核磁共振代谢组学方法研究雷公藤红素对大鼠糖尿病溃疡促愈合作用机制

采用腹腔注射链脲佐菌素结合打孔器皮肤烫伤构建SD大鼠糖尿病溃疡模型,随机分为正常创面生理盐水处理组、糖尿病溃疡模型生理盐水处理组、糖尿病溃疡模型雷公藤红素处理组,溃疡创面持续给药处理14 d,利用基于核磁共振的代谢组学方法研究雷公藤红素处理后大鼠糖尿病溃疡组织代谢特征,并结合形态观察计算溃疡愈合率及组织病理学检测(HE染色、Masson染色)技术研究大鼠糖尿病溃疡的愈合情况,揭示雷公藤红素对大鼠糖尿病溃疡的治疗作用及其机制。研究结果表明,雷公藤红素可诱导大鼠溃疡创面上皮再生化,调节炎性细胞浸润和胶原纤维分布,促进溃疡创面愈合。经偏最小二乘法分析,筛选并鉴定出20种潜在内源性差异代谢物。代谢通路分析表明,雷公藤红素能够显著提高溃疡组织三羧酸循环水平,促进其能量供应,加快蛋白合成,改善线粒体功能和氧化应激水平,加速皮肤组织的自我修复能力,促进糖尿病溃疡的愈合。本研究为雷公藤红素作为治疗糖尿病溃疡新药研究提供了参考。     

中药柴胡总皂苷急性肝毒性的代谢组学研究

运用UPLC-MS联用技术对空白对照组、柴胡总皂苷(SS)组大鼠在给药后第3天和第5天的尿液样本进行分析检测,获得了以质荷比和保留时间为变量的矩阵数据。据此建立各组大鼠的尿液代谢轮廓图,并利用主成分分析(PCA)建立了SS的代谢组学毒性模型。结合肝组织病理,探讨了柴胡总皂苷的急性肝毒性。对于SS组与正常组,大鼠尿液代谢轮廓图表现出了一定的差异。在代谢组学毒性模型中,给药第3天和第5天的PCA分类均偏离正常组,而第5天的偏离程度强于第3天。同时,在第5天的肝组织病理切片中发现了组织病变。由此表明,给药组大鼠的代谢组偏离了正常组,这种偏离正是肝急性毒性的表现,且给药到第5天的肝毒性强于第3天,表明肝毒性与给药累积剂量呈正相关,表现出了明显的急性和累积肝毒性。     

尿液代谢组学方法研究人参总皂苷治疗糖尿病心肌病大鼠作用机制

利用基于质谱的代谢组学方法考察了人参总皂苷(TG)治疗糖尿病心肌病(DCM)大鼠的效应机制;建立了糖尿病心肌病大鼠模型,并连续12周口服人参总皂苷,采用快速高分辨液相色谱/四级杆-飞行时间/质谱(RRLC/Q-TOF/MS)技术对糖尿病心肌病模型组(DCM组)和人参总皂苷治疗组(TG组)大鼠尿样的尿液代谢物进行分析,采用主成分分析(PCA)对两组代谢物进行分类,并寻找潜在生物标记物,同时检测心肌病理超微结构、血液生化指标和心肌氧化应激水平。RRLC/Q-TOF/MS检测结果表明,DCM组和TG组大鼠的尿液代谢物谱能得到很好的区分,发现并鉴定了3种生物标记物。TG降低了DCM大鼠心肌超微结构损伤并改善其血脂、血糖及心肌氧化应激水平,代谢组学研究结果表明:作用机制可能是TG对柠檬酸循环、脂肪酸代谢和氧化应激水平的调节作用。     

五味子治疗大鼠糖尿病肾病作用机制的血清代谢组学研究

采用基于超高效液相色谱与串联四级杆飞行时间质谱仪(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS)联用技术的代谢组学方法,通过分析大鼠血清内源性代谢物的变化,研究五味子治疗糖尿病肾病的作用机制。利用高脂高糖饲料喂养并腹腔注射链脲佐菌素(STZ)建立糖尿病大鼠模型。给药12周后,采用试剂盒方法测定尿蛋白、尿肌酐的含量,结果表明五味子水提取物可以显著降低模型动物的尿蛋白含量(p<0.05),对糖尿病大鼠肾病并发症具有一定的改善作用。采用UPLC/Q-TOF-MS方法分析了五味子对糖尿病肾病大鼠的血清代谢轮廓,分析了健康组、模型组和五味子给药组的大鼠血清,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)进行数据分析。PLS-DA得分图显示健康组、模型组和五味子组的代谢轮廓有显著差别,根据正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,将对各组分离贡献大的化合物的串联质谱分析数据,经Human Metabolome Database(HMDB)等数据库检索,进行质谱信息匹配,鉴定出黄尿酸、油酰胺、棕榈酰胺、尿酸、5-羟基己酸、硫酸对甲酚、对甲酚葡萄糖苷酸7种内源性代谢物为生物标记物。研究结果表明五味子通过影响色氨酸代谢、嘌呤代谢、肠内菌代谢、脂肪酸代谢等通路对糖尿病肾病发挥治疗作用,其中嘌呤代谢、肠内菌代谢通路可能是五味子发挥治疗作用的重要途径。     

基于气相色谱-质谱联用的代谢组学用于黄连治疗II型糖尿病的机理探索

将代谢组学的方法用于研究黄连治疗II型糖尿病的机理。II型糖尿病造模采用对大鼠灌胃脂肪乳并腹腔注射40 mg/kg链脲佐菌素的方法,大鼠分为正常组、模型组、黄连给药治疗组(10 g/kg)、二甲双胍给药治疗组(0.08 g/kg)。大鼠给药30天后,采集血样用于生化指标的检测,采集24 h尿样用于代谢组学的分析。与模型组相比,糖尿病大鼠给药黄连30天后,空腹血糖值(FBG)显著降低了59.26%,总胆固醇(TC)降低了58.66%,甘油三酯(TG)降低了42.18%。采用气相色谱-质谱联用技术(GC-MS)对大鼠尿样中的内源性物质进行了相对含量测定,主成分分析结果表明,正常组与模型组显著分离,黄连组处于正常组与模型组之间,更接近于正常组。发现12个代谢物与糖尿病有关,包括4-甲基苯酚、苯甲酸、氨基丙二酸等。给药黄连后,其中的7个代谢物发生显著性回调,与氧化应激状态相关的氨基丙二酸和L-抗坏血酸出现向正常组显著性调节的趋势。这些结果表明,黄连不仅具有降糖和降血脂的作用,而且具有抗氧化作用,在一定程度上可能会抑制糖尿病合并症的发生和发展。     

非靶向尿液代谢组学方法研究五味子治疗精尿病并发症作用机制

采用超高效液相色谱-串联四级杆飞行时间质谱联用技术(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS),结合主成分分析技术(Principal component analysis,PCA),进行了五味子治疗糖尿病并发症大鼠尿液代谢组学研究,对五味子治疗糖尿病并发症大鼠后尿液生物标志物变化进行了考察.采用UPLC/Q-TOF-MS方法分析了健康组、糖尿病并发症模型组和五味子给药组的大鼠尿液,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)和正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,通过分析对各组分离贡献较大(VIP>1,P°0.05)化合物的串联质谱数据,再经Human Metabolome Data-base(HMDB)等数据库检索,进行质谱信息匹配,尿液中共鉴定出28种潜在生物标记物,其中正谱13种,负谱15种.这些生物标记物主要影响戊糖和葡糖醛酸相互转化通路、核黄素代谢、泛酸和CoA合成、精氨酸和脯氨酸代谢、肠内菌代谢、嘌吟代谢、Vc代谢胆酸合成、色氨酸代谢等通路,五味子从能量代谢、氨基酸代谢、肠内菌代谢、脂类代谢等各个角度发挥治疗糖尿病并发症的作用.从各个通路的相关生物功能分析,五味子治疗糖尿病肾病的作用较强,此外还具有降脂、抗氧化的功效.     

天然本草添加卷烟烟气暴露大鼠肺组织代谢组学研究

应用代谢组学方法,比较研究了普通卷烟和含有天然本草添加剂卷烟对大鼠肺组织代谢的影响。运用液相色谱-质谱联用技术分别分析了对照组、普通卷烟及某品牌天然本草添加卷烟烟气暴露7,14,30 d时的大鼠肺组织样品,对所得数据用偏最小二乘判别分析(PLS-DA)进行模式识别并筛选生物标志物。结果显示,吸食普通卷烟和某品牌卷烟,均会损伤大鼠肺组织,造成磷脂代谢、脂肪酸代谢紊乱,其中有2只普通卷烟暴露组大鼠的肺组织样品显著异常,受到明显的氧化损伤。综合来看,特别是长时间烟气暴露状态下,吸食某品牌卷烟造成的肺组织损伤低于普通卷烟,表明烟草中添加天然本草在一定程度上可减轻烟气对机体的损伤。     

基于液相色谱-质谱联用技术的代谢组学方法研究薄荷烟对大鼠代谢的影响

将基于液相色谱-质谱联用(LC-MS)技术的代谢组学分析平台用于薄荷烟对大鼠代谢影响的研究。分析了3组大鼠的尿样,包括对照大鼠、吸食普通烟大鼠和吸食薄荷烟大鼠,并采用主成分分析(PCA)方法对数据进行模式识别。PCA得分图表明吸食薄荷烟大鼠与对照组大鼠尿样的代谢差异要小于吸食普通烟大鼠。从PCA载荷图中找到并鉴定了犬尿喹啉酸等8种重要代谢物。通过考察代谢物在对照大鼠、吸食薄荷烟大鼠和吸食普通烟大鼠尿样中的相对含量变化,进一步说明了烟草中添加薄荷醇可减少烟草对大鼠代谢的影响。     

阿卡波糖对Ⅱ型糖尿病大鼠尿液代谢轮廓的影响

采用超高效液相色谱-质谱联用(UPLC-MS/MS)方法研究了阿卡波糖对Ⅱ型糖尿病大鼠代谢轮廓的影响, 分析了健康组、 糖尿病模型组和糖尿病给予阿卡波糖组的大鼠尿样, 采用主成分分析法(PCA)和偏最小二乘法-判别分析(PLS-DA)对数据进行分析. PCA得分图表明, 健康组、 糖尿病组和阿卡波糖组的代谢轮廓有显著差别, 根据PLS-DA载荷图筛选, 将对各组分离贡献大的化合物的串联质谱分析数据经Human Metabolome Database(HMDB)和Mass Bank.jp等数据库检索, 进行质谱信息匹配, 鉴定出苯乙酰甘氨酸、 肌酐及葡萄糖酸等8种内源性代谢物为潜在生物标记物.     

格列美脲治疗的2型糖尿病大鼠的尿液代谢组学研究

采用快速高分离度液相色谱-质谱技术(RRLC-MS)检测经格列美脲治疗的2型糖尿病大鼠尿液中代谢物的变化, 对糖尿病组、 给药组和健康对照组大鼠的尿液代谢物谱进行了分析. 采用主成分分析(PCA)对3组大鼠进行分类并寻找潜在生物标记物. 结果表明, 3组大鼠的尿液代谢物谱得到了很好的区分, 发现并鉴定了2个潜在生物标记物, 分别为4-脱氧三羟基丁酸和4-胍基丁酸. 格列美脲对2型糖尿病大鼠的药物作用可能体现为对氨基酸代谢的调节作用.     

Dose–Response Relationship of Photobiomodulation Therapy on Matrix Metalloproteinase in Healing Dynamics of Diabetic Neuropathic Ulcers—An in vivo Study

Individuals with diabetic foot ulcers have overlapped the inflammatory, proliferative and remodeling phase, making the tissue vulnerable to delayed healing responses. We aimed to establish the dose–response relationship of photobiomodulation therapy of different doses and matrix metalloproteinases in the healing dynamics of diabetic neuropathic ulcers. Diabetes was induced in 126 Albino Wistar rats, and neuropathy was induced to the hind paw by a sciatic nerve injury method. An excisional wound was created on the neuropathy-induced leg. Photobiomodulation therapy of dosages 4, 6, 8, 10, 12 and 15 J cm⁻² and wavelength 655 nm and 808 nm was irradiated. Photobiomodulation therapy of dosages 4, 6 and 8 J cm⁻² showed better wound healing properties with optimized levels of matrix metalloproteinases-1 and 8. We observed a strong dose response in the experimental group treated with 6 and 8 J cm⁻². The findings from the present study conclude that photobiomodulation therapy of dosages 4, 6 and 8 J cm⁻² is suggestive of usefulness in diabetic neuropathic ulcer healing. Markers like matrix metalloproteinases may give a clear direction on response to the therapy. Based on the findings from the present study, we recommend to validate the findings for safety and efficacy in future through human prospective randomized controlled clinical trials.     

Topical propolis improves wound healing in patients with diabetic foot ulcer: a randomized controlled trial

In this randomized controlled trial, diabetic patients with foot ulcers (Wagner grades 1 and 2) were randomly assigned to conventional therapies for diabetic foot ulcer plus topical propolis ointment (5%; twice daily) or conventional therapies alone. The process of ulcer healing was observed during 4 weeks and compared between the two groups regarding the size, erythema, exudates, white blood cell (WBC) count and erythrocyte sedimentation rate (ESR). The process of ulcer size reduction during the four-week period of study was significantly different between the groups. However, this difference was not significant between the third and fourth weeks. There was no significant difference between two groups regarding erythema and exudate reduction as well as WBC count and ESR. Administration of topical propolis ointment in addition to the conventional treatments of diabetic foot ulcer could reduce the size of ulcers with Wagner grades 1 and 2.     

Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach

Type-2 diabetes mellitus (T2DM), the leading global health burden of this century majorly develops due to obesity and hyperglycemia-induced oxidative stress in skeletal muscles. Hence, developing novel drugs that ameliorate these pathological events is an immediate priority. The study was designed to analyze the possible role of Stevioside, a characteristic sugar from leaves of Stevia rebaudiana (Bertoni) on insulin signaling molecules in gastrocnemius muscle of obesity and hyperglycemia-induced T2DM rats. Adult male Wistar rats rendered diabetic by administration of high fat diet (HFD) and sucrose for 60 days were orally administered with SIT (20 mg/kg/day) for 45 days. Various parameters were estimated including fasting blood glucose (FBG), serum lipid profile, oxidative stress markers, antioxidant enzymes and expression of insulin signaling molecules in diabetic gastrocnemius muscle. Stevioside treatment improved glucose and insulin tolerances in diabetic rats and restored their elevated levels of FBG, serum insulin and lipid profile to normalcy. In diabetic gastrocnemius muscles, Setvioside normalized the altered levels of lipid peroxidase (LPO), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH*), antioxidant enzymes (CAT, SOD, GPx and GSH) and molecules of insulin signaling including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt mRNA levels. Furthermore, Stevioside enhanced glucose uptake (GU) and oxidation in diabetic muscles by augmenting glucose transporter 4 (GLUT 4) synthesis very effectively in a similar way to metformin. Results of molecular docking analysis evidenced the higher binding affinity with IRS-1 and GLUT 4. Stevioside effectively inhibits oxidative stress and promotes glucose uptake in diabetic gastrocnemius muscles by activating IR/IRS-1/Akt/GLUT 4 pathway. The results of the in silico investigation matched those of the in vivo study. Hence, Stevioside could be considered as a promising phytomedicine to treat T2DM.     

Thymoquinone Lowers Blood Glucose and Reduces Oxidative Stress in a Rat Model of Diabetes

The aim of the present study was to assess the short-term effects of Thymoquinone (TQ) on oxidative stress, glycaemic control, and renal functions in diabetic rats. DM was induced in groups II and III with a single dose of streptozotocin (STZ), while group I received no medication (control). The rats in groups I and II were then given distilled water, while the rats in group III were given TQ at a dose of 50 mg/kg body weight/day for 4 weeks. Lipid peroxidase, nitric oxide (NO), total antioxidant capacity (TAC), glycated haemoglobin (HbA1c), lipid profiles, and renal function were assessed. Moreover, the renal tissues were used for histopathological examination. STZ increased the levels of HbA1c, lipid peroxidase, NO, and creatinine in STZ-induced diabetic rats in comparison to control rats. TAC was lower in STZ-induced diabetic rats than in the control group. Furthermore, rats treated with TQ exhibited significantly lower levels of HbA1c, lipid peroxidase, and NO than did untreated diabetic rats. TAC was higher in diabetic rats treated with TQ than in untreated diabetic rats. The histopathological results showed that treatment with TQ greatly attenuated the effect of STZ-induced diabetic nephropathy. TQ effectively adjusts glycaemic control and reduces oxidative stress in STZ-induced diabetic rats without significant damaging effects on the renal function.     

The hypoglycemic and antioxidant activities of garden cress (Lepidium sativum L.) seed on alloxan-induced diabetic male rats

The hypoglycemic and antioxidants activities of the methanol extract of Lepidium sativum seeds was tested in alloxan-induced diabetic male rats. Thirty male albino rats weighing 190–200 g were divided into five groups as follows: negative control, positive control and three diabetic groups treated with three concentrations of L. sativum methanol extract for four weeks. Induction of hyperglycemia in the positive control group increased blood glucose, glycated hemoglobin A1c, immunoglobulins, liver enzyme, lipid peroxide and kidney function, total cholesterol, low-density lipoproteins, very low-density lipoproteins and decreased antioxidants and high-density lipoproteins compared with the negative control. Furthermore, pancreas tissues showed pathological changes compared with the negative control. Treating the diabetic rats with L. sativum methanol extract decreased blood sugar and restored all biochemical and histological changes to the normal. It could be concluded that L. sativum methanol extract succeeded in controlling diabetes, increasing antioxidants and ameliorating lipid profile.     

Effect of andrographolide on cysteamine-induced duodenal ulcer in rats

The aim of this study was to evaluate the gastroprotective efficacy of andrographolide isolated from Andrographis paniculata in rats induced with duodenal ulcers. Duodenal ulcers were induced by cysteamine administration in rats pretreated with 3?mg?kg?1 BW?day?1 of andrographolide for 30 days. Ulcer score, myeloperoxidase activity, TBARS level, GSH/GSSG ratio and enzyme antioxidants were measured in the duodenal tissue. Brush border and basolateral membranes were isolated to assay sucrase, maltase, alkaline phosphatase and total ATPases. Ulcer score was significantly minimised in rats pretreated with andrographolide. Elevation in myeloperoxidase and TBARS levels were found to be minimised significantly due to andrographolide treatment. Membrane-bound enzyme activities and the thiol redox status of glutathione were significantly maintained in duodenal mucosa of rats that received andrographolide. This study reveals that the major component of A. paniculata, andrographolide, has potent antiulcer properties that are most likely caused by minimising inflammatory changes, counteracting free radical formation and maintaining the thiol redox status in the duodenum.     

快速高分辨液相色谱-质谱联用技术分析2型糖尿病大鼠口服四君子汤的药代动力学

研究四君子汤在2型糖尿病大鼠体内的药代动力学。 将四君子汤水提物以18 g/kg给正常大鼠和2型糖尿病大鼠灌胃,分别在不同时间点取尿液和粪便,检测其成分及代谢产物的含量;比较正常大鼠和2型糖尿病大鼠的药代动力学参数。 采用快速高分辨液相色谱-质谱联用技术对人参皂苷Rc、甘草甜素及其脱糖代谢物进行表征。 结果表明,四君子汤中人参皂苷Rc和甘草甜素在2型糖尿病大鼠体内的累计排泄率均高于正常大鼠,大部分人参皂苷通过尿液排出体外、粪便代谢为稀有人参皂苷;大部分甘草甜素在尿液和粪便中转化为甘草次酸,2型糖尿病大鼠与正常大鼠人参皂苷Rc和甘草甜素的药代动力学参数存在明显差异。 初步总结了四君子汤在体内的药代动力学机制和代谢途径。本分析方法具有高度自动化,灵敏性和准确性,这将有助于四君子汤的临床研究的不断发展。     

Ganoderma lucidum polysaccharides exert anti-hyperglycemic effect on streptozotocin-induced diabetic rats through affecting β-cells

Previous studies have demonstrated that Ganoderma lucidum polysaccharides (Gl-PS) exhibited potential antihyperglycemic effect in rats. The aim of the present study was to investigate the mechanism of the hypoglycemic effect of a low- molecular-weight Gl-PS in streptozotocin (STZ)-induced diabetic Sprague-Dawley (SD) rats. Gl-PS was extracted and purified from Ganodema lucidum fruiting body. 50 male SD rats were included in the study; 10 were taken as healthy controls; 40 were induced to diabetes by a single injection of 65 mg/kg STZ, of which 30 were selected as successful diabetic rat models. The 30 diabetic rats were divided into three groups: Gl-PS (200 mg/kg Gl-PS), metformin (100 mg/kg metformin) and diabetic control (n = 10 per group). After eight weeks' oral administration, plasma concentrations of fasting glucose, triacylglyceride, total cholesterol and nitric oxide were significantly decreased in Gl-PS and metformin groups. Pancreatic superoxide dismutase, catalase and glutathione peroxidase were significantly increased in Gl-PS and metformin groups. Histopathological results showed that Gl-PS and metformin had protective effect on β-cells. The mRNA expressions of Bcl-2 and PDX-1 in pancreas were up-regulated, but Bax, iNOS and Casp-3 down-regulated in Gl- PS and metformin groups compared to diabetic control group. The present results suggested that Gl-PS had a hypoglycemic effect in STZ-induced diabetic rats through preventing apoptosis of pancreatic β-cells and enhancing β-cells regeneration.     

质谱法分析2型糖尿病大鼠神经视网膜差异表达蛋白

采用二维电泳(2DE)分离了正常SD大鼠和2型糖尿病模型大鼠神经视网膜组织总蛋白, 并用Image Master 5.0软件分析比较了正常组和糖尿病组2DE图像, 正常组检测到 3122±37(n=3)个蛋白质点; 糖尿病组检测到2702±21(n=3)个蛋白质点. 约150个蛋白质点的表达水平在两组之间存在明显差异(P<0.05). 在糖尿病组中表达上调的点68个, 下调的点82个. 选择20个差异表达蛋白质点进行肽质量指纹谱(PMF)或串联质谱鉴定, 其中7个蛋白已有报道与糖尿病视网膜病变(DR)相关, 10个蛋白尚未见有报道.     

Angiotensin II receptor blocker attenuates overexpression of vascular endothelial growth factor in diabetic podocytes

VEGF expressed in glomerular podocytes, is known to increase vascular permeability to macromolecules. Angiotensin II can stimulate the release of VEGF, and the protective effects of angiotensin II antagonist against diabetic glomerular injury suggest that the angiotensin II-induced VEGF is an important pathogenetic mechanism in the development of proteinuria during diabetic nephropathy although this mechanism is not fully understood. In this study, the changes of VEGF expression was examined in the experimental diabetic nephropathy to determine whether these changes were modified by renoprotective intervention by blockers of angiotensin II receptors. The streptozotocin- induced diabetic rats were treated with L-158,809, a blocker of angiotensin II receptors, for 12 weeks. Age-matched rats with L-158,809 served as controls. RT-PCR and immunohistochemistry were used to assess and quantify gene and protein expression of VEGF. A progressive increase in urinary protein excretion was observed in diabetic rats. Glomerular VEGF expression was significantly higher in diabetic rats than in the control groups, with a significant reduction in glomerular VEGF expression and proteinuria in L-158,809- treated diabetic rats. VEGF mRNA was also significantly higher in diabetic kidneys than in the control groups, with a significant reduction in VEGF mRNA in L-158,809-treated diabetic kidneys. These results demonstrates that VEGF expression is significantly increased in diabetic podocytes, and angiotensin II receptor antagonist attenuated these changes in VEGF expression and prevented the development of proteinuria in vivo. Attenuation of increased VEGF expression in podocytes could contribute to the renoprotective effects of angiotensin II receptor antagonists in diabetic nephropathy.