广藿香醇对幽门螺杆菌酸抵抗能力的影响及其机制探讨

目的观察广藿香醇对于幽门螺杆菌(Hp)酸抵抗能力的影响,并探讨其机制。方法体外培养并鉴定Hp后随机分为5组,受试药高、中、低剂量组分别加入20、10、5mg/L广藿香醇,阳性对照组加入尿素酶(Ure)抑制剂AHA,空白对照组加入等体积DMSO;干预0.5h后应用琼脂稀释法测定Hp菌落数,观察其抗酸能力。通过大肠杆菌系统表达和纯化得到Hp尿素酶A(UreA)、UreB、UreG,取UreA/UreB蛋白混合溶液体外模拟Ure成熟过程,将UreG蛋白单体溶液和镍离子(Ni^(2+))溶液混合,均分别以20、10、5mg/L广藿香醇(受试药高、中、低剂量组)和等体积DMSO处理(对照组);采用Berthelot显色法检测Ure活性,判断Ure成熟度;以原子吸收光谱法检测Ni^(2+)浓度,判断UreG携带Ni^(2+)的能力。结果在中性环境中,受试药各剂量组和阳性对照组与空白对照组Hp菌落数比较无统计学差异;在酸性环境中,受试药各剂量组和阳性对照组均较空白对照组Hp菌落数减少(P均<0.01),且受试药高剂量组、阳性对照组<受试药中剂量组<受试药低剂量组(P均<0.01)。受试药高、中、低剂量组和对照组Ure活性分别为54.89%±11.47%、63.99%±9.46%、83.88%±11.81%、101.53%±2.55%,Ni^(2+)浓度分别为(0.77±0.24)、(0.80±0.22)、(1.12±0.35)、(1.56±0.42)μmol/L,受试药高、中剂量组<受试药低剂量组<对照组(P均<0.01)。结论广藿香醇可以降低Hp的酸抵抗能力,可能与阻断Ni^(2+)的传递途径和Ure的成熟过程进而抑制Ure活性有关。

AMPK/mTOR通路参与脓毒症亮氨酸抵抗的研究进展

脓毒症可导致机体蛋白质合成显著抑制,产生亮氨酸抵抗。蛋白质合成由m TOR磷酸化核糖体蛋白p70S6激酶1(p70S6K1)和真核细胞翻译抑制因子4E结合蛋白1(e IF4E-BP1)进行调控,AMPK作为负向调节m TOR的重要信号分子,在脓毒症状态下存在活性异常。文中就AMPK/m TOR通路与脓毒症亮氨酸抵抗的相关研究进展进行综述。

Effect of Milrinone Induced Insulin Resistance on Glucose and Lipid Metabolism in Rats

Aim To investigate the effects of milrinone (a selective phosphodiesteraseIII inhibitor PDE_3 ) on insulin secretion, blood glucose, plasma free fatty acids (FFA) anddose-response relationship, and assess possible effects of milrinone on glucose metabolism andinsulin sensitivity in conscious rats. Methods The catheterized nonstressed rats were administeredvarious doses of milrinone (1, 5, 25μmoL·kg^(-1)) and were compared with controls. Ahyperinsulinaemic-eugly-caemic clamp was established in counscious rats, andmilrinone(25μmoL·kg^(-1)) and 25% dimethyl sulfoxide (DMSO, as a control) were given at 120 minduring hyperinsulinaemic-euglycaemic clamping. Glucose turnover was determind with by gaschromatograph mass spectrometer (GC-MS). Results After dosing, plasma FFA levels in 3 milrinonegroups significantly increased, compared with the controls and before dosing. The percentages ofelevation of FFA by the different milrinone doses were very similar, 50%, and 52% , 55% for 1, 5,and 25 μmoL·kg^(-1), repectively, at 2 min after dosing. Plasma insulin levels were significantlyelevated in the 5 and 25 μmoL·kg^(-1) groups, and the effect of milrione on glucose concentrationwas detectable only in 25μmoL·kg^(-1) group. During hyperinsulinaemic clamping, there weresignificant increase, in plasma FFA (from 173 +- 15 to 634 +- 87μmoL·kg^(-1)) and hepatic glucoseproduction (HGP), and a significant decrease in glucose infusion rates (GIR) to about 21% and aslight increase in plasma insulin after milrinone treatment. Conclusion Milrinone impaires theability of insulin to suppress lipolysis and HGP, and insulin-mediated glucose utilization inperipheral tissue. Therefore, milrinone administration may induce an acute insulin resistance invivo.

Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ

AIM: To investigate the effects and molecular mechanisms of berberine on improving insulin resistance induced by free fatty acids (FFAs) in 3T3-L1 adipocytes. METHODS: The model of insulin resistance in 3T3-L1 adipocytes was established by adding palmic acid (0.5 mmol/L) to the culture medium. Berberine treatment was performed at the same time. Glucose uptake rate was determined by the 2-deoxy-[3H]-D-glucose method. The levels of IkB kinase beta (IKKβ) Ser181 phosphorylation, insulin receptor substrate-1(IRS-1) Ser307 phosphorylation, expression of IKKβ, IRS-1, nuclear transcription factor kappaB p65 (NF-κB p65), phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 4 (GLUT4) proteins were detected by Western blotting. The distribution of NF-κB p65 proteins inside the adipocytes was observed through confocal laser scanning microscopy (CLSM). RESULTS: After the intervention of palmic acid for 24 h, the insulin-stimulated glucose transport in 3T3-L1 adipocytes was inhibited by 67%. Meanwhile, the expression of IRS-1 and PI-3K p85 protein was reduced, while the levels of IKKβ Ser181 and IRS-1 Ser307 phosphorylation, and nuclear translocation of NF-κB p65 protein were increased. However, the above indexes, which indicated the existence of insulin resistance, were reversed by berberine although the expression of GLUT4, IKKβ and total NF-κB p65 protein were not changed during this study. CONCLUSION: Insulin resistance induced by FFAs in 3T3-L1 adipocytes can be improved by berberine. Berberine reversed free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ.

PLASMA RESISTIN LEVELS AND SINGLE-NUCLEOTIDE POLYMORPHISMS IN RESISTIN GENE 5 FLANKING REGION IN PATIENTS WITH STROKE

Objective To analyze the role of resistin in insulin resistance (IR) through investigating the variation of plasma resistin levels and single-nucleotide polymorphisms (SNPs) in resistin gene 5’ flanking region in stroke patients.Methods In 103 atherothrombotic cerebral infarction (ACI) patients, 85 lacunar infarction (LI) patients, 70 intracerebral hemorrhage (ICH) patients, and 86 healthy controls, plasma resistin and insulin levels were measured by ELISA , SNPs in resistin gene 5’ flanking region were detected by PCR and direct DNA sequencing. The subjects’ body height and weight, the body mass index, quantitative insulin sensitivity check index (QUICKI), blood pressure, and the concentration of fasting plasma glucose, triglyceride, total cholesterol, creatinine, low-density lipoprotein, and high-density lipoprotein were also determined. Results QUICKI was significantly lower in the ACI and ICH patients (0.316±0.037 and 0.309±0.032, respectively) than that in the controls (0.342±0.043, P<0.001), while plasma resistin level was significantly higher in the ACI and ICH patients (6.36±3.79 and 7.15±4.27 ng/mL, respectively) than that in the controls (5.28±2.56 ng/mL, P<0.05), but such difference was not observed in the LI patients compared with controls. There was a statistically negative correlation between plasma resistin level with QUICKI (r=-0.228, P<0.001). The distributions of allele and genotype frequencies of resistin gene -420C>G and -537A>C SNPs were not significantly different among the different groups, and those SNPs were not correlated with other clinical and biochemical parameters.Conclusions Plasma resistin is associated with stroke by participating in the development of IR. The SNPs in resistin gene 5’ flanking region has no impact on the plasma resistin level.

New real-time-PCR method to identify single point mutations in hepatitis C virus

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

Emerging role of obeticholic acid in the management of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease(NAFLD) is the commonest chronic liver disease and its prevalence is increasing driven by the pandemic of obesity and type 2 diabetes mellitus. NAFLD can progress to cirrhosis and is associated with increased risk for cardiovascular disease and hepatocellular cancer. Diet and exercise are limited by suboptimal long-term adherence in patients with NAFLD. On the other hand, current pharmacological treatment of NAFLD has limited efficacy and unfavorable safety profile. In this context, obeticholic acid(OCA), a selective agonist of the farnesoid X receptors, might represent a useful option in these patients. Preclinical studies suggest that OCA improves hepatic steatosis, inflammation and fibrosis. A proof-of-concept study and the randomized, placebo-controlled Farnesoid X Receptor Ligand Obeticholic Acid in non-alcoholic steatohepatitis Treatment(FLINT) trial also showed improvements in liver histology in patients with NAFLD who received OCA. Weight loss and reduction in blood pressure were also observed. However, the effects of OCA on insulin resistance are conflicting and the lipid profile is adversely affected by this agent. In addition, pruritus is frequently observed during treatment with OCA and might lead to treatment discontinuation. However, given the limitations of existing treatments for NAFLD, OCA might represent a useful therapeutic option in selected patients with NAFLD.

Fatty Acid Composition Analyses of the DCMU Resistant Mutants of Nannochloropsis oculata(Eustigmatophyceae)

Ultraviolet mutagenesis was applied to Nannochloropsis oculata and three mutants resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were isolated. The cellular chlorophyll a and total lipid content of the wild are higher in the medium supplemented with DCMU than in the control without DCMU. Without DCMU, the growth rates and chlorophyll a contents of the mutants are similar to those of the wild. Significant changes of fatty acid content and composition have occurred in DCMU-resistant mutants growing in the medium supplemented with DCMU. The total lipid, palmitic acid (16∶0), palmitoleic acid (16∶1ω9) and oleic (18∶1ω9) contents decrease significantly, while the vaccenic acid (18∶1ω11) increases significantly and the EPA content of dried powder increases slightly in the mutants. The study may provide a basis to improve EPA content in Nannochloropsis oculata in the future.

Effect of puerarin on the P13K pathway for glucose transportation and insulin signal transduction in adipocytes

To explore the effect of puerarin on insulin receptor （IR）, insulin receptor substrate-1 （IRS-1） and protein expression of protein kinase B （PKB） in the P13K pathway of the glucose consumption, transportation and insulin signal transduction in 3T3-L1 adipoeytes with insulin resistance. The insulin resistance 3T3-L1 adipocytes model was established by free fatty acid induction. The model cells were managed with puerarin in different concentrations. Glucose consumption was detected with glucose oxidase method, glucose transportation rate was determined by 2-deoxy-^3H glucose ingesting method, and the IR, IRS-1 and PKB expression were determined by Western blot. Glucose consumption and transportation were significantly decreased in the model adipoeytes, but increased after treated with puerarin （P 〈 0. 01 ）. Moreover, the level of tyrosine phosphorylation of IR subunit β was higher in the puerarin treated groups, and that of IRS-1 was higher in the group treated with low dose puerarin than that in the model group. The 3T3-L1 adipocytes of insulin resistance model could be induced by free fatty acid successfully, puerarin could promote the glucose utilization in them to alleviate the in- sulin resistance, which may be related with the action in advancing the tyrosine phosphorylation of IR and IRS-1.

Saturated Fatty Acid Induces Insulin Resistance Partially Through Nucleotide-binding Oligomerization Domain 1 Signaling Pathway in Adipocytes

Objective To investigate the potential role of nucleotide-binding oligomerization domain 1 （NOD1）, a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes. Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB （NF-κB） activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[SH] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA （siRNA） targeting NOD 1 upon fatty acids treatment were analyzed. Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-R, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD 1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake. Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

Analysis and clinical applications of metabolic syndrome risk factors for cardiovascular

Metabolic syndrome had many different names, including syndrome X, insulin resistance syndrome. At present the cause of metabolic syndrome is unclear, it may come from two aspects： First, acquired, including being overweight or obese, reduced physical activity and excessive carbohydrate diet; Second, genetic factors, involving multiple genes, not yet fully elucidated. The syndrome is generally believed to be the collection of a variety of cardiovascular risk factors caused by poor lifestyle under the genetic background, including hypertension, dyslipidemia, abdominal obesity, hyperinsulinemia, microalbuminuria, hypercoagulable state, hyperhomocysteinemia and so on. Hyperinsulinemia and insulin resistance is the central link, which is closely related to dyslipidemia, impaired glucose tolerance, and abdominal obesity. Metabolic syndrome may eventually lead to atherosclerosis： coronary artery disease, myocardial infarction, stroke, peripheral vascular disease and endothelial dysfunction. In 1999, the working definition of World Health Organization （WHO） to the metabolic syndrome is： glucose regulation impairment or diabetes, and / or insulin resistance, accompanied by the other two items or more ingredients, such as hypertension, high triglycerides esters hyperlipidemia and / or low HDL cholesterol, central obesity or microalbuminuria.

Research progress in the heat resistance, toughening and filling modification of PLA

Due to its high strength, high modulus, excellent clarity, good biodegradability and biocompatibility, poly(lactic acid)(PLA), a bio-based thermoplastic polyester, has evolved into a competitive commodity material with potential to replace conventional petrochemical-based polymers. However, the wide applications of PLA have been hampered by its native drawbacks, such as low heat distortion temperature(HDT), inherent brittleness and relatively high cost. In recent years, researchers have devoted to breaking above-mentioned bottleneck and attempted to extend the application of PLA. This review will summarize recent work about the modification of PLA, especially focusing on enhancing HDT, toughening and reducing cost.