Effects of Pioglitazone on Renal Mitochondrial calcium and cytochrome C levels in Early Diabetic Nephropathy Rats

Objective: To study the effect of pioglitazone on mitochondrial Ca2+ and cytochrome c (cyt c) in early diabetic rats, and to explore its mechanism of protecting kidney. Methods: 72 DM rats were randomly divided into negative control group (NC group), negative control+ pioglitazone intervention group (NCP group), diabetes group (DM group) and diabetes +pioglitazone intervention group (DMP group). In NCP and DMP groups, pioglitazone was administered to the stomach, blood glucose, renal mass index, 24-h urine protein, glomerular morphologic indice, glomerular base-membrane thickness and other indexes were measured, and the contents of Ca2+ and Cyt C in renal tissue were measured. Results: (1) the renal metabolism index, glomerular morphologic indice, glomerular base-membrane thickness of DM group was significantly higher than that of NC group (P < 0.01). The renal metabolism index, glomerular morphologic indice, glomerular base-membrane thickness of DMP group was significantly lower than that of DM group, the difference between the two groups was significant(P<0.05). (2) in DM group, mitochondrial Ca2+ and cytoplasmic cytc were higher than those in NC group, while mitochondrial cytc was lower than that in control group. There was significant difference between the two groups (P < 0.05). In DMP group, mitochondrial Ca2+ and cytoplasmic cytc were lower than those in DM group, while mitochondrial cytc was higher than that in control group. There was significant difference between the two groups (P <0.05). Conclusion: Pioglitazone treatment can reduce the release of mitochondrial cytochrome C and maintain mitochondrial calcium homeostasis.

Effects of angiotensin-Ⅱ receptor blockers on β-catenin expression in a rat model of experimental streptozotocin-induced early-stage of diabetic nephropathy

Objective： Diabetic nephropathy （DN） is one of the most common causes of end-stage renal failure. The pathogenesis of progressive renal injury is multifactorial and the mechanism by which hyperglycemia causes microangiopathy is still poorly understood. The WNT pathway is activated in DN and regulating β-catenin protein levels is referred to as the canonical Wntβ-catenin pathway. Because the renin angiotensin system has been reported to be an important contributory factor in the pathophysiology of DN, exogenous administration of angiotensin Ⅱ receptor antagonist may be beneficial in counteracting some biochemical or functional changes of DN. The aim of the study was to determine the β-catenin expression and the possible protective effects of irbesartan, an angiotensin Ⅱ type 1 receptor blocker （ARB） in a rat model of streptozotocin（STZ）-induced diabetic nephropathy. Methods： STZ-induced DN in rats was assessed biochemically by measuring urine volume, protein and creatinine clearance as well as Kidney weight/body weight （KW/BW） and the index of mesangial expansion. Three groups of male Sprague-dawley rats were used. The first group consisted of non-diabetic control rats （control）. The second group was the untreated diabetic rats（STZ＋vehicle）. The third group consisted of diabeti rats treated with irbesartan, 50 mg/kg for 12 weeks （STZ＋irbesartan）. Immunohistochemical stainings and real time PCR for β-catenin were performed in renal cortex of rat modals. Results： Marked hyperglycemia, polyuria, proteinuria, renal hypertrophy, mesangial matrix expansion and glomerular hyperfiltration were observed in STZ diabetic rats. The levels of microalbuminuria and KW/BW in the STZ＋irbesartan group were lower than those in the STZ＋vehicle group （P〈0.05）. The up-regulated immunostaining and mRNA expression of β-catenin were decreased in renal cortic of the Irbesartan-treated diabetic group, but there was no significant difference compared to the untreated diabetic group. Conclusion： The data suggest that irbesartan ameliorates proteinuria and renal hypertrophy, charactered damages of STZ-induced early-stage DN in rats, but its effective drug target is not to inhibit the up-regulated expressions of β-catenin.

Renal Protective Activity of Hsian-tsao Extracts in Diabetic Rats

Objective To investigate the renal protective activity of Hsian-tsao Mesona procumbens Hemsl. water extracts in diabetic rats. Methods Thirty Sprague-dawley female rats were randomly divided into three groups （n=10 each）, ＂control group＂ with intraperitoneal saline injection, ＂diabetic group＂ with 60 mg of intraperitoneal streptozotocin injection per kg of body weight and ＂Hsian-tsao group＂ with intragastric administration of Hsian-tsao extraction everyday for 4 weeks after intraperitoneal streptozotocin injection. The body weight and blood sugar were measured before and after model induction in the three groups. Thrombospondin-1 （TSP-1） expressions in the kidney were monitored by immunohistochemistry. Kidney ultrastructural changes were also analyzed by using transmission electron microscopy. Results Before diabetic model induction, there were no significant differences among the three groups in body weight and blood sugar. Four weeks after the induction of diabetes, the differences became statistically significant. Electron microscopy also revealed disruption of the foot processes of the podocytes and other damages in diabetic group. These damages were significantly less severe in Hsian-tsao group when compared with the diabetic group. TSP-1 expressions in the kidney were significantly increased in both the diabetic group and Hsian-tsao group, but it was relatively lower in Hsian-tsao group than in diabetic group. Conclusion Our results showed that Hsian-tsao treatment in the diabetic rats effectively prevented the pathological alterations in the kidney and decreased the TSP- 1 expression. It was suggested that Hsian-tsao had protective effect on the kidneys of the diabetic rats.

Effect of liraglutide on endoplasmic reticulum stress in the renal tissue of type 2 diabetic rats

BACKGROUND Liraglutide is a glucagon-like peptide 1 receptor agonist analog that has been found to have a therapeutic effect in diabetes.In addition to its ability to treat diabetes,liraglutide has beneficial effects on the cardiovascular system and kidney as well as other beneficial effects,but its specific mechanism is not clear.In this study,a rat model of type 2 diabetes was established by administration of a high-sugar,high-fat diet combined with low-dose streptozotocin(STZ)to observe the effect of liraglutide on the kidneys of type 2 diabetes rats and the possible underlying mechanisms.AIM To explore whether liraglutide has a protective effect on type 2 diabetic rat kidneys and the underlying mechanisms.METHODS Eight-week-old male Sprague-Dawley rats were randomly divided into a control group,model group,low-dose liraglutide group,and high-dose liraglutide group.Control rats were fed a standard diet,while model group and intervention group rats were fed high-sugar,high-fat feed for 1 mo and then intraperitoneally injected with 40 mg/kg STZ to induce type 2 diabetes.The low-dose and highdose intervention groups received 100μg/kg and 200μg/kg liraglutide,respectively,once daily by subcutaneous injection.The control and model groups were given an equivalent volume of physiological saline for 8 wk.Pathological changes in renal tissues were observed by hematoxylin and eosin staining and periodic acid-Schiff staining,and GRP78 and caspase-12 expression was detected by Western blot and reverse transcription-polymerase chain reaction(RT-PCR).RESULTS Western blot analysis showed that GRP78 and caspase-12 protein expression in kidney tissue was significantly higher in model rats than in normal rats and lowerin the liraglutide-treated groups than in the model group,with a more significant decrease being observed in the high-dose group than in the low-dose group.RTPCR showed that the mRNA expression of GRP78 and caspase-12 was higher in model rats than in control rats and lower in the liraglutide-treated groups than in the model group,with the high-dose group exhibiting a more significant decrease than the low-dose group.CONCLUSION Liraglutide may delay the progression of diabetic nephropathy by reducing endoplasmic reticulum stress and protect the kidneys in a dose-dependent manner.

Expression of p27(Kip1), a Cyclin-Dependent Kinase Inhibitor, in Human Peripheral Blood Mononuclear Cells Is Inversely Associated with Potential Carcinogenic Risk in Obese Type 2 Diabetic Individuals Relative to Lean Normal Controls

Introduction: The consensus report issued jointly by the American Diabetes Association and the American Cancer Society stated that “type 2 diabetes and cancer share many risk factors, but potential biologic links between the two diseases are incompletely understood”. Interestingly, however, a recent report suggested that the expression of p27(Kip1), a cell cycle repressor protein, in the rodent liver was inversely associated with potential carcinogenic risk in the genetic rodent models of diabetic obesity. p27 is a cyclin-dependent kinase inhibitor that, when down-regulated, allows the progression of the cell cycle from G1 to S phase, thereby increasing the risk of developing cancer. Objective: The objective of the study described below was to extend the results of the recent report on the expression of p27 in the livers of obese, diabetic rodents to the humans and investigate whether the expression of p27 in the human peripheral blood mononuclear cells (PBMCs) might also be inversely associated with potential carcinogenic risk in obese type 2 diabetic individuals relative to the lean normal controls. Methods: Western immunoblot analysis was performed to evaluate the expression of p27 and the two most relevant upstream molecular signaling pathways of the expression of p27, namely 4E-BP1 and MNK1, in human PBMCs obtained from obese type 2 diabetic individuals relative to the lean normal controls. Results: First, expression of p27 in human PBMCs was significantly down-regulated in obese type 2 diabetic individuals relative to the lean normal controls. Secondly, expression of p27 in human PBMCs was also significantly down-regulated in obese type 2 diabetic African Americans relative even to the obese type 2 diabetic Caucasian Americans. Conclusions: Expression of p27 in human PBMCs was inversely associated with potential carcinogenic risk in obese type 2 diabetes relative to the lean normal controls.

Resveratrol对糖尿病大鼠肾皮质4E-BP1和S6磷酸化表达的影响

目的:探讨Resveratrol对糖尿病(DM)大鼠早期肾脏肥大的影响及其可能的机制。方法:雄性SD大鼠13只,应用链脲佐菌素诱导建立DM大鼠模型,6只正常SD大鼠作为对照(NC)。DM大鼠于第11周随机分为DM组7只和Resveratrol(DR)组6只,DR组给予Resveratrol 10 mg/(kg.d)灌胃,共4周。第14周收集3组大鼠24 h尿测尿微量白蛋白。处死大鼠,心脏取血测血肌酐。取肾组织,制石蜡切片做HE染色,免疫组化观察肾皮质4E-BP1磷酸化蛋白表达量的变化。Western blot检测肾皮质S6磷酸化蛋白表达的改变。结果:与NC组相比,DR和DM组大鼠血糖、24 h尿微量白蛋白和血肌酐明显升高,而体重明显降低(P均<0.01),而用Resveratrol干预后DR组大鼠血糖、24 h尿微量白蛋白及血肌酐比DM组明显好转(P<0.05)。肾脏HE染色结果显示,DM组大鼠肾小球系膜基质中度增生,系膜细胞增生,系膜区明显扩大,肾小球体积增大。DR组肾小球系膜细胞和基质增生及肾小球系膜区扩张较DM组明显减轻。肾皮质免疫组化结果显示,4E-BP1磷酸化蛋白在DM组表达呈强阳性,在DR组表达呈弱阳性。DM和DR组大鼠血管组织中S6磷酸化蛋白的表达明显高于NC组(P<0.01),而DR组明显低于DM组(P<0.01)。结论:DM大鼠早期存在肾脏肥大和mTOR信号通路下游的4E-BP1和S6磷酸化蛋白表达增高。Resveratrol可能通过抑制4E-BP1和S6的磷酸化来减轻DM大鼠早期肾脏肥大,延缓糖尿病肾病的发展。

基于CNA35靶向液态氟碳纳米粒的超声分子成像检测糖尿病肾病大鼠肾脏纤维化的实验研究

目的制备一种由胶原结合蛋白35(CNA35)介导的靶向液态氟碳纳米粒(PFP-NPs),探讨其在超声分子成像检测糖尿病肾病(DN)大鼠肾脏纤维化中的应用价值。方法制备经荧光标记的非靶向PFP-NPs和CNA35靶向PFP-NPs,经转化生长因子-β诱导人肾小管上皮HK-2细胞间质转化,发生胶原沉积,然后分别将非靶向PFP-NPs和CNA35靶向PFP-NPs与细胞共同孵育,于倒置荧光显微镜下观察细胞上PFP-NPs荧光信号。建立DN大鼠模型,分为非靶向组和靶向组,每组各10只,分别静脉注射非靶向PFP-NPs与CNA35靶向PFP-NPs,应用超声分子成像检测两组DN大鼠静脉注射PFP-NPs后10 min、30 min肾脏超声分子成像信号强度;然后处死DN大鼠并取肾脏组织,检测肾脏组织PFP-NPs荧光信号强度及Ⅰ型胶原荧光信号强度;免疫组织化学检测肾脏组织Ⅰ型胶原染色面积占比,分析肾脏超声分子成像信号强度与Ⅰ型胶原染色面积占比的相关性。结果CNA35靶向PFP-NPs孵育的人肾小管上皮HK-2细胞上的荧光信号强度高于非靶向PFP-NPs孵育的细胞(388.21±15.28 vs.25.79±3.62),差异有统计学意义(P<0.05)。靶向组DN大鼠肾脏组织PFP-NPs荧光信号强度高于非靶向组(946.02±83.55 vs.73.69±21.72),差异有统计学意义(P<0.05);靶向组Ⅰ型胶原荧光信号强度与非靶向组比较差异无统计学意义(969.07±96.67 vs.943.39±106.18),且CNA35靶向PFP-NPs荧光信号与Ⅰ型胶原荧光信号区域重合。体内超声分子成像显示,靶向组DN大鼠静脉注射PFPNPs后10 min、30 min肾脏组织超声分子成像信号强度均高于非靶向组(8.37±1.27 vs.1.92±0.25、9.73±1.25 vs.2.08±1.32),差异均有统计学意义(均P<0.05)。相关性分析显示,DN大鼠肾脏超声分子成像信号强度与Ⅰ型胶原染色面积占比呈正相关(r=0.838,P<0.001)。结论成功制备了CNA35靶向PFP-NPs,其能够靶向结合大鼠肾脏胶原高表达部位,实现了对DN大鼠肾脏纤维化的靶向超声分子成像。

隐丹参酮对STZ诱导的糖尿病肾病模型大鼠的肾脏保护作用及机制

目的探讨隐丹参酮对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病肾病(diabetic nephropa-thy,DN)模型大鼠肾脏保护作用及可能作用机制.方法60只大鼠按随机数字表法分为对照组,DN模型组,隐丹参酮低、中、高剂量组(15、30、60 mg·kg^(-1)·d^(-1))和二甲双胍组(200 mg·kg^(-1)·d^(-1)),除对照组,其余均以STZ诱导制成DN大鼠模型.给药结束24 h后,检测大鼠的空腹血糖(FPG)、甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白中胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、24h尿蛋白定量、血尿素氮(BUN)、血清肌酐(Scr)、超氧化物歧化酶(SOD)、丙二醛(MDA)、活性氧(ROS)指标;Western blot检测TGF-β1/Smad3信号通路相关蛋白表达水平.结果生化指标检测结果显示:经药物干预后,二甲双胍组和隐丹参酮中、高剂量组大鼠FPG、尿蛋白、BUN、Scr、MDA、ROS、TG、TC、LDL-C水平、全血高切黏度、全血中切黏度、全血低切黏度及血浆黏度水平均较DN模型组降低,而SOD、HDL-C水较DN模型组升高.Western blot结果显示:经药物干预后,二甲双胍组和隐丹参酮中、高剂量组大鼠TGF-β1蛋白表达水平、p-Smad3/Smad3比值较DN模型组降低,而p-Smad7/Smad7比值较DN模型组升高.结论隐丹参酮可改善STZ诱导的DN模型大鼠的糖脂代谢、氧化应激及血液流变学情况,提升大鼠的肾功能,其保护作用可能与激活TGF-β1/Smad3信号通路有关.

基于AMPK/mTOR自噬信号通路研究达格列净对糖尿病肾病模型大鼠肾损伤改善的作用机制

目的研究达格列净对糖尿病肾病(diabetic nephropathy,DN)模型大鼠肾损伤的改善机制。方法40只清洁级Wistar雄性大鼠分为空白对照组(蒸馏水灌胃)、模型组(蒸馏水灌胃)、达格列净低剂量组[达格列净片1 mg/(kg·d)灌胃]、达格列净高剂量组[达格列净片10 mg/(kg·d)灌胃]各10只,干预12周。观察各组大鼠体质量、24 h尿量、24 h尿蛋白、随机血糖、血清尿素氮(BUN)、肌酐(Scr)的差异;苏木精-伊红染色观察各组大鼠肾脏组织病理变化;免疫组化检测各组大鼠肾组织腺苷酸活化蛋白激酶(AMPK)及哺乳动物雷帕霉素靶蛋白(mTOR)表达;实时荧光PCR(qRT-PCR)检测各组大鼠肾组织AMPK、mTOR mRNA表达。结果空白对照组、达格列净高剂量组、达格列净低剂量组、模型组大鼠体质量依次降低,24 h尿量、随机血糖、24 h尿蛋白、BUN、Scr依次升高,组间两两比较,差异均有统计学意义(P<0.01)。模型组、达格列净低剂量组、达格列净高剂量组大鼠肾脏组织病理变化比较,肾小球大小逐渐恢复,细胞排列依次紧密有序,肾小管上皮细胞空泡样变性逐渐减小,细胞排列整齐度依次增加,出血减少。空白对照组、达格列净高剂量组、达格列净低剂量组、模型组大鼠肾脏组织AMPK蛋白表达[(0.36±0.03)、(0.27±0.02)、(0.21±0.02)、(0.18±0.02)]依次降低(P<0.01),mTOR表达[(0.13±0.02)、(0.17±0.02)、(0.26±0.02)、(0.31±0.03)]依次升高(P<0.01),AMPK mRNA相对表达量[(1.00±0.08)、(0.77±0.07)、(0.52±0.05)、(0.26±0.03)]依次降低(P<0.01),mTOR mRNA相对表达量[(1.00±0.06)、(1.17±0.08)、(1.76±0.13)、(2.31±0.16)]依次升高(P<0.01)。结论达格列净可激活AMPK/mTOR自噬信号通路,保护肾小球滤过屏障,减少蛋白尿,延缓DN大鼠的疾病进程。

白藜芦醇对糖尿病肾病大鼠JAK/STAT信号通路的影响

目的研究白藜芦醇对糖尿病肾病大鼠JAK/STAT信号通路的影响。方法高糖高脂饲料喂养健康SD大鼠8周后,用链脲佐菌素45 mg/kg腹腔注射造模。将40只成模大鼠随机分为模型组、白藜芦醇低剂量组、白藜芦醇中剂量组、白藜芦醇高剂量组,每组10只。白藜芦醇低、中、高剂量组分别给予10 mg/kg、20 mg/kg、40 mg/kg白藜芦醇灌胃治疗,正常组和模型组则给予等容量蒸馏水。连续灌胃4周后,以HE染色观察肾脏组织形态学改变,并比较各组大鼠血糖、血清胆固醇、甘油三酯、尿素氮和肌酐水平。采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)和白介素-6(interleukin-6,IL-6)水平;荧光定量PCR法检测肾组织两面神激酶2(Janus kinase 2,JAK2)、信号传导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)、细胞因子信号抑制物1(suppressor of cytokine signaling 1,SOCS1)、ICAM-1和IL-6的mRNA水平;Western blot法检测肾组织磷酸化两面神激酶2(phosphorylated Janus kinase 2,p-JAK2)、磷酸化信号传导与转录激活因子3(phosphorylated signal transducer and activator of transcription 3,p-STAT3)、SOCS1、ICAM-1和IL-6的蛋白表达。结果经白藜芦醇治疗后,白藜芦醇各剂量组肾脏病理损伤减轻。与正常组比较,模型组大鼠血糖、血清胆固醇、甘油三酯、尿素氮、肌酐均升高(P均<0.05)。与模型组相比,白藜芦醇中、高剂量组大鼠血糖、血清胆固醇、甘油三酯、尿素氮和肌酐均降低(P均<0.05)。白藜芦醇各剂量组大鼠血清ICAM-1和IL-6均较模型组显著降低(P均<0.05),且白藜芦醇中、高剂量组大鼠血清ICAM-1和IL-6显著低于低剂量组(P均<0.05)。白藜芦醇各剂量组肾组织JAK2和STAT3的mRNA水平无明显变化(P>0.05),而p-JAK2和p-STAT3的蛋白表达均较模型组下降(P均<0.05);ICAM-1和IL-6的mRNA水平和蛋白表达均较模型组降低(P均<0.05),而SOCS1的mRNA水平和蛋白表达均较模型组升高(P均<0.05)。结论白藜芦醇能减轻糖尿病肾病大鼠的肾脏损害,控制其血糖、血脂,降低血清尿素氮和肌酐水平,其机制可能与抑制JAK/STAT信号通路有关。

三黄益肾胶囊调控NF-κB信号通路减轻糖尿病肾病大鼠炎症反应的机制研究

目的:旨在探究三黄益肾胶囊治疗糖尿病肾病(DN)可能的炎症反应机制。方法:按照随机数字表法将60只大鼠分为正常组、模型组、厄贝沙坦组及三黄益肾低、中、高剂量组,每组10只。除正常组外,其余各组大鼠用高糖高脂饲料联合链脲佐菌素(STZ)注射建立DN模型。正常组和模型组给予生理盐水2 mL灌胃;厄贝沙坦组给予厄贝沙坦11.51 mg/kg灌胃;三黄益肾低、中、高剂量组分别给予三黄益肾胶囊0.41、0.81、1.62 g/kg灌胃。各组大鼠均1次/d,连续给药4周。比较各组大鼠空腹血糖(FBG)、体质量、24 h尿白蛋白、肾功能指标、肾组织病理学变化、肾组织抗氧化相关指标(SOD、GSH-Px活性及MDA水平)、肾组织炎症介质[白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)]、肾组织诱导型一氧化氮合酶(iNOS)、肾组织CD68、CC趋化因子受体2(CCR2)表达、肾组织CC趋化因子配体2(CCL2)、NF-κB P65、p-NF-κB P65、IκB、p-IκB表达。结果:与模型组比较,各三黄益肾胶囊组大鼠体质量高,FBG、血清肌酐(Cr)、尿素氮(BUN)及24 h尿蛋白水平低,肾脏组织病理变化减轻,三肾组织中SOD、GSH-Px活性高,MDA水平低,IL-6、IL-1β、TNF-α水平低,肾组织中iNOS水平低,肾组织CCR2与CD68共定位水平低,CCL2、p-NF-κB P65、p-IκB蛋白表达低。结论:三黄益肾胶囊治疗DN的机制可能与抑制肾组织中NF-κB信号通路激活,减轻肾脏氧化应激及炎症反应有关。

基于脂噬介导的mTOR/TFEB信号通路探讨当归红芪超滤物干预糖尿病肾病作用机制

目的以当归红芪超滤物干预糖尿病肾病模型大鼠,探讨其对糖尿病肾病脂噬的效应机制。方法50只SPF级雄性Wistar大鼠随机选取7只为空白组,其余43只采用一次性大剂量腹腔注射链脲佐菌素联合高脂高糖饲料喂养制备糖尿病肾病模型。将成模大鼠随机分为模型组、厄贝沙坦组(17.9 mg/kg)和中药低、中、高剂量组(1.5、3、6g/kg),分别灌胃相应溶液,连续12周。检测大鼠随机血糖、体质量及24h尿蛋白含量,生化检测糖化血清蛋白(GSP)、血肌酐(SCr)、血尿素氮(BUN)、三酰甘油(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、游离脂肪酸(FFA)含量,HE染色观察肾组织形态变化,Masson染色观察肾组织纤维化情况,透射电镜观察肾组织超微结构,RT-qPCR检测肾组织哺乳动物雷帕霉素靶蛋白(mTOR)、微管相关蛋白1轻链3(LC3B)mRNA表达,Westernblot检测肾组织mTOR、转录因子EB(TFEB)、p-TFEB、LC3B蛋白表达。结果与空白组比较,模型组大鼠随机血糖显著升高(P<0.01),体质量显著减少(P<0.01),24h尿蛋白含量显著升高(P<0.01),GSP、SCr、BUN、TG、TC、LDL-C、FFA含量显著升高(P<0.01),HDL-C含量显著降低(P<0.01);肾小管上皮细胞空泡变性、胞质空泡化、有大量炎性细胞浸润,胶原纤维增多且分布杂乱,脂滴数量增加,自噬体数量减少;肾组织mTORmRNA表达显著升高(P<0.01),LC3BmRNA表达显著降低(P<0.01),mTOR、p-TFEB蛋白表达显著升高(P<0.01),TFEB、LC3BⅡ蛋白表达显著降低(P<0.01)。与模型组比较,各给药组大鼠给药12周随机血糖、24h尿蛋白含量降低,体质量增加,GSP、SCr、BUN、TG、TC、LDL-C、FFA含量降低,HDL-C含量升高;肾组织炎性细胞浸润、纤维化、脂滴沉积有不同程度缓解,肾组织mTORmRNA表达降低,LC3BmRNA表达升高,mTOR、p-TFEB蛋白表达降低,TFEB、LC3BⅡ蛋白表达升高,其中厄贝沙坦组和中药高剂量组差异均有统计学意义(P<0.01,P<0.05)。结论当归红芪超滤物能改善糖尿病肾病模型大鼠糖脂代谢紊乱、拮抗肾脏异位脂质沉积、促进脂噬、延缓糖尿病肾病进程,其机制可能与mTOR/TFEB信号通路有关。

复方鱼腥草提取物对2型糖尿病大鼠肾脏组织中Sirt1/PGC-1α通路的影响

目的探讨复方鱼腥草提取物(牛蒡子及鱼腥草的挥发油、水提物、醇提物的混合物)对2型糖尿病大鼠肾脏组织中Sirt1/PGC-1α通路的影响。方法将SD大鼠随机分为正常组、模型组、罗格列酮组(0.3 mg·kg^(-1))、白藜芦醇组(0.12 g·kg^(-1))及复方鱼腥草提取物组(4.5 g·kg^(-1)),每组10只。采用高糖高脂饲料喂养4周联合小剂量腹腔注射1%链脲佐菌素(STZ,30 mg·kg^(-1))复制2型糖尿病大鼠模型。检测各组大鼠24 h尿蛋白、24 h尿白蛋白、尿肌酐以及血清肌酐水平,计算肌酐清除率、肾脏指数。采用HE染色法观察肾脏组织病理形态学变化;免疫组化法检测肾脏组织中Sirt1表达水平;Western Blot法检测大鼠肾皮质中Sirt1、PGC-1α蛋白表达水平。结果与正常组比较,给药4、8周后,模型组大鼠的24 h尿蛋白及尿白蛋白水平、肌酐清除率、肾脏指数均明显升高(P<0.05);肾小球体积明显增大,可见肾间质伴有炎性细胞浸润,系膜区可见明显扩张;免疫组化显示肾脏组织中的Sirt1表达显著下调(P<0.01);肾皮质中Sirt1、PGC-1α蛋白表达均明显下调(P<0.05)。与模型组比较,复方鱼腥草提取物组大鼠的24 h尿蛋白及尿白蛋白水平、肌酐清除率、肾脏指数均明显降低(P<0.05);肾小球增大、系膜增生与基质聚积均有明显改善;免疫组化显示肾脏组织中的Sirt1表达显著上调(P<0.01);肾皮质中Sirt1、PGC-1α蛋白表达均明显上调(P<0.05,P<0.01)。结论复方鱼腥草提取物对2型糖尿病大鼠的早期肾脏损伤具有保护作用,其机制可能与上调肾皮质中Sirt1/PGC-1α通路有关。

抵当汤含药血清对高糖诱导大鼠肾小球内皮细胞损伤的影响

目的观察抵当汤含药血清对高糖诱导损伤大鼠肾小球内皮细胞(RGECs)的保护作用。方法大鼠灌胃给予蒸馏水或抵当汤制备空白血清或抵当汤含药血清,CCK8法筛选葡萄糖造模浓度及含药血清给药浓度。将RGECs分为空白组、模型组、抵当汤组(10%抵当汤含药血清)、达格列净组(空白血清+2μmol/L达格列净)、抵当汤+达格列净组(10%抵当汤含药血清+2μmol/L达格列净),药物处理24 h后,RT-qPCR法和Western blot法检测细胞Nrf2、HO-1、NQO-1 mRNA和蛋白表达,免疫荧光法检测细胞Nrf2荧光表达,比色法检测细胞SOD活性及MDA水平。结果与空白组比较,模型组RGECs中Nrf2、HO-1、NQO-1 mRNA和蛋白表达以及SOD活性降低(P<0.01),MDA水平升高(P<0.01);与模型组比较,各给药组Nrf2、HO-1、NQO-1 mRNA和蛋白表达以及SOD活性升高(P<0.05,P<0.01),MDA水平降低(P<0.01)。结论抵当汤含药血清可以通过激活Nrf2信号通路从而抑制氧化应激,对高糖损伤的RGECs起到保护作用。

基于网络药理学研究灯盏细辛治疗糖尿病肾病的作用机制

利用网络药理学研究中草药灯盏细辛治疗糖尿病肾病(Diabetic Nephropathy,DN)的作用机制。通过TCMSP平台、ETCM数据库、PubChem数据库筛选出灯盏细辛的有效活性成分及作用靶点;利用DisGenet及GeneCards获取与DN相关靶点基因;筛选灯盏细辛活性成分作用靶点与疾病靶点的共同靶点基因。运用Cytoscape 3.9.1软件构建“成分-靶点-疾病”网络图;借助STRING在线数据库构建蛋白互作(PPI)网络图,使用Cytoscape 3.9.1软件插件ClueGo对共同靶点进行KEGG及GO分析,筛选出潜在的信号通路,并分析其作用机制。通过筛选得到灯盏细辛的有效活性成分9个,其作用于DN的靶点为79个。GO富集分析结果示,灯盏细辛-糖尿病肾病交集基因的生物功能主要涉及跨膜受体蛋白酪氨酸激酶信号通路正调控、细胞增殖、细胞凋亡过程的负调控等生物学过程。KEGG通路富集结果显示,交集基因主要分布在PI3K-AKT信号通路上。灯盏细辛的作用机制呈现多成分、多靶点、多通路的特点,本研究从药理作用及网络药理学两个方面研究灯盏细辛治疗DN的作用机制,有利于更加深入全面地了解灯盏细辛的药用价值,为其开发和利用提供理论参考。

糖尿病肾病大鼠原代肾小管上皮细胞的提取及体外培养

目的探讨糖尿病肾病(DN)大鼠原代肾小管上皮细胞(RTECs)的提取及体外培养方法。方法取SD雄性大鼠25只,随机分为正常组(n=5)和模型组(n=20)。对模型组大鼠构建DN大鼠模型,不给予正常组任何处理。获取两组大鼠双肾组织,一部分组织用于HE染色及Masson染色以观察肾组织结构,另一部分用于RTECs的提取及培养。RTECs的提取、培养方法:将肾皮质组织在80目筛网上研磨,经100目筛网过滤、Ⅰ型胶原酶消化25 min后,使用含10%胎牛血清、1%胰岛素⁃转铁蛋白⁃硒⁃乙醇胺添加剂和1%青链霉素双抗溶液的DMEM/F-12完全培养基培养细胞。用免疫荧光染色法检测细胞角蛋白18的表达情况以鉴定所获得的细胞。结果(1)建模后模型组大鼠出现多饮、多食、多尿、体重下降、血糖升高等特征,尿蛋白定性呈阳性,肾组织病理检测提示肾小球结构不完整且出现明显缺损,RTECs明显肿胀、变性,肾小管管腔萎缩,肾小管间质纤维化严重。(2)培养72 h后,两组RTECs均已经贴壁并呈岛屿状聚集生长,培养5~8 d后细胞呈多边鹅卵石样。模型组RTECs高表达细胞角蛋白18,阳性率>95%。结论采用在80目筛网上研磨、100目筛网过滤、Ⅰ型胶原酶消化25 min的方法可以得到数量较多、活性良好、纯度较高的DN大鼠模型RTECs,且在形态与贴壁情况方面与正常大鼠RTECs无明显差异。

人脐带间充质干细胞对糖尿病肾病大鼠肾脏HIF-1α表达的影响

目的观察人脐带间充质干细胞(HUMSC)对糖尿病肾病大鼠肾脏缺氧诱导因子(HIF-1α)表达的影响。方法50只健康清洁级8周龄雄性SD大鼠,随机选取20只为健康对照组,其余30只采用链脲菌素复制糖尿病肾病大鼠模型(模型组),两组大鼠饲养12周。第12周,随机选取两组大鼠各3只分别尾静脉注射DiR-HUMSCs,代谢12~16 h后在小动物活体光学3D成像系统下观察DiR-HUMSCs在大鼠体内的分布。随机选取9只模型组大鼠进行HUMSCs移植(HUMSCs移植组)。HUMSCs移植采用尾静脉注射方式移植浓度为1×10^(6)个/mL HUMSCs 500μL至大鼠体内,每周1次,连续4周,共28 d。检测3组大鼠的24 h尿蛋白定量(24 h UPro)、血清肌酐(Scr)、血尿素氮(BUN)、尿肌酐(Ucr)、尿白蛋白与肌酐比值(UACR)水平;酶联免疫吸附试验(ELISA)检测3组大鼠血清HIF-1α水平;采用PAS和Masson染色进行肾脏组织病理检测;免疫荧光法检测3组大鼠肾组织HIF-1α、Slc12A3和Aquaporin1蛋白的表达。结果移植HUMSCs治疗4周后,与健康对照组比较,模型组和HUMSCs移植组大鼠Ucr水平降低(P<0.05),Scr、24 h UPro、BUN、UCAR均升高(P<0.05);与模型组比较,HUMSCs移植组大鼠Ucr水平差异无统计学意义(P>0.05),Scr、24 h UPro、BUN、UCAR均降低(P<0.05)。糖尿病肾病大鼠病理损伤缓解,系膜增生和基底膜增厚改善,小管空泡变性减少,间质纤维化减轻。模型组大鼠血清HIF-1α水平较健康对照组升高(P<0.05),HUMSCs移植组血清HIF-1α水平较模型组下降(P<0.05)。与健康对照组比较,模型组大鼠肾脏远端小管中HIF-1α蛋白水平增加(P<0.05);HUMSCs移植组的HIF-1α蛋白水平较模型组降低(P<0.05)。模型组远端小管标记蛋白Slc12A3水平低于健康对照组(P<0.05),HUMSCs移植组Slc12A3水平较模型组升高(P<0.05)。HUMSCs移植组的Aquaporin1蛋白水平较模型组和健康对照组均降低(P<0.05)。糖尿病肾病大鼠近端小管中无HIF-1α表达。结论HIF-1α主要在糖尿病肾病大鼠肾脏远端小管表达,且HUMSCs可通过抑制HIF-1α的表达修复肾小管的损伤。

贝那普利灌胃对糖尿病肾病大鼠肾组织连接蛋白表达的影响

目的探讨贝那普利对于糖尿病肾病大鼠肾脏连接蛋白(Cxs)表达的影响。方法将SD雄性大鼠分为对照组、糖尿病组、干预组,糖尿病组及干预组予以STZ 65 mg/kg一次性左下腹注射建立Ⅰ型糖尿病模型,对照组予以相同剂量的枸橼酸盐缓冲液腹腔注射。造模成功后第2天开始,干预组予贝那普利10 mg/(kg·d)每日定时灌胃,对照组和糖尿病组给予等量0.9%氯化钠溶液灌胃,治疗8周,测量各组体质量,采用酶电解层析法检测空腹血糖,采用双抗体夹心法检测尿微量白蛋白;分别应用肌氨酸氧化酶法、酶偶联速率法检测血肌酐、血尿素。8周后处死大鼠,应用SABC法观察连接蛋白Cx37、Cx40、Cx43在肾脏病理组织的分布,免疫组化法检测连接蛋白Cx37、Cx40、Cx43表达,RT-qRCR法检测Cx37、Cx40、Cx43 mRNA表达。结果与对照组相比,糖尿病组及干预组大鼠体质量较前明显下降,血糖较前明显升高(P均<0.05);干预组血尿素、血肌酐、尿微量白蛋白较糖尿病组均明显下降(P均<0.05)。糖尿病组Cx37、Cx43在出入球小动脉、近端肾小管分布明显减弱,干预组Cx37、Cx43在近端肾小管表达明显升高,糖尿病组可见Cx40在多处入球小动脉、致密斑分布明显减弱,干预组Cx40在致密斑分布增强。与糖尿病组比较,干预组Cx37、Cx40表达升高,差异有统计学意义(P均<0.05);干预组Cx43表达高于糖尿病组,差异无统计学意义(P>0.05)。Cx37 C_(T)值在糖尿病组明显升高,且对照组Cx37 C_(T)值高于干预组(P均<0.05)。三组Cx40 C_(T)值差异无统计学意义,干预组Cx40 C_(T)值较糖尿病组明显减小(P<0.05)。与对照组比较,糖尿病组Cx43 C_(T)值明显下降,干预组Cx43 C_(T)值明显上升,三组Cx43 C_(T)值差异均有统计学意义(P均<0.05)。结论贝那普利可上调糖尿病肾病大鼠肾脏连接蛋白表达,改善糖尿病肾病大鼠肾脏病理损伤,保护肾脏,延缓疾病进展。

甲状腺激素与糖尿病肾病严重程度及肾功能的关系

目的分析甲状腺激素与糖尿病肾病(DN)严重程度及肾功能的关系。方法实验时间2021年9月—2022年9月,选取SPF级健康雄性SD大鼠40只,采用随机数字表法分为4组,每组10只,将仅注射等量的枸橼酸盐缓冲液的大鼠纳入空白对照组,将注射浓度为50 mg/kg链脲佐菌素溶液的大鼠纳入A组,注射浓度为60 mg/kg链脲佐菌素溶液的大鼠纳入B组,注射浓度为70 mg/kg链脲佐菌素溶液的大鼠纳入C组。在造模过程,B组大鼠死亡1只,C组大鼠死亡3只。观察4组大鼠活动、精神状态,比较4组大鼠摄食量、摄水量、体质量、肾功能指标[血肌酐(SCr)、血尿素氮(BUN)、24 h尿微量白蛋白(24 hmAlb)]、肾肥大指数、甲状腺激素[三碘甲状腺原氨酸(T3)、甲状腺素(T4)、促甲状腺激素(TSH)],采用Spearman秩相关分析探讨A、B、C组大鼠甲状腺激素与其肾功能指标间的相关性。结果建模后第6周,空白对照组大鼠精神状态评分为0分,A组为1分,B组为1.22分,C组为1.43分。A、B、C组建模后第6周连续7 d的摄食量、摄水量均多于空白对照组(P<0.05);摄食量上,B、C组少于A组,C组少于B组(P<0.05);摄水量上,B、C组多于A组,C组多于B组(P<0.05)。A、B、C组建模后第6周体质量低于空白对照组(P<0.05);C组体质量低于A、B组(P<0.05)。A、B、C组SCr水平、BUN水平、24 hmAlb、肾肥大指数均高于空白对照组(P<0.05),且B、C组高于A组,C组高于B组(P<0.05)。4组T4水平比较,差异无统计学意义(P>0.05)。A、B、C组T3水平低于空白对照组,TSH水平高于空白对照组(P<0.05);B、C组T3水平低于A组,TSH水平高于A组(P<0.05),且C组T3水平低于B组,TSH水平高于B组(P<0.05)。Spearman秩相关分析结果显示,A、B、C组T3水平与SCr、BUN、24 hmAlb均呈负相关(P<0.05),T4、TSH水平与SCr、BUN、24 hmAlb均无直线相关性(P>0.05)。结论随着DN病情加重,T3水平随之明显降低,TSH水平随之明显升高,且T3水平与不同严重程度DN大鼠肾损伤呈负相关。

Relationship between urinary nephrin and urinary albumin changes in diabetic rats and effects of Yiqiyangyinhuayutongluo Recipe

OBJECTIVE:To investigate the dynamic changes of urinary nephrin,and the relationship between it and urinary albumin excretion rate(UAER) in a diabetic rat model,as well the effects of yiqiyangyinhuayutongluo recipe.METHODS:Diabetic model was induced by high fat diet combined with low-dose Streptozotocin(STZ) in rats.Normal group(NG),model group(MG),and yiqiyangyinhuayutongluo recipe treated group(YHTG) were set.Gastrointestinal Yiqiyangyinhuayutongluo recipe was administered once daily for 32 w.At the end of the 2nd w(2w),8w,16w,and 32w,fasting blood glucose(FBG),UAER and 24h urinary nephrin(U-nephrin) were detected.RESULTS:Compared with NG,FBG in MG increased notably(P<0.05).Compared with MG,FBG of YHTG reduced slowly,and the difference was significant(P<0.05) since 16w.U-nephrin and UAER in MG increased significantly from 2w,peaked at 16w,lessened in different degree at 32w,but were still higher than NG.The correlation analysis showed that there was a significant positive correlation between U-nephrin and UAER at different time,the correlation coefficient as r>0.9,and P<0.05.Compared with MG,U-nephrin and UAER in YHTG decreased markedly(P<0.05) except for U-nephrin at 8w.CONCLUSIONS:U-nephrin and UAER in diabetic rat model have a positive linear correlation.Yiqiyangyinhuayutongluo recipe can reduce UAER markedly,and preventing the lose of nephrin in urine maybe one of the mechanisms.