Anti-infection effects of heparin on SARS-CoV-2 in a diabetic mouse model

Severe acute respiratory syndrome coronavirus 2(SARSCo V-2)infection can result in more severe syndromes and poorer outcomes in patients with diabetes and obesity.However,the precise mechanisms responsible for the combined impact of coronavirus disease 2019(COVID-19)and diabetes have not yet been elucidated,and effective treatment options for SARS-Co V-2-infected diabetic patients remain limited.To investigate the disease pathogenesis,K18-h ACE2 transgenic(h ACE2^(Tg))mice with a leptin receptor deficiency(h ACE2-Lepr^(-/-))and high-fat diet(h ACE2-HFD)background were generated.The two mouse models were intranasally infected with a 5×10^(5) median tissue culture infectious dose(TCID_(50))of SARSCo V-2,with serum and lung tissue samples collected at 3days post-infection.The h ACE2-Lepr^(-/-)mice were then administered a combination of low-molecular-weight heparin(LMWH)(1 mg/kg or 5 mg/kg)and insulin via subcutaneous injection prior to intranasal infection with1×10^(4) TCID_(50)of SARS-Co V-2.Daily drug administration continued until the euthanasia of the mice.Analyses of viral RNA loads,histopathological changes in lung tissue,and inflammation factors were conducted.Results demonstrated similar SARS-Co V-2 susceptibility in h ACE2^(Tg)mice under both lean(chow diet)and obese(HFD)conditions.However,compared to the h ACE2-Lepr^(+/+)mice,h ACE2-Lepr^(-/-)mice exhibited more severe lung injury,enhanced expression of inflammatory cytokines and hypoxia-inducible factor-1α(HIF-1α),and increased apoptosis.Moreover,combined LMWH and insulin treatment effectively reduced disease progression and severity,attenuated lung pathological changes,and mitigated inflammatory responses.In conclusion,preexisting diabetes can lead to more severe lung damage upon SARS-Co V-2 infection,and LMWH may be a valuable therapeutic approach for managing COVID-19patients with diabetes.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4

BACKGROUND Diabetic kidney disease(DKD)is a major complication of diabetes mellitus.Renal tubular epithelial cell(TEC)damage,which is strongly associated with the inflammatory response and mesenchymal trans-differentiation,plays a significant role in DKD;However,the precise molecular mechanism is unknown.The recently identified microRNA-630(miR-630)has been hypothesized to be closely associated with cell migration,apoptosis,and autophagy.However,the association between miR-630 and DKD and the underlying mechanism remain unknown.AIM To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats.METHODS Streptozotocin was administered to six-week-old male rats to create a hypergly cemic diabetic model.In the second week of modeling,the rats were divided into control,DKD,negative control of lentivirus,and miR-630 overexpression groups.After 8 wk,urine and blood samples were collected for the kidney injury assays,and renal tissues were removed for further molecular assays.The target gene for miR-630 was predicted using bioinformatics,and the association between miR-630 and toll-like receptor 4(TLR4)was confirmed using in vitro investigations and double luciferase reporter gene assays.Overexpression of miR-630 in DKD rats led to changes in body weight,renal weight index,basic blood parameters and histopathological changes.RESULTS The expression level of miR-630 was reduced in the kidney tissue of rats with DKD(P<0.05).The miR-630 and TLR4 expressions in rat renal TECs(NRK-52E)were measured using quantitative reverse transcription polymerase chain reaction.The mRNA expression level of miR-630 was significantly lower in the high-glucose(HG)and HG+mimic negative control(NC)groups than in the normal glucose(NG)group(P<0.05).In contrast,the mRNA expression level of TLR4 was significantly higher in these groups(P<0.05).However,miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Furthermore,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and IL-6 were significantly higher in the HG and HG+mimic NC groups than in NG group(P<0.05).However,the levels of these cytokines were significantly lower in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Notably,changes in protein expression were observed.The HG and HG+mimic NC groups showed a significant decrease in E-cadherin protein expression,whereas TLR4,α-smooth muscle actin(SMA),and collagen IV protein expression increased(P<0.05).Conversely,the HG+miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4,α-SMA,and collagen IV protein expression than in the HG+mimic NC group(P<0.05).The miR-630 targets TLR4 gene expression.In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC.Additionally,rats treated with miR-630 agomir showed significant reductions in urinary albumin,blood glucose,TLR4,and proinflammatory markers(TNF-α,IL-1β,and IL-6)expression levels(P<0.05).Moreover,these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells.CONCLUSION MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4,and has a protective effect on DKD.

Effect of periocular injection of celecoxib and propranolol on ocular level of vascular endothelial growth factor in a diabetic mouse model

AIM： To investigate the effects of periocular injection of propranolol and celecoxib on ocular levels of vascular endothelial growth factor （VEGF） in a diabetic mouse model. METHODS： Forty 4-6wk BALB-C male mice weighing 20-25 g were used. The study groups included： nondiabetic control （group 1）, diabetic control （group 2）, diabetic propranolol （group 3）, and diabetic celecoxib （group 4）. After induction of type 1 diabetes by streptozotocin, propranolol （10 μg） and celecoxib （200 μg dissolved in carboxymethylcellulose 0.5%） were injected periocularly. The ocular level of VEGF was measured in all the study groups using enzyme-linked immuno sorbent assay （ELISA） method. RESULTS： Ocular VEGF level was significantly increased （1.25 fold） in the diabetic control group when compared to the non-diabetic group one week after induction with streptozotocin （P=0.002）. Both periocular propranolol and celecoxib significantly reduced ocular VEGF levels （P=0.047 and P〈0.001, respectively）. The effect was more pronounced with celecoxib, CONCLUSION： The periocular administration of propranolol and celecoxib can significantly reduce ocular VEGF levels in a diabetic mouse model.

Diabetic mouse models

The number of patients with lifestyle-related diseases, such as cardiovascular disease, diabetes mellitus, hypertension, atherosclerosis, and cancer, is increasing all over the world, and that of diabetics is increasing especially rapidly. Diabetic animal models have played a key role in elucidating the etiology of diabetes and developing anti-diabetic drugs. In this review, we overviewed characteristics of diabetic mouse models and pharmacological evaluation using the diabetic models.

Therapeutic effect of folic acid combined with decitabine on diabetic mice

AIM:To evaluate the therapeutic effect of folic acid combined with decitabine on diabetic mice.METHODS:The diabetic model of db/db mice were randomly divided into model group,folic acid group,decitabine group,folic acid combined with decitabine group,and C57 mice as normal control group.The density of retinal blood vessels and retinal thickness were detected by fundus photography and optical coherence tomography,respectively.Pathological changes of retina were observed by hematoxylin-eosin(HE)staining.The homocysteine(Hcy)in serum was detected by enzyme linked immunosorbent assay(ELISA).TdT-mediated dUTP nick-end labeling(TUNEL)was used to detect apoptosis in retinal tissue.Evans blue dye was used to detect the permeability of retinal blood vessels.The platelet endothelial cell adhesion molecule-1(CD31)and vascular endothelial growth factor receptor(VEGFR)protein were detected by Western blot.The 3-nitrotyrosine(3-NT)and 4-hydroxynonanine(4-HNE)were detected by immunohistochemistry.RESULTS:The density of retinal blood vessels,retinal thickness,retinal vascular permeability and the proportion of apoptotic cells of retinal tissue in the model group increased significantly than control group(P<0.05).The Hcy in serum and the levels of CD31,VEGFR,3-NT,and 4-HNE in retinal tissue increased significantly in the model group(P<0.01).Folic acid and decitabine both reversed these changes significantly,and the combination of the folic acid and decitabine worked best.CONCLUSION:The combination of folic acid and decitabine has a more significant protective effect on the retina in diabetic mice.

Assessing the host genetic background effects on type 2 diabetes and obesity development in response to mixed–oral bacteria and high-fat diet using the collaborative cross mouse model

Background: Host genetic background and sex, play central roles in defining the pathogenesis of type 2 diabetes(T2 D), obesity and infectious diseases. Our previous studies demonstrated the utilization of genetically highly diverse inbred mouse lines, namely collaborative cross(CC), for dissecting host susceptibility for the development of T2 D and obesity, showing significant variations following high-fat(42% fat) diet(HFD). Here, we aimed to assessing the host genetic background and sex effects on T2 D and obesity development in response to oral-mixed bacterial infection and HFD using the CC lines.Materials and Methods: Study cohort consists of 97 mice from 2 CC lines(both sexes), maintained on either HFD or Standard diet(CHD) for 12 weeks. At week 5 a group of mice from each diet were infected with Porphyromonas gingivalis(Pg) and Fusobacterium nucleatum(Fn) bacteria(control groups without infection). Body weight(BW) and glucose tolerance ability were assessed at the end time point of the experiment.Results: The CC lines varied(P <.05) at their BW gain and glucose tolerance ability(with sex effect) in response to diets and/or infection, showing opposite responses despite sharing the same environmental conditions. The combination of diet and infection enhances BW accumulation for IL1912, while restraints it for IL72. As for glucose tolerance ability, only females(both lines) were deteriorated in response to infection.Conclusions: This study emphasizes the power of the CC mouse population for the characterization of host genetic makeup for defining the susceptibility of the individual to development of obesity and/or impaired glucose tolerance.

Sodium glucose co-transporter 2 inhibition reduces succinate levels in diabetic mice

BACKGROUND Type 1 diabetes(T1D) is associated with major chronic microvascular complications which contribute significantly to diabetes associated morbidity.The protein primarily responsible for glucose reabsorption in the kidney is sodium glucose co-transporter 2(SGLT2). Presently, SGLT2 inhibitors are widely used in diabetic patients to improve blood glucose levels and prevent cardiovascular and renal complications. Given the broad therapeutic application of SGLT2 inhibitors, we hypothesised that SGLT2 inhibition may exert its protective effects via alterations of the gut microbiome and tested this in a type 1 diabetic mouse model of diabetic retinopathy.AIM To determine whether the treatment with two independent SGLT2 inhibitors affects gut health in a type 1 diabetic mouse model.METHODS The SGLT2 inhibitors empagliflozin or dapagliflozin(25 mg/kg/d) or vehicle dimethylsulfoxide(DMSO) were administered to C57 BL/6 J, Akita, Kimba and Akimba mice at 10 wk of age for 8 wk via their drinking water. Serum samples were collected and the concentration of succinate and the short chain fatty acid(SCFA) butyric acid was measured using gas chromatography-mass spectrometry. Enzyme-linked immunosorbent assay(ELISA) was performed to determine the concentration of insulin and leptin. Furthermore, the norepinephrine content in kidney tissue was determined using ELISA. Pancreatic tissue was collected and stained with haematoxylin and eosin and analysed using brightfield microscopy.RESULTS Due to the presence of the Akita allele, both Akita and Akimba mice showed a reduction in insulin production compared to C57 BL/6 J and Kimba mice.Furthermore, Akita mice also showed the presence of apoptotic bodies within the pancreatic islets. The acinar cells of Akita and Akimba mice showed swelling which is indicative of acute injury or pancreatitis. After 8 wk of SGLT2 inhibition with dapagliflozin, the intermediate metabolite of gut metabolism known as succinate was significantly reduced in Akimba mice when compared to DMSO treated mice. In addition, empagliflozin resulted in suppression of succinate levels in Akimba mice. The beneficial SCFA known as butyric acid was significantly increased in Akita mice after treatment with dapagliflozin when compared to vehicle treated mice. The norepinephrine content in the kidney was significantly reduced with both dapagliflozin and empagliflozin therapy in Akita mice and was significantly reduced in Akimba mice treated with empagliflozin.In non-diabetic C57 BL/6 J and Kimba mice, serum leptin levels were significantly reduced after dapagliflozin therapy.CONCLUSION The inhibition of SGLT2 reduces the intermediate metabolite succinate, increases SCFA butyric acid levels and reduces norepinephrine content in mouse models of T1 D. Collectively, these improvements may represent an important mechanism underlying the potential benefits of SGLT2 inhibition in T1 D and its complications.

PTPN22 and islet-specific autoimmunity:What have the mouse models taught us?

An allelic variant of the protein tyrosin phosphatase non-receptor 22(PTPN22) gene, PTPN22 R620 W, constitutes the strongest non-HLA genetic risk factor for the development of type 1 diabetes(T1D). A numberstudies using mouse models have addressed how PTPN22 predisposes to T1D. PTPN22 downmodulation, overexpression or expression of the variant gene in genetically manipulated mice has generated controversial results. These discrepancies probably derive from the fact that PTPN22 has differential effects on innate and adaptive immune responses. Moreover, the effects of PTPN22 are dependent on other genetic variables. Here we discuss these findings and try to explain the discrepancies. Exploring the mechanism by which PTPN22 contributes to islet-specific autoimmunity could help us understand its role in T1D pathogenesis and exploit it as a potential therapeutic target to prevent the disease.

High-fat diet and oral infection induced type 2 diabetes and obesity development under different genetic backgrounds

Background:Type 2 diabetes(T2D)is an adult-onset and obese form of diabetes caused by an interplay between genetic,epigenetic,and environmental components.Here,we have assessed a cohort of 11 genetically different collaborative cross(CC)mouse lines comprised of both sexes for T2D and obesity developments in response to oral infection and high-fat diet(HFD)challenges.Methods:Mice were fed with either the HFD or the standard chow diet(control group)for 12 weeks starting at the age of 8 weeks.At week 5 of the experiment,half of the mice of each diet group were infected with Porphyromonas gingivalis and Fusobacterium nucleatum bacteria strains.Throughout the 12-week experimental period,body weight(BW)was recorded biweekly,and intraperitoneal glucose tolerance tests were performed at weeks 6 and 12 of the experiment to evaluate the glucose tolerance status of mice.Results:Statistical analysis has shown the significance of phenotypic variations between the CC lines,which have different genetic backgrounds and sex effects in different experimental groups.The heritability of the studied phenotypes was estimated and ranged between 0.45 and 0.85.We applied machine learning methods to make an early call for T2D and its prognosis.The results showed that classification with random forest could reach the highest accuracy classification(ACC=0.91)when all the attributes were used.Conclusion:Using sex,diet,infection status,initial BW,and area under the curve(AUC)at week 6,we could classify the final phenotypes/outcomes at the end stage of the experiment(at 12 weeks).

Effect of Micardis on the Expression of Renal Medulla Aquaporin-2 in Diabetic Mice

In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 （AQP2） was investigated in the renal medullary collecting duct of mice with diabetic nephropathy （DN）. Mice were divided into three groups： normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group （2.39±0.11 vs 3.16±0.16, P〈0.05）. Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system （RAS） is implicated in the pathogenesis of DN by regulating the expression of AQP2.

鬼箭羽对糖尿病肾病小鼠肾脏结构及功能的影响

目的:探讨鬼箭羽对糖尿病肾病小鼠肾脏形态结构及功能的影响。方法:10只7周龄db/m小鼠为空白组;60只db/db小鼠随机分为模型组,鬼箭羽低、中、高剂量[1.5、3.0、6.0 g/(kg·d)]组和厄贝沙坦[20 mg/(kg·d)]组,每组12只;空白组和模型组灌胃生理盐水,余4组分别灌胃相应药物,连续给药12周。实验期间观察小鼠一般状态,给药前后检测空腹血糖、24 h尿量和饮水量。末次给药后检测尿白蛋白/肌酐(UACR)、血肌酐(SCr)、血尿素氮(BUN);处死小鼠,称重双肾,计算肾脏指数(KI);取左肾组织,HE、PAS、Masson染色,观察组织学表现;分离右肾皮质,透射电镜下观察超微结构。结果:与模型组比较,鬼箭羽高剂量组及厄贝沙坦组小鼠KI、空腹血糖、SCr、BUN、UACR均降低(P均<0.05),系膜基质百分比及胶原面积百分比降低(P<0.05),肾脏病理损害减轻,肾脏基底膜厚度减少(P<0.05),足突融合改善。结论:高剂量鬼箭羽可有效降低糖尿病肾病小鼠的血糖,修复肾脏功能及结构,对糖尿病肾病有较好的治疗作用。

灵芝螺旋藻复方胶囊辅助降血糖功能

该文探究灵芝螺旋藻复方胶囊对Ⅱ型糖尿病小鼠的降血糖作用。采用高热能饲料结合腹腔注射链脲佐菌素的方法,建立Ⅱ型糖尿病小鼠模型,随机将小鼠分为对照组、模型组和低、中、高剂量组。灵芝螺旋藻复方胶囊连续干预4周,测定小鼠体质量、脏器指数、血液相关指标并观察肝、胰腺、盲肠病理组织切片。结果显示,低、中、高剂量(相当于人体推荐用量5、10、30倍)灵芝螺旋藻复方胶囊能不同程度改善糖尿病小鼠脏器指数、血糖水平以及葡萄糖耐量;从病理组织切片观察到经过3个剂量组干预的肝细胞体积明显恢复且排列整齐、脂滴空泡减少,肝细胞的损伤与炎症明显改善;胰岛细胞边界随剂量的增大而变得逐渐清晰且有序;低、中剂量两个组除绒毛有少部分呈圆状环绕外,3个剂量组盲肠组织外膜、肌层、黏膜、绒毛形态均有明显改善。综上,低、中、高3个剂量灵芝螺旋藻复方胶囊均可有效改善Ⅱ型糖尿病小鼠的糖脂代谢,有助于维持血糖健康水平。

普鲁士蓝纳米颗粒促进糖尿病皮肤创面愈合

背景:炎症、氧化应激及细菌感染是糖尿病创面难愈合的主要原因,近年来各种无机纳米材料以其抗菌活性被广泛应用于皮肤创面愈合的治疗,但抗氧化和抗炎方面的作用有限。目的:考察普鲁士蓝纳米颗粒在抗氧化、抗炎和光热抗菌多方面的糖尿病创伤修复的效果。方法:制备普鲁士蓝纳米颗粒并进行表征。(1)体外实验:采用MTT法检测不同浓度普鲁士蓝纳米颗粒的生物相容性;在过氧化氢条件下,检测普鲁士蓝纳米颗粒的细胞保护作用及活性氧荧光表达;检测普鲁士蓝纳米颗粒分解过氧化氢和超氧阴离子自由基的能力;考察普鲁士蓝纳米颗粒抑制脂多糖诱导巨噬细胞炎症的作用;采用平板菌落计数法检测普鲁士蓝纳米颗粒的光热抗菌能力。(2)体内实验:腹腔注射链脲佐菌素建立糖尿病ICR小鼠模型,使用打孔器在背部建立直径6 mm全厚皮肤创面,分对照组(未给予治疗)、普鲁士蓝组及普鲁士蓝光照组干预,观察创面愈合与组织形态学变化。结果与结论:(1)体外实验:普鲁士蓝纳米颗粒在25-200μg/mL质量浓度下对细胞无毒性;普鲁士蓝纳米颗粒具有极强的抗氧化能力,能够抑制氧化应激条件下过度活性氧的产生及对细胞的杀伤,对过氧化氢有降解活性且具有很强的清除超氧阴离子自由基的能力;普鲁士蓝纳米颗粒还显示出显著的抗炎活性,并且在光照后显示出极强的抗菌能力。(2)体内实验:造模14 d后,普鲁士蓝组、普鲁士蓝光照组创面明显缩小,其中普鲁士蓝光照组创面愈合速度最快。苏木精-伊红和Masson染色显示,普鲁士蓝组、普鲁士蓝光照组创面可见大量的肉芽组织形成及胶原沉积,其中以普鲁士蓝光照组最多;免疫荧光染色显示,与对照组比较,普鲁士蓝组和普鲁士蓝光照组α-SMA和CD31表达明显增多(P<0.05),F4/80表达明显减少(P<0.05),其中以普鲁士蓝光照组改善更明显。(3)结果表明,普鲁士蓝纳米颗粒通过发挥抗炎、抗氧化及抗菌作用促进糖尿病小鼠模型皮肤创面的愈合。

天花粉凝集素对2型KK-Ay糖尿病小鼠血糖、血脂的调节作用

目的:探讨天花粉凝集素(Trichosanthes kirilowii lectin.TKL)对自发性2型KK-Ay糖尿病小鼠血糖、血脂的调节作用.方法:选取KK-Ay小鼠,随机分为模型组、阳性对照组(罗格列酮,0.003g/kg)、TKL(0.1,0.2,0.3g/kg)剂量组,以同龄C57BL/6J小鼠为正常组,连续灌胃30d,检测小鼠体质量、饮食量、饮水量、空腹血糖(FBG)、口服糖耐量、血清胰岛素(INS)及血脂等指标.结果:与模型组比较,各给药组体质量、饮食量、饮水量、FBG及INS值有显著下降(p<0.05,p<0.01),小鼠糖耐量异常也有所改善;30d后,TKL(0.3g/kg)组血清中TC、TG有明显改善(p<0.05),LDL-C明显降低,HDL-C明显升高(p<0.01);TKL(0.2g/kg)组血清中TG、LDL-C、HDL-C值有明显变化(p<0.05).结论:TKL对2型KK-Ay糖尿病小鼠有一定的降血糖、调血脂作用.

复方黄连降糖片对KK-ay小鼠骨骼肌GLUT-4基因表达的影响

为探讨复方黄连降糖片(主药:大黄、黄连、肉桂等)对胰岛素抵抗型KK ay小鼠骨骼肌葡萄糖转运蛋白4(glucosetransporter 4,GLUT 4)基因表达的影响,将雄性KK ay小鼠,随机分为模型组、中药低剂量组、中药高剂量组、西药组,饲以高脂饮食;选用健康的雄性C57BL小鼠作对照,饲以普通饮食。用药两周后处死动物,测定各组的空腹血糖、血脂、血清胰岛素、GLUT 4mRNA表达。结果:KK ay小鼠在饲以高脂饮食后,血糖、血脂、血清胰岛素显著升高,同时骨骼肌GLUT 4mRNA的表达明显下降。治疗后血糖降低,血甘油三酯降低,骨骼肌GLUT 4mRNA表达明显增加。结论:复方黄连降糖片能改善胰岛素抵抗型KK ay小鼠的糖脂代谢紊乱,增加KK ay小鼠的骨骼肌GLUT 4mRNA表达。

糖尿病诱导的小鼠室旁核催产素和加压素阳性神经元FOS表达

目的:探索糖尿病(DM)不同状态下小鼠下丘脑室旁核(PVN)催产素和加压素阳性神经元的FOS表达变化。方法:腹腔注射溶媒或链脲佐菌素(STZ)制备对照组或糖尿病小鼠模型,根据机械痛测试区分糖尿病痛(DNP组)与非痛组(DWP组)。采用免疫组化和免疫荧光染色技术检测PVN内FOS、催产素(OXT)和加压素(VP)阳性神经元的分布及双标情况,并进行计数和比较。结果:7 d时三组(Control组、DNP组和DWP组)小鼠PVN内均有较多的FOS表达,而28 d时DWP和DNP组FOS的表达几近于无,与7 d组相比差异显著(P<0.05或0.001)。与对照组相比,DNP组和DWP组动物的VP和OXT荧光染色同样存在随着造模时间延长染色强度减弱的趋势(P<0.05)。VP、OXT与FOS的双标染色计数结果显示,在DWP 7 d组,VP/FOS双标细胞数为74.33±22.10,占VP阳性细胞数的(56.64±7.52)%,而OXT/FOS的双标率则仅为(10.44±3.14)%。在DNP 7 d组,OXT/FOS双标细胞数为51.00±31.80,占OXT阳性细胞数的(18.50±9.51)%,而VP/FOS的双标率仅为(9.34±3.27)%。与此相对,28 d组FOS的表达锐减,几乎无双标细胞。结论:下丘脑PVN内的VP和OXT阳性神经元在糖尿病痛与非痛,疾病发展的不同阶段存在明显不同的可塑性变化特点,理解这些变化对于揭示糖尿病及其并发症的神经机制具有重要意义。

SMYD2介导高糖诱导小鼠肾成纤维细胞活化的机制研究

目的 探讨组蛋白赖氨酸甲基转移酶2(SMYD2)介导体外高糖环境下小鼠肾成纤维细胞(NRK-49F)活化的可能机制。方法 复制糖尿病(DM)小鼠模型,经全自动生化分析仪检测各组小鼠血糖(BG)、血肌酐(Scr)水平,经苏木精-伊红、Masson染色观察小鼠肾组织病理改变,经Western blotting检测各组小鼠肾组织纤维连接蛋白(Fibronectin)、Ⅰ型胶原蛋白(CollagenⅠ)、α-平滑肌肌动蛋白(α-SMA)、组蛋白H3赖氨酸4三甲基化(H3K4me3)、SMYD2、核转录因子-κB p65(NF-κB p65)及磷酸化NF-κB p65(NF-κB p-p65)蛋白表达,经免疫荧光检测各组小鼠肾组织α-SMA及SMYD2表达。复制体外DM细胞模型,使用正常糖(含5.5mmol/L葡萄糖)或高糖(含30.0 mmol/L葡萄糖)处理NRK-49F细胞,经Western blotting检测SMYD2、α-SMA及细胞外基质(ECM)成分蛋白表达;经CCK-8法检测SMYD2特异性抑制剂AZ505的细胞毒性;在AZ505处理/不处理的条件下,经Western blotting检测SMYD2、α-SMA、H3K4me3、Fibronectin、CollagenⅠ、NF-κB p65、NF-κB p-p65蛋白表达,经免疫荧光检测α-SMA、SMYD2及NF-κB p-p65在细胞中的表达及空间定位情况。结果 DM组血清BG、Scr水平高于NC组(P <0.05)。DM组肾组织α-SMA、Fibronectin、CollagenⅠ、SMYD2、H3K4me3、NF-κB p-p65蛋白相对表达量高于NC组(P <0.05),两组NF-κB p65蛋白相对表达量比较,差异无统计学意义(P>0.05)。HG刺激不同时间点NRK-49F细胞α-SMA、Collagen I、Fibronectin、SMYD2蛋白相对表达量比较,差异均有统计学意义(P <0.05)。40μmol/L组、60μmol/L组NRK-49F细胞存活率较0μmol/L组低(P <0.05)。HG组NRK-49F细胞Fibronectin、CollagenⅠ、SMYD2蛋白相对表达量较NG组高(P <0.05),HG+AZ505(20μmol/L)组较HG组低(P <0.05)。HG组NRK-49F细胞Fibronectin、CollagenⅠ、α-SMA、H3K4me3、SMYD2蛋白相对表达量较NG组高(P <0.05),HG+AZ505组较HG组低(P <0.05)。HG组NRK-49F细胞NF-κB p-p65蛋白相对表达量较NG组高(P <0.05),HG+AZ505组较HG组低(P <0.05),各组小鼠NRK-49F细胞NF-κB p65蛋白相对表达量比较,差异无统计学意义(P>0.05)。结论SMYD2可介导高糖诱导的NRK-49F细胞活化,其机制可能与激活NF-κB细胞信号通路有关。

Comparison of the pathological characteristics of uninjured skin tissues of diabetic rodents and diabetic patients

Objective To study uninjured diabetic skin structure and compare the pathological characteristics of skin tissue from mouse and rat models of diabetes mellitus(DM)and humans with DM.Methods Establishing the DM C57BL/6J mice skin tissue models and db/db mice skin tissue models,as well as DM rat skin tissue models.Skin tissues were collected from the animal models and DM patients.The biopsy specimens were stained with hematoxylin and eosin(HE)staining,Masson trichrome staining and periodic acid-Schiff(PAS)staining.Results(1)Skin from mice with streptozotocin(STZ)-induced DM was thinner and had less hair than skin from control mice.The epidermis had abnormal keratinization and dermal collagen and elastin fibers were dense and significantly atrophied.(2)Compared with the control group,skin tissue from db/db mice was thinner and had less hair.There was no aberrant keratinization in the epidermis,and dermal collagen and elastin fibres were loose.(3)Skin tissue from rats with STZ-induced DM was significantly thinner than control skin.The epidermis had mildly abnormal keratinization,and the dermis layer was atrophied and partly loose.Some focal lipofuscin was deposited.(4)Whole skin tissue from patients with diabetes was significantly thinner than skin from subjects without DM.The epidermis had mild hyper keratinization,and the dermal elastin fibers were atrophied,loose,irregular,and showed focal mucoid degeneration.Conclusions Uninjured DM skin tissues were thinner and more irregular than normal skin.Structural changes in rat DM skin were most like those seen in the skin of patients with DM.

芪卫颗粒对自发性2型糖尿病KK-Ay小鼠肾组织转化生长因子β_1蛋白及mRNA表达的影响

目的探讨芪卫颗粒对KK-Ay小鼠肾组织TGF-β1蛋白及mRNA表达的影响。方法采用自发性2型糖尿病KK-Ay小鼠模型。将KK-Ay小鼠随机分为模型组、中药组、西药组,以C57BL/6J小鼠作为正常组,每组15只,连续治疗12周,用HE、PAS及Masson染色法观察肾脏病理改变。采用免疫组织化学法及实时荧光定量PCR法,检测肾组织中转化生长因子β1(TGF-β1)的蛋白及mRNA表达。结果模型组小鼠肾脏病理改变呈现肾小球肥大,系膜基质区增宽,系膜细胞增多,肾小管上皮细胞空泡样变性,肾小管扩张,灶状萎缩,偶见坏死,可见较多蛋白管型,间质内炎性细胞浸润,间质纤维化表现。肾脏细胞内的TGF-β1的蛋白及mRNA表达较正常组明显增多。经芪卫颗粒、缬沙坦处理后,小鼠肾脏病理改变较模型组明显减轻,TGF-β1的蛋白及mRNA表达明显降低(P<0.05)。结论芪卫颗粒可通过抑制TGF-β1的蛋白及mRNA表达起到防治糖尿病肾病的作用。

达格列净拮抗糖尿病肾脏纤维化作用研究

目的探讨达格列净对糖尿病小鼠肾脏以及棕榈酸诱导的小鼠肾小球系膜细胞(SV40)的保护作用。方法将36只SPF级C57BL/6小鼠随机分为正常组、糖尿病组、达格列净组,体外培养SV40并分为正常组、棕榈酸组、正常对照组、棕榈酸+达格列净组。Masson染色检测小鼠肾脏组织中肌纤维与胶原纤维表达情况,Western blot检测小鼠肾脏组织与SV40中Ⅰ型胶原、Ⅲ型胶原和转化生长因子β1(TGF-β1)蛋白表达,qPCR检测小鼠肾脏组织与SV40中Ⅰ型胶原、Ⅲ型胶原、α-平滑肌肌动蛋白(α-SMA)mRNA含量变化,CCK-8法检测SV40增殖水平。结果达格列净可明显改善糖尿病小鼠肾脏纤维化,抑制SV40细胞增殖,降低糖尿病小鼠肾脏组织与SV40中Ⅰ型胶原、Ⅲ型胶原、TGF-β1与α-SMA表达。结论达格列净通过减少胶原沉积,下调TGF-β1与α-SMA,发挥对糖尿病肾脏纤维化的保护机制。