypes, validated the effect of EA, and explored the role of hypothalamic DNA methylation by analysing the methylation of global DNA and selected candidate genes. The PCOS rats presented with reproductive dysfunctions such as lack of regular oestrous cyclicity, metabolic disorders such as increased body weight and insulin resistance, and depression and anxiety-like behaviours. EA improved the reproductive functions, decreased body weight and improved experimental depressive behaviour. Furthermore, global DNA methylation and the expression of DNA methyltransferases (DNMTs) were increased in PCOS rats compared to the control group, and EA decreased the global DNA methylation and the expression of DNMT3b. In addition, pyrosequencing showed that the DNA methylation of certain CpG sites in targeted genes (Plcg1, Camk2b, Esr2 and Pgr) was increased in the PCOS group, but the DNA methylation of Camk2b and Ar was decreased after EA treatment. These results indicate that hypothalamic DNA methylation might be correlated with the development of PCOS and that EA has an effect on hypothalamic DNA methylation in PCOS rats.

Associations of TNFRSF1A Polymorphisms with Autoimmune Thyroid Diseases: A Case-Control Study.

Previous studies have shown associations of polymorphisms in the tumor necrosis factor (TNF) receptor super family member 1A (TNFRSF1A) gene with several groups of inflammatory and autoimmune related diseases, but associations of TNFRSF1A polymorphisms with autoimmune thyroid diseases (AITD), mainly including two sub-types of Hashimoto's thyroiditis (HT) and Graves' disease (GD), in the Chinese Han population is unclear. A case-control study of 1812 subjects (965 AITD patients and 847 unrelated healthy controls) was conducted to assess AITD associations with five single nucleotide polymorphisms (SNPs), including rs4149576, rs4149577, rs4149570, rs1800693, and rs767455 in the TNFRSF1A gene locus. Genotyping was performed and evaluated using the platform of ligase detection reaction. No significant difference was observed in the allele and genotype frequencies between HT or GD patients and controls in any of the five SNPs in the TNFRSF1A gene (all p values >0.05). However, a moderate association of rs4149570 with HT was found after adjusting for age and gender [odds ratio (OR)=1.40, p=0.03]. No obvious difference was found in the haplotype distribution of any of the five SNPs in the TNFRSF1A gene between the AITD patients and controls. These data suggest that these five SNPs in the TNFRSF1A gene are not associated with AITD in the Chinese Han population, but rs4149570 shows a weak association with HT after adjusting for gender and age.

Co-circulation of multiple genotypes of influenza A (H7N9) viruses in eastern China, 2016-2017.

Five epidemic waves of human infection with influenza A (H7N9) virus have emerged in China since spring 2013. We previously described the epidemiological characterization of the fifth wave in Jiangsu province. In this study, 41 H7N9 viruses from patients and live-poultry markets were isolated and sequenced to further elucidate the genetic features of viruses of the fifth wave in Jiangsu province. Phylogenetic analysis revealed substantial genetic diversity in the internal genes, and 18 genotypes were identified from the 41 H7N9 virus strains. Furthermore, our data revealed that 41 isolates from Jiangsu contained the G186V and Q226L/I mutations in their haemagglutinin (HA) protein, which may increase the ability of these viruses to bind the human receptor. Four basic amino acid insertions were not observed in the HA cleavage sites of 167 H7N9 viruses from Jiangsu, which revealed that highly pathogenic avian influenza (HPAI) H7N9 viruses did not spread to Jiangsu province in the fifth wave. These findings revealed that multiple genotypes of H7N9 viruses co-circulated in the fifth wave in Jiangsu province, which indicated that the viruses have undergone ongoing evolution with genetic mutation and reassortment. Our study highlights the need to constantly monitor the evolution of H7N9 viruses and reinforce systematic influenza surveillance of humans, birds, and pigs in China.

Pink1/Parkin-mediated mitophagy play a protective role in cisplatin induced renal tubular epithelial cells injury.

Cisplatin often causes acute kidney injury (AKI) in the treatment of a wide variety of malignancies. Mitochondrial dysfunction is one of the main reasons for cisplatin nephrotoxicity. Previous study showed that Pink1 and Parkin play central roles in regulating the mitophagy, which is a key protective mechanism by specifically eliminating dysfunctional or damaged mitochondria. However, the mechanisms that modulate mitophagy in cisplatin induced nephrotoxicity remain to be elucidated. The purpose of this study was to investigate the effects of Pink1/Parkin pathway in mitophagy, mitochondrial dysfunction and renal proximal tubular cells injury during cisplatin treatment. In cultured human renal proximal tubular cells, we found that knockdown of Pink1/Parkin induced the aggravation of mitochondrial function, leading to the increase of cell injury through inhibition of mitophagy. Additionally, the overexpression of Pink1/Parkin protected against cisplatin-induced mitochondrial dysfunction and cell injury by promoting mitophagy. Our results provide clear evidence that Pink1/Parkin-dependent mitophagy has identified potential targets for the treatment of cisplatin-induced AKI.

Plasma heparanase is associated with blood glucose levels but not urinary microalbumin excretion in type 2 diabetic nephropathy at the early stage.

AIM: To explore the possible correlations between plasma heparanase and albuminuria, glucose and lipid metabolism in the type 2 diabetic nephropathy patients at the early stage. METHODS: One hundred and forty patients with type 2 diabetic nephropathy at early stage were recruited into the study. Plasma heparanase and the characterized advanced glycation end products (AGEs), carboxymethyllysine (CML) were measured by enzyme-linked immunosorbent assay. RESULTS: Plasma heparanase was positively associated with fasting blood glucose (R = 0.24, p = .01) while heparanase was not significantly correlated with the urinary microalbumin to creatinine ratio (urinary mAlb/Cr) (R = 0.05, p = .58) and CML (R = 0.16, p = .26). On stepwise linear regression analysis, fasting blood glucose was the main independent determinants of plasma heparanase concentration. CONCLUSION: Plasma heparanase is not significantly associated with urinary mAlb/Cr while it is correlated positively with blood glucose levels in the early stage of diabetic nephropathy. Plasma heparnase might be regarded as a marker for vascular endothelial cells injury in diabetic patients.

Activation of Cold-Sensitive Channels TRPM8 and TRPA1 Inhibits the Proliferative Airway Smooth Muscle Cell Phenotype.

PURPOSE: Airway smooth muscle cell (ASMC) phenotypic modulation is one of the key factors contributing to asthma. Temperature changes may induce asthma, and these changes are known to be related to the temperature-sensitive transient receptor potential channels (TS-TRPs). The present study was designed to investigate the cellular functions of cold-sensitive channels, TRPM8 and TRPA1, in the phenotypic modulation of ASMCs. METHODS: A rat asthma model was constructed and the expression of TS-TRPs in ASM was tested. Using the agonists and antagonists for both TRPM8 and TRPA1, the effects of cold-sensitive channels on the phenotypic modulation of ASMCs were evaluated by measurement of contractile protein expression and cell proliferation and migration. Signaling pathways and matrix metalloproteinase-2 (MMP-2) activity were assayed with Western blotting and gelatin zymography. RESULTS: TRPM8 and TRPA1 were decreased in the ASM of the rat asthma model. Icilin and menthol, agonists for TRPM8 and TRPA1, inhibited ASMC proliferation and migration induced by fetal bovine serum (FBS) or platelet-derived growth factor (PDGF). Moreover, icilin reversed the FBS-induced inhibition of the expression of contractile phenotype markers, smooth muscle α-actin, and SM22α. Icilin also antagonized the activation of p38 and MMP-2 and the repression of p21 caused by FBS. CONCLUSIONS: Our findings show, for the first time, that the activation of TRPM8 and TRPA1 inhibits ASMC proliferative phenotype. These data suggest that TRPM8 and TRPA1 agonists may be promising new therapies for asthma.

Endoplasmic reticulum stress and its effects on renal tubular cells apoptosis in ischemic acute kidney injury.

Ischemia is the most frequent cause of acute kidney injury (AKI), which is characterized by apoptosis of renal tubular cell. A common result of ischemia in AKI is dysfunction of endoplasmic reticulum (ER), which causes the protein-folding capacity to lag behind the protein-folding load. The abundance of misfolded proteins stressed the ER and results in induction of the unfolded protein response (UPR). While the UPR is an adaptive response, over time it can result in apoptosis when cells are unable to recover quickly. Recent research suggests that ER stress is a major factor in renal tubular cell apoptosis resulting from ischemic AKI. Thus, ER stress may be an important new progression factor in the pathology of ischemic AKI. In this article, we review UPR signaling, describe pathology and pathophysiology mechanisms of ischemic AKI, and highlight the dual function of ER stress on renal tubular cell apoptosis.

TNFSF4 Gene Variations Are Related to Early-Onset Autoimmune Thyroid Diseases and Hypothyroidism of Hashimoto's Thyroiditis.

The aim of the current study was to examine whether the polymorphism loci of the tumor necrosis factor superfamily member 4 (TNFSF4) gene increase the risk of susceptibility to autoimmune thyroid diseases (AITDs) in the Han Chinese population, and a case-control study was performed in a set of 1,048 AITDs patients and 909 normal healthy controls in the study. A total of four tagging single nucleotide polymorphisms (SNPs) in the TNFSF4 region, including rs7514229, rs1234313, rs16845607 and rs3850641, were genotyped using the method of ligase detection reaction. An association between GG genotype of rs3850641 in TNFSF4 gene and AITDs was found (p = 0.046). Additionally, the clinical sub-phenotype analysis revealed a significant association between GG genotype in rs7514229 and AITDs patients who were ≤18 years of age. Furthermore, rs3850641 variant allele G was in strong association with hypothyroidism in Hashimoto's thyroiditis (HT) (p = 0.018). The polymorphisms of the TNFSF4 gene may contribute to the susceptibility to AITDs pathogenesis.

[Treatment of Early Diabetic Retinopathy by Liuwei Dihuang Pill Combined Ginkao Leaf Tablet].

OBJECTIVE: To observe the prevention and clinical efficacy of combination of Liuwei Dihuang Pill (LDP) and Ginkgo Leaf Tablet (GLT) for early diabetic retinopathy (DR). METHODS: Using randomized, double-blind, double simulation, parallel controlled clinical trial, 140 type 2 diabetes mellitus (T2DM) outpatients were recruited and assigned to the treatment group and the control group, 70 in each group. All patients received basic Western medicine treatment (such as blood glucose and pressure control). Patients in the treatment group took LDP (8 pills each time, 3 times per day) and GLT (19.2 mg each time, 3 times per day), while those in the control group took LDP placebos and GLT placebos. All treatment lasted for 24 consecutive months. All subjects were followed-up every month. The general clinical data as sex, age, and metabolic data such as blood glucose, blood pressure, blood lipid, and DR prevalence rate were collected and statistically analyzed. RESULTS: There was no significant difference in levels of blood glucose, blood pressure, or blood lipid between the two groups (P > 0.05). After treatment the DR incidence rate was significantly lower in the treatment group than in the control group [3.1% (2/64) vs 18.6% (11/59), P < 0.05)]. Meanwhile, the DR prevalence rate of the treatment group was also significantly lower than that of the control group [6.3% (4/64) vs 20.0% (13/59), P < 0.05]. CONCLUSION: Combination of LDP and GLT could effectively prevent and treat the development of DR in T2DM patients.

Endothelial PFKFB3 plays a critical role in angiogenesis.

OBJECTIVE: Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3) is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. APPROACH AND RESULTS: Under hypoxic conditions or after treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. In addition, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated protein kinase B was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. CONCLUSIONS: The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis.

Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase.

Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward para-nitrophenyl phosphate, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase-specific sequence of STEP were required for ERK interaction. In addition to the N-terminal kinase-specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. Regulation of phospho-ERK by STEP underlies important neuronal activities. A detailed enzymologic characterisation and cellular studies of STEP revealed that specific residues in KIM and active site mediated ERK recognition. Structural differences between the KIM-ERK interfaces and the active site among different ERK phosphatases could be targeted to develop specific STEP inhibitor, which has therapeutic potential for neurological disorders. PKA, protein kinase A & NGF, nerve growth factor.

Platelet IκB kinase-ß deficiency increases mouse arterial neointima formation via delayed glycoprotein Ibα shedding.

OBJECTIVE: On the luminal surface of injured arteries, platelet activation and leukocyte-platelet interactions are critical for the initiation and progression of arterial restenosis. The transcription factor nuclear factor-κB is a critical molecule in platelet activation. Here, we investigated the role of the platelet nuclear factor-κB pathway in forming arterial neointima after arterial injury. METHODS AND RESULTS: We performed carotid artery wire injuries in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice with a platelet-specific deletion of IκB kinase-ß (IKKß) (IKKß(fl/fl)/PF4(cre)/LDLR(-/-)) and in control mice (IKKß(fl/fl)/LDLR(-/-)). The size of the arterial neointima was 61% larger in the IKKß(fl/fl)/PF4(cre)/LDLR(-/-) mice compared with the littermate control IKKß(fl/fl)/LDLR(-/-) mice. Compared with the control mice, the IKKß(fl/fl)/PF4(cre)/LDLR(-/-) mice exhibited more leukocyte adhesion at the injured area. The extent of glycoprotein Ibα shedding after platelet activation was compromised in the IKKß-deficient platelets. This effect was associated with a low level of the active form of A Disintegrin And Metalloproteinase 17, the key enzyme involved in mediating glycoprotein Ibα shedding in activated IKKß-deficient platelets. CONCLUSIONS: Platelet IKKß deficiency increases the formation of injury-induced arterial neointima formation. Thus, nuclear factor-κB-related inhibitors should be carefully evaluated for use in patients after an arterial intervention.