LC-MS/MS定量检测小鼠尿液RNA氧化标志物8-oxoGsn

目的建立快速高效的液相色谱-串联质谱法(LC-MS/MS)用于测定小鼠尿液中RNA氧化标志物8-氧化鸟苷(8-oxoGsn)水平。方法小鼠尿液37℃孵育离心后,用70%甲醇溶液(含30%10 mmol/L醋酸铵)1∶10稀释尿液上清,加入同位素内标[13C,15N2]8-oxoGsn,用Agilent ZORBOX SB-Aq色谱柱(100 mm×3 mm,1.8μm)分离,以含0.1%甲酸的10 mmol/L醋酸铵水溶液及甲醇溶液为流动相梯度洗脱,柱温35℃,流速0.3 mL/min,进样量5μL,电喷雾离子源正离子模式,多反应监测。结果8-oxoGsn响应和水平的线性关系良好,R^(2)为0.996,定量检测限为0.25 ng/mL,加标回收率86.21%~112.56%,基质效应为-5.01%,日内和日间变异系数分别为3.38%~7.17%、2.74%~9.52%,满足生物样品的分析要求;小鼠尿液检测结果显示C57小鼠对乙酰氨基酚(APAP)处理组8-oxoGsn水平明显高于C57对照组(P<0.01);乙型肝炎病毒转基因(HBV-Tg)小鼠APAP处理组8-oxoGsn水平明显高于HBV-Tg小鼠对照组(P<0.001)。结论建立的LC-MS/MS灵敏度高,准确可靠,适用于小鼠尿液中8-oxoGsn水平的测定。

小鼠尿中沙美特罗及其代谢物的LC/MS研究

采用较大剂量小鼠灌胃给药 ,得到的尿样经固相提取后用 LC/ APCI/ MS分析 ,根据代谢知识及比较空白尿和阳性尿的响应数据 ,得出了沙美特罗的原型和四种代谢产物。结果表明 :小鼠尿中沙美特罗可能的主要代谢途径为羟基化、甲氧基化以及两者的加合。

降解苯丙氨酸的工程益生菌构建及其喂养苯丙酮尿症小鼠的效果分析

苯丙酮尿症(phenylketonuria,PKU)是一种常染色体隐性遗传病,低蛋白饮食是目前临床治疗的主要方法,而采用工程益生菌治疗尚处于研发阶段。为了探究工程益生菌对PKU的治疗效果,该研究基于大肠埃希菌Nissle 1917(EcN)菌株,构建了一株表达苯丙氨酸裂解酶的工程益生菌EcN-PAL。通过λ-Red同源重组系统在EcN中导入经优化设计的苯丙氨酸裂解酶基因,并通过体外降解实验和小鼠喂养实验验证其功能性。实验结果表明,工程益生菌EcN-PAL成功表达苯丙氨酸裂解酶,并且可降解苯丙氨酸和有效降低PKU小鼠的血液苯丙氨酸浓度。该研究成功构建的工程益生菌EcN-PAL对苯丙氨酸有良好的降解效果,动物实验证实可有效治疗PKU小鼠,为用于PKU患者的益生菌治疗奠定了基础。

Effects of binuclear copper(Ⅱ)threonine complex on blood glucose, lipids and protection of the hearts and kidneys in diabetic mice

Aim To study the effects of binuclear copper （Ⅱ） threonine complex （Cu2 （Thr）4） as analogue of superoxide dismutase （SOD） on blood glucose, blood lipids and vessels of hearts and kidneys in diabetic mice. Methods Diabetic mouse model was established by intraperitioneal injection of alloxan. Low, middle, and high doses of Cu2（Thr）4 at 0.002%, 0.02% and 0.1% were given respectively to diabetic mice following lavage. The fasting blood glucose was determined after the diabetic mice were given Cu2 （Thr）4 for 0, 30, and 45 d. The diabetic mice were killed on the 45th day. Then glycosylated hemoglobin （HbAlc） and blood lipids were assayed, and pathologic changes in hearts and kidneys stained with HE were observed. Results Compared with the control group in which the diabetic mice were given distilled water, the value of blood glucose reduced significantly in middle dose group （P 〈 0.01 ）, followed by that in low dose group （P 〈 0.05）. TC level reduced markedly and HDL level increased significantly in all three treatment groups （P 〈 0.05）. Especially in middle dose group, cardiac muscle fibers were neatly arranged, nucleus and cytoplasm well distributed, glomeruli showing normal structure, cells well distributed and staining being normal. Conclusion Cu2 （Thr）4 reduces blood glucose, regulates blood lipids, and play protective action on the vessels of hearts and kidneys in diabetic mice. The effects of it in middle dose were better than those of other doses.

Effect of vitamin E on oxidative stress status in small intestine of diabetic rat

AIM:To investigate the effect of vitamin E on oxidative stress status in the small intestine of diabetic rats. METHODS:Twenty-four male Wistar rats were randomly divided into three groups:Control (C),non-treated diabetic (NTD) and vitamin E-treated diabetic (VETD) groups. The increases in lipid peroxidation,protein oxidation and superoxide dismutase (SOD) in these three groups was compared after 6 wk. RESULTS:There was no significant difference in catalase activity between NTD and control rats. Compared to NTD rats,the treatment with vitamin E significantly decreased lipid peroxidation and protein oxidation,and also increased catalase activity and SOD. CONCLUSION:The results revealed the occurrence of oxidative stress in the small intestine of diabetic rats. Vitamin E,as an antioxidant,attenuates lipid peroxidation and protein oxidation,and increases antioxidant defense mechanism.

Effects of mycophenolate mofetil vs cyclosporine administration on graft survival and function after islet allotransplantation in diabetic rats

AIM: To develop an experimental model of islet allotransplantation in diabetic rats and to determine the positive or adverse effects of MMF as a single agent. METHODS: Thirty-six male Wistar rats and 18 male Lewis rats were used as recipients and donors respectively. Diabetes was induced by the use of streptozotocin (60 mg/kg) intraperitoneally. Unpurified islets were isolated using the collagenase digestion technique and transplanted into the splenic parenchyma. The recipients were randomly assigned to one of the following three groups: group A (control group) had no immunosuppression; group B received cyclosporine (CsA) (5 mg/kg); group C receivedmycophenolate mofetil (MMF) (20 mg/kg). The animalswere killed on the 12th d. Blood and grafted tissues were obtained for laboratory and histological assessment. RESULTS: Median allograft survival was significantly higher in the two therapy groups than that in the controls (10 and 12 d for CsA and MMF respectively vs 0 d for the control group, P＜0.01). No difference in allograft survival between the CsA and MMF groups was found. However,MMF had less renal and hepatic toxicity and allowed weight gain.CONCLUSION: Monotherapy with MMF for immunosu ppression was safe in an experimental model of islet allotransplantation and was equally effective with cyclosporine, with less toxicity.

Attenuation of graft ischemia-reperfusion injury by urinary trypsin inhibitor in mouse intestinal transplantation

AIM: Ischemia/reperfusion (I/R) injury is one of the major obstacles for intestinal transplantation (ITx). Urinary trypsin inhibitor (Ulinastatin, UTI) suppresses proteases and stabilizes lysosomal membranes. We supposed that Ulinastatin would diminish I/R injury of intestinal graft.METHODS: UTI- treated group and untreated control group were investigated by histological assessment at 1.5, 4, 24, and 72 h after ITx. Myeloperoxidase (MPO)activity was used as the activity of neutrophils, and malondialdehyde (MDA) was used as an index of lipid peroxidation. TNFα and i-NOS mRNA expression in graft tissue were measured by semi-quantitative RT-PCR.CD11b+ Gr1+ cells in graft lamina propria were analyzed by flow cytometry.RESULTS: Histological scores of the graft showed that the tissue injury was markedly attenuated by UTI treatment at different time points after ITx, with reduced MPO and MDA value in the grafts. The expression of TNFα and i-NOS mRNA was profoundly inhibited, while the infiltration of CD11b+ Gr1+ cells into the intestinal graft was decreased in UTI group.CONCLUSION: Urinary trypsin inhibitor attenuates I/R injury in mouse intestinal transplantation by reducing monocytes infiltration and down-regulation of TNFα and i-NOS mRNA expression.

Helicobacter pylori arginase mutant colonizes arginase Ⅱ knockout mice

AIM: To investigate the role of host and bacterial arginases in the colonization of mice by Helicobacter pylori (H.pylori).METHODS: H.pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium,which neutralizes acid.Urease is absolutely essential to H.pylori pathogenesis;therefore,the urea substrate must be in ample supply for urease to work efficiently.The urea substrate is most likely provided by arginase activity,which hydrolyzes L-arginine to L-ornithine and urea.Previous work has demonstrated that H.pylori arginase is surprisingly not required for colonization of wild-type mice.Hence,another in vivo source of the critical urea substrate must exist.We hypothesized that the urea source was provided by host arginase Ⅱ,since this enzyme is expressed in the stomach,and H.pylori has previously been shown to induce the expression of murine gastric arginase Ⅱ.To test this hypothesis,wild-type and arginase (rocF) mutant H.pylori strain SS1 were inoculated into arginase Ⅱ knockout mice.RESULTS: Surprisingly,both the wild-type and rocF mutant bacteria still colonized arginase Ⅱ knockout mice.Moreover,feeding arginase Ⅱ knockout mice the host arginase inhibitor S-(2-boronoethyl)L-cysteine (BEC),while inhibiting > 50% of the host arginase Ⅰ?activity in several tissues,did not block the ability of the rocF mutant H.pylori to colonize.In contrast,BEC poorly inhibited H.pylori arginase activity.CONCLUSION: The in vivo source for the essential urea utilized by H.pylori urease is neither bacterial arginase nor host arginase Ⅱ;instead,either residual host arginase Ⅰ?or agmatinase is probably responsible.

Effect of lysozyme chloride on betel quid chewing aggravated gastric oxidative stress and hemorrhagic ulcer in diabetic rats

AIM： To evaluate the protective effect of lysozyme chloride on betel quid chewing （BQC） aggravated gastric oxidative stress and hemorrhagic ulcer in rats with diabetes mellitus （DM）. METHODS： Male Wistar rats were challenged intravenously with streptozotocin （65 mg/kg） to induce DM. Rats were fed with regular pellet food or BQC-containing diets. Afcer 90 d, rats were deprived of food for 24 h. Rat stomachs were irrigated for 3 h with normal saline or simulated gastric juice. Rats were killed and gastric specimens were harvested. RESULTS： An enhancement of various gastric ulcerogenic parameters, including acid back-diffusion, mucosal lipid peroxide generation, as well as decreased glutathione levels and mucus content, were observed in DM rats. Afcer feeding DM rats with BQC, an exacerbation of these ulcerogenic parameters was achieved. Gastric juice caused a further aggravation of these ulcerogenic parameters. Daily intragastric lysozyme chloride dose-dependently inhibited exacerbation of various ulcerogenic parameters in those BQC-fed DM rats. CONCLUSION： （1） Gastric juice could aggravate both DM and BQC-fed DM rat hemorrhagic ulcer; （2） BQC exacerbated gastric hemorrhagic ulcer in DM rats via enhancing oxidative stress and reducing defensive factors; （3） lysozyme chloride effectively protected BQC aggravated gastric damage in DM rats.

Ultrastructure and histochemistry of rat myocardial capillary endothelial cells in response to diabetes and hypertension

Insufficient growth and rarefaction of capillaries, followed by endothelial dysfunction may represent one of the most critical mechanisms involved in heart damage. In this study we examined histochemical and ultrastructural changes in myocardial capillary endothelium in two models of heart failure streptozotocin-induced diabetes mellitus （STZ） and NO-deficient hypertension in male Wistar rats. Diabetes was induced by a single i.v. dose of STZ （45 mg/kg） and chronic 9-week stage was analysed. To induce NO-deficient hypertension, animals were treated with inhibitor of NO synthase Lnitroarginine methylester （L-NAME） （40 mg/kg） for 4 weeks. Left ventricular tissue was processed for enzyme catalytic histochemistry of capillary alkaline phosphatase （A1Ph）, dipeptidyl peptidase IV （DPP IV）, and endothelial NO synthase/NADPH-diaphorase （NOS） and for ultrastructural analysis. In diabetic and hypertensive rats, lower/absent A1Ph and DPP IV activities were found in focal micro-areas. NOS activity was significantly reduced and persisted only locally. Quantitative evaluation demonstrated reduction of reaction product intensity of A1Ph, DPP and NOS by 49.50%,74.36%, 20.05% in diabetic and 62.93%, 82.71%, 37.65% in hypertensive rats. Subcellular alterations of endothelial cells were found in heart of both groups suggesting injury of capillary function as well as compensatory processes. Endothelial injury was more significant in diabetic animals, in contrast the adaptation was more evident in hypertensive ones. Concluding： both STZ-induced diabetes- and NO-deficient hypertension-related cardiomyopathy were accompanied by similar features of structural remodelling of cardiac capillary network manifested as angiogenesis and angiopathy. The latter was however, predominant and may accelerate disappearance of capillary endothelium contributing to myocardial dysfunction.

Screening and functional evaluation of the glucose-lowering active compounds of total saponins of Baibiandou(Lablab Semen Album)

Objective To screen forα-glucosidase inhibitor active compounds in the total saponins of Baibiandou(Lablab Semen Album)based on UHPLC-Q-Exactive Orbitrap MS technology and to evaluate its hypoglycemic activity in vivo.Methods Acarbose was used as the positive control,and the median inhibitory concentration(IC50)was used as the evaluation index ofα-glucosidase inhibitory activity to establish an in vitroα-glucosidase inhibition model.Further,UHPLC-Q-Exactive Orbitrap MS technology was used to screen and identify the active compounds ofα-glucosidase inhibitors in the total saponins of Baibiandou(Lablab Semen Album)in order to further verify the activity of the main active monomer and to perform homologous modeling and molecular docking of yeast-derivedα-glucosidase and human-derivedα-glucosidase,while the hypoglycemic activity was evaluated in diabetic mice.Results This study successfully identified 15 compounds with potentialα-glucosidase inhibitory activity,including Chikusetsusaponin IVa,from the total saponins of Baibiandou(Lablab Semen Album).Simultaneously,we verified the activity of the main active monomer Chikusetsusaponin IVa,and showed that it has strongα-glucosidase inhibitory activity.Theα-glucosidase inhibitory concentration IC50 was(565.2±1.026)μg/m L,and the IC50 of acarbose,which was lower than the positive control,was(706.6±1.058)μg/m L.The docking energies of Chikusetsusaponin IVa were–6.1 and–7.7 kcal/mol with yeast-derivedα-glucosidase and human-derivedα-glucosidase molecules,respectively.Both showed strong binding activity,and the levels of alanine aminotransaminase(ALT),aspartate aminotransaminase(AST),UREA,Creatinine(CREA),and cholesterol(CHO)were significantly decreased by Chikusetsusaponin IVa(P<0.05).In addition,it could repair damaged liver and pancreas cells of diabetic mice to some extent.Conclusion This study provides a basis for screeningα-glucosidase inhibitors and structural modifications of the total saponins of Baibiandou(Lablab Semen Album).

DIFFERENTIAL RENAL GENE EXPRESSION IN EXPERIMENTAL DIABETIC RATS AFTER ASTRAGALUS MEMBRANACEUS TREATMENT

Objective To find the genes involved in pathogenesis of diabetic nephropathy using gene chip technology. Methods We established a type I diabetic rat model by streptozotocin injection and divided these diabetic rats into two groups： diabetic rats group（ D group） and diabetic rats group treated with Astragalus Membranaceus （ DA group）. The renal tissue was collected and total RNA was extracted for gene chips. With the help of gene chip, we tried to discover the differential-displayed genes between these two groups. Results Totally 201 differential-displayed genes were found between the two groups, among which 126 genes were up-regulated and 75 genes were down-regulated in the rat renal tissue. Conclusion With gene chip results, we find several genes which are associated with diabetes in the rat renal tissue. The further research on the function of these genes will be helpful to understand the mechanism of diabetic nephropathy.

Effects of 95%ethanol extract of Aquilaria sinensis leaves on hyperglycemia in diabetic db/db mice

The leaves of Aquilaria sinensis（Lour.） Gilg have been used in folk medicine for the treatment of diabetes in Guangdong Province,China.In this study,effects of ethanol extract of Aquilaria sinensis leaves on hyperglycemia were investigated in diabetic db/db mice.After 4 weeks of administration,the 95%ethanol（EtOH） extract of Aquilaria sinensis leaves （AE）,especially at high dose level（600 mg/kg）,activated AMP-activated protein kinase（AMPK）,resulting in reduced fasting blood glucose and glycosylated hemoglobin levels in db/db mice.In addition,the oral glucose tolerance test（OGTT test） showed that AE could remarkably improve insulin resistance.Compared to Thiazolinediones（TZDs）,no weight gain was observed after AE administration,which is a severe side effect of TZDs.The data suggested that AE could act as an activator of AMPK,and might be used as an alternative to TZDs in the management of obesity-related diabetes.

Urine metabonomic study for blood-replenishing mechanism of Angelica sinensis in a blood-deficient mouse model

This study aimed at determining the effects of Angelica sinensis(AS) on urinary metabolites in blood deficiency mice and exploring its replenishing blood mechanism. Gas chromatography–mass spectrometry(GC-MS) was applied to detect metabolites in the urine samples in different collection periods. Principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used to investigate the differences in metabolic profiles among control group(CG), blood deficiency model group(MG), AS groups, and Colla Corii Asini group(CCAG). The potential biomarkers were identified based on the variable importance in the projection(VIP), T-test, and National Institute of Standards and Technology(NIST) and mass spectra library. The metabolites were analyzed using metabolomics pathway analysis(Met PA) to build the metabolic pathways. Our results indicated that, on the seventh day, the levels of glucose, lactic acid, pyruvic acid, alanine, acetoacetic acid, and citric acid changed significantly in blood deficiency mice. However, these metabolic deviations came to closer to normal levels after AS intervention. The reversing blood-deficiency mechanism of AS might involve regulating synthesis and degradation of ketone bodies, Pyruvate metabolism, TCA cycle, and Glycolysis / Gluconeogenesis. In conclusion, metabonomics is a robust and promising means for the identification of biomarkers and elucidation of the mechanisms of a disease, thereby highlighting its importance in drug discovery.

SpinalP2X7R contributes to streptozotocin-induced mechanical allodynia in mice

Painful diabetic neuropathy(PDN)is a diabetes mellitus complication.Unfortunately,the mechanisms underlying PDN are still poorly understood.Adenosine triphosphate(ATP)-gated P2X7 receptor(P2X7R)plays a pivotal role in non-diabetic neuropathic pain,but little is known about its effects on streptozotocin(STZ)-induced peripheral neuropathy.Here,we explored whether spinal cord P2X7R was correlated with the generation of mechanical allodynia(MA)in STZ-induced type 1 diabetic neuropathy in mice.MA was assessed by measuring paw withdrawal thresholds and western blotting.Immunohistochemistry was applied to analyze the protein expression levels and localization of P2X7R.STZ-induced mice expressed increased P2X7R in the dorsal horn of the lumbar spinal cord during MA.Mice injected intrathecally with a selective antagonist of P2X7R and P2X7R knockout(KO)mice both presented attenuated progression of MA.Double-immunofluorescent labeling demonstrated that P2X7R-positive cells were mostly co-expressed with Iba1(a microglia marker).Our results suggest that P2X7R plays an important role in the development of MA and could be used as a cellular target for treating PDN.

Intermittent protein restriction protects isletβcells and improves glucose homeostasis in diabetic mice

Diabetes is caused by the interplay between genetics and environmental factors, tightly linked to lifestyle and dietary patterns. In this study, we explored the effectiveness of intermittent protein restriction(IPR)in diabetes control. IPR drastically reduced hyperglycemia in both streptozotocin-treated and leptin receptor-deficient db/db mouse models. IPR improved the number, proliferation, and function of β cells in pancreatic islets. IPR reduced glucose production in the liver and elevated insulin signaling in the skeletal muscle. IPR elevated serum level of FGF21, and deletion of the Fgf21 gene in the liver abrogated the hypoglycemic effect of IPR without affecting β cells. IPR caused less lipid accumulation and damage in the liver than that caused by continuous protein restriction in streptozotocin-treated mice. Single-cell RNA sequencing using mouse islets revealed that IPR reversed diabetes-associated β cell reduction and immune cell accumulation. As IPR is not based on calorie restriction and is highly effective in glycemic control and β cell protection, it has promising translational potential in the future.

Inhibition mechanism of compound ethanol extracts from Wuweizi(Fructus Schisandrae Chinensis) on renal interstitial fibrosis in diabetic nephropathy model mice

OBJECTIVE：To evaluate inhibition effect and mech- anism of compound ethanol extracts from Wuweizi （Fructus Schisandrae Chinensis）, Chuanxiong （Rhi- zoma Chuanxiong） and Muli （Cocha Ostreae） （FRC） on glomerular and tubular interstitial fibrosis in streptozocin （STZ）-induced diabetic nephropathy （ND） model mice. METHODS： Twenty-seven male C57BL/6 mice were divided randomly into 3 groups： nondibetic （ND）, STZ-induced diabetic （D）, and STZ-induced diabetic that were treated with .5 g. kg1. daylof FRC by oral gavage （DFRc）, with 9 in each group. The protein ex- pressions of E-cadherin, a-smooth muscle actin （a-SMA）, Plasminogen Activator Inhibitor-1 （PAl-l） in renal tissues were investigated by Western blot- ting. The expressions of fibronectin （FN） and o-SMA were detected by immunohistochemical method. The morphological changes of renal tissues were observed under a microscope. RESULTS： Renal tissues in the DFRC group showed a lessened degree of fibrosis. Meanwhile, the expres- sions of FN, o-SMA and PAl-lwere significantly lower in the DrRc group than those in the D group （all P〈0.05）.CONCLUSION： FRC can ameliorate the DN in the C57BL/6 mice, and its mechanism may relate to in- hibition on the epithelial to mesenchymal transdif- ferentiation, endothelial-myofibroblast transition and PAl-1 expression.

Pretreatment with Jieduxiezhuo decoction impedes elevations in serum uric acid levels in mice

OBJECTIVE: To investigate whether Jieduxiezhuo decoction(JDXZD) can prevent serum uric acid elevations in mice. METHODS: Hyperuricemia in mice was induced by intraperitoneally administering uric acid(250 mg/ kg). Concentrations of uric acid in serum were determined using the uric acid enzyme method. Mice were treated with JDXZD for 4 days before uric acid was administered. RESULTS: After intraperitoneal injection of uric acid, serum uric acid concentrations in mice significantly increased. However, the levels of uric acid in groups pretreated with 16.25 or 4.06 g/kg of JDXZD were significantly lower than those in the model and normal groups. CONCLUSION: Pretreatment with JDXZD slowed increases in serum uric acid levels in mice intraperito-neally administered uric acid.

Effect of curcumin on rats/mice with diabetic nephropathy: a systematic review and Meta-analysis of randomized controlled trials

OBJECTIVE: To assess the renal protective effects of curcumin administration ondiabetic rats/mice.METHODS: Databases were searched electronically and conference papers searched manually for search terms to find relevant studies. Articles were assessed independently by two reviewers. Review Manager 5.1 was used fordata analysis.RESULTS: Fourteen randomized controlled experiments were included. Meta-analysis demonstrated that blood sugar levels and kidney weight to body weight ratios in the model group were higher than those in the normal group, and the curcumin group had significantly lower mesangial area to glomerular area ratios compared with the model group, and also lower levels of urinary protein,blood urea nitrogen and serum creatinine.CONCLUSION: Curcumin shows protective effects on the kidneys of rats/mice with diabetes.