Efficacy and Safety of Clindamycin Phosphate 1.2% and Tretinoin 0.025% Gel for the Treatment of Acne and Acne-induced Post-inflammatory Hyperpigmentation in Patients with Skin of Color

Objective: To assess the efficacy and safety of a topical gel containing clindamycin 1.2% and tretinoin 0.025% for the treatment of acne and acne-induced postinflammatory hyperpigmentation (PIH) in darker skinned patients. Design: Randomized, double-blind, placebo-controlled study. Setting: Two United States clinical sites. Participants: Thirty-three patients 12 years of age or older with skin types IV to VI, mild-to-moderate facial acne, and PIH were enrolled. Measurements: Patients applied clindamycin phosphate/tretinoin gel or a nonmedicated vehicle each evening and a sun protection factor 30 sunscreen daily. Changes in skin erythema and hyperpigmentation were measured using a chromameter and photographic images. Efficacy was assessed using the Evaluators Global Acne Severity Scale, lesion counts, Post-inflammatory Hyperpigmentation Severity Scales and Patient's Global Assessment Scale. Safety and tolerability were assessed by adverse event reports and a Safety Assessment Scale. Results: The mean (SD) baseline inflammatory lesion count was 11.9 (11.1) in clindamycin/tretinoin-treated patients, decreasing by 5.5 (6.56) after 12 weeks while the mean baseline inflammatory lesion count was 13.6 (11.15) in placebo-treated patients, decreasing by 4.1 (11.36) (p=0.05 for change from baseline, clindamycin/tretinoin vs. placebo). Clindamycin/tretinoin-treated patients generally demonstrated superior efficacy versus placebo treatment. The clindamycin/tretinoin topical gel was well tolerated, causing little or no irritation, although one patient withdrew due to periorbital edema of moderate severity possibly related to clindamycin/tretinoin gel. Conclusion: Although limited by small sample size, the results of this pilot study suggest clindamycin phosphate 1.2% and tretinoin 0.025% topical gel is a safe and effective option for treating mild-to-moderate acne in patients with skin of color.     

Aminated β-1,3-d-glucan improves wound healing in diabetic db/db mice

Delayed wound healing in diabetes is caused by neuropathy, vascular changes, and impaired cellular response to the injury. Macrophages are crucial in normal wound healing, and impaired functions of these cells have been shown in diabetes. beta-1,3-D-glucans stimulate macrophage function. This open-label study was performed to see if aminated beta-1,3-D-glucan (AG) stimulates wound healing in diabetes. Four groups (1-4) of diabetic db/db mice and one nondiabetic control group, db/+(5) were studied: group 1 (n=11): topical AG; group 2 (n=10): topical AG and subcutaneous insulin; group 3 (n=14): topical placebo and subcutaneous insulin; group 4 (n=10): diabetic control (placebo); group 5 (n=12): normal control (placebo). At the end of the experiments fasting blood glucose and A1C were (mean +/- SE) as follows: Group 1: 30.5 +/- 1.9 mmol/L and 11.3 +/- 0.6%; group 2: 12.0 +/- 1.7 mmol/L and 8.0 +/- 0.6%; group 3: 15.4 +/- 2.4 mmol/L and 7.4 +/- 0.3%; group 4: 32.6 +/- 2.6 mmol/L and 12.3 +/- 0.6%; group 5: 7.2 +/- 0.4 mmol/L and 3.9 +/- 0.04%, respectively. The closed wound area was the same in group 1 (AG alone) and group 2 (AG plus insulin) after 17 days, 57.3 +/- 4.7 vs. 50.1 +/- 4.9% (p=0.7).The results of these two groups were superior to group 3 (insulin treatment alone, 32.0 +/- 4.3%, p<0.001) and diabetic controls (38.2 +/- 5.1%, p=0.001). The macrophage-stimulant AG improves wound healing in db/db mice.     

Absence of Degradation of Tretinoin When Benzoyl Peroxide is Combined with an Optimized Formulation of Tretinoin Gel (0.05%)

Background: Clinicians have been reluctant to prescribe benzoyl peroxide concurrently with topical tretinoin due to a belief that the benzoyl peroxide may cause oxidation and degradation of the tretinoin molecule, thereby reducing its effectiveness. However, benzoyl peroxide-induced degradation of tretinoin may not necessarily apply to all topical tretinoin formulations. Objective: To evaluate the potential for benzoyl peroxide-induced degradation of an optimized aqueous gel formulation of tretinoin (0.05%). Methods: Tretinoin gel (0.05%) and benzoyl peroxide gel (6.26% premix concentration to produce 5% benzoyl peroxide in a fixed combination clindamycin product) were mixed together (1:1) at 32oC and samples assayed after 1, 2, 3, 5, and 7 hours. Each sample was analyzed for tretinoin (expressed as % tretinoin remaining) and its degradation product content. Results: No loss of tretinoin was observed over the seven-hour time period. When tretinoin gel (0.05%) was combined with benzoyl peroxide, 100 percent of the initial tretinoin concentration remained after seven hours. There was no increase in the degradation products of tretinoin. Conclusions: There was no benzoyl peroxide-induced degradation of tretinoin when the optimized formulation of tretinoin gel (0.05%) was admixed with benzoyl peroxide gel (6.26%). Although the direct clinical significance of these results is unknown, clinicians may feel comfortable using this particular combination concurrently without concerns about tretinoin oxidation and degradation.     

microRNA-215在糖尿病肾病小鼠肾组织中的表达及意义

目的 探讨microRNA-215( miR-215)在糖尿病肾病(DN)小鼠肾组织中的表达变化规律及在DN发病中的作用.方法 选择4周龄的2型糖尿病肾病db/db小鼠(实验组)和db/m小鼠(对照组),采用实时荧光定量PCR法动态检测8、12及16周龄时肾组织miR-215的表达变化;实时荧光定量PCR和Western印迹法、免疫组化法测定连环蛋白β互动蛋白1( CTNNBIP1)的mRNA及蛋白的表达;双荧光素酶报告法确证miR-215对CTNNBIP1表达的直接调控作用.结果 (1)随着周龄的增加,db/db小鼠肾小球逐渐肥大、节段性系膜细胞增生和系膜基质积聚.(2)与同周龄的db/m小鼠比较,8、12及16周龄的db/db小鼠体质量(BW)、血糖( Glu)及24 h尿白蛋白排泄量(UAE)均显著增加(均P＜0.05).(3)随着周龄的增加,db/db小鼠肾脏组织miR-215表达显著高于同周龄的db/m小鼠(P＜0.05).(4)与同周龄的db/m小鼠比较,db/db小鼠肾脏组织CTNNBIP1 mRNA和蛋白均显著降低(均P＜0.05).(5)应用双荧光素酶报告法证实,miR-215可显著抑制CTNNBIP1的表达(P＜0.01).结论 miR-215表达上调可能通过抑制CTNNBIP1的表达参与DN的发生、发展.     

Acceleration of wound healing in healing-impaired db/db mice with a photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2

Application of ultraviolet light irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) results within 30 seconds in an insoluble, flexible hydrogel. The FGF-2 molecules retained in the chitosan hydrogel remain biologically active and are released from the chitosan hydrogel upon in vivo biodegradation of the hydrogel. To evaluate the accelerating effect on wound healing of this hydrogel, full-thickness skin incisions were made in the backs of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. The mice were later killed, and histological sections of the wound were prepared. The degree of wound healing was evaluated using several histological parameters such as the rate of contraction, epithelialization, and tissue filling. Application of the chitosan hydrogel significantly advanced the rate of contraction on Days 0 to 2 in db/db and db/+ mice. Although the addition of FGF-2 into the chitosan hydrogel in db/+ mice had little effect, application of the chitosan hydrogel-containing FGF-2 further accelerated the adjusted tissue filling rate (Days 2 to 4 and Days 4 to 8) in db/db mice. Furthermore, the chitosan hydrogel-containing FGF-2 markedly increased the number of CD-34-positive vessels in the wound areas of db/db mice on Day 4. Thus, the application of chitosan hydrogel-containing FGF-2 onto a healing-impaired wound induces significant wound contraction and accelerates wound closure and healing.     

Regulation of melatonin on Toll-like receptor 4 signaling in diabetic db/db mice kidneys

Objective To investigate the regulation of melatonin (MT) on Toll-like receptor 4 (TLR4) signaling in diabetic db/db mice kidneys. Methods The 48 10-week-old male db/db mice were randomly divided into db/db group, db/db+MT 50 μg/kg group, db/db+MT 100 μg/kg group and db/db+MT 200 μg/kg group, each consisting of 12 mice. These mice received i.p. injections of MT These mice received i.p. injections of MT [dissoved in phosphate buffer solution (PBS)/ dimethylsulfoxide (DMSO) solution, given every day]. Alternatively, 12 db/m mice served as the control group. db/m and db/db group were injected i.p. with the same volume of PBS/DMSO solution. The animals were sacrificed after 12 weeks of dosage administration. Blood glucose (BG), body weight (BW), kidney weight (KW) and 24 h urinary albumin excretion rate (UAER) were determined; Kidney pathological lesions were evaluated by renal pathological staining. Immunohistochemistry of renal TLR4, NF-κB p65, and ED-1 was performed to determine the immunoreactivity. Western blotting was used to detect the expression of renal TLR4, myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF-3) and NF-κB p65, while the mRNA expressions of renal tumor necrosis factor -α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were much higher in db/db mice group (P＜0.01), while KW in db/db+MT (100, 200 μg/kg) groups and UAER level in db/db+MT (50, 100, 200 μg/kg) groups were distinctly decreased compared with those in db/db group (P＜0.01). In week 12 db/db mice, the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with those in db/m mice (P＜0.01). The above kidney histopathologic lesions were distinctly ameliorated by 50, 100, 200 μg/kg MT (P＜0.05). Immunohistochemistry intensity of renal TLR4, NF-κB p65 and ED-1 displayed obvious differences between db/m mice and db/db mice (P＜0.01), and that were remarkably decreased in db/db+MT (50, 100, 200 μg/kg) mice compared with db/db mice (P＜0.05). Western blotting showed that the protein expression of renal TLR4, MyD88, TRIF, IRF-3 and NF-κB p65 were stronger in db/db group compared with those in db/m group (P＜0.05) and weaker in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.05). Futhermore, the mRNA expressions of renal MCP-1 and TNF-α were higher in db/db group compared with those in db/m group (P＜0.01) and lower in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.01). Conclusion Melatonin may partly down-regulate TLR4 signaling pathway to inhibit Inflammatory reaction and alleviate kidney injury in diabetic db/db mice.     

L-arginine reduces glomerular basement membrane collagen N epsilon-carboxymethyllysine in the diabetic db/db mouse.

The present study was carried out to examine the effect of L-arginine on advanced stage nonenzymatic glycosylation end products in glomerular basement membrane (GBM) as represented by carboxymethyllysine (CML). Twelve db/db mice were given a solution containing a daily dosage of L-arginine of 50 mg/kg body weight orally. Twelve db/db mice served as controls. At the end of the 4-months study period treated animals had significantly lower concentrations of CML (0.084 +/- 0.008, 0.071-0.098 nmol/mumol OH-proline; mean +/- SD, range, p less than 0.01) compared to untreated controls (0.11 +/- 0.018, 0.095-0.152 nmol/mumol OH-proline). In addition, there was a significant positive correlation between GBM thickness and concentrations of CML (r = 0.86, p less than 0.001). We conclude that reduction of CML concentrations in treated db/db mice possibly reflects a beneficial effect of L-arginine on advanced stage nonenzymatic glycosylation end-products in GBM. In addition, measuring CML concentrations might have future clinical implications as a noninvasive parameter for basement membrane thickening.     

PPARalpha agonist fenofibrate improves diabetic nephropathy in db/db mice

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARalpha by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARalpha, decreased type I collagen production. Taken together, the PPARalpha agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARalpha by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy.     

Tretinoin photostability: comparison of micronized tretinoin (0.05%) gel and tretinoin (0.025%) gel following exposure to ultraviolet a light

Background: Various formulations of tretinoin (gel, liquid, cream) have been reported to be unstable on the skin under bright artificial light or sunlight. This photodegradation can potentially influence treatment regimens and possibly modify efficacy. The maximum light energy absorption of tretinoin is in the ultraviolet A region. Objective: To compare the photostability of a micronized formulation of tretinoin (0.05%) aqueos gel with tretinoin (0.025%) gel following exposure to ultraviolet A light for eight hours. Methods: Micronized tretinoin (0.05%) gel and tretinoin (0.025%) gel were then exposed to ultraviolet A light with an integrated intensity from 315 to 400nm of 22watt/m(2). Samples of both products were prepared and analyzed for tretinoin and its degradation products using a high-performance liquid chromatography method. Additional duplicate samples were similarly prepared and analyzed after 2,4,6, and 8 hours. Results: There was a nine-percent degradation of micronized tretinoin in the 0.05% aqueous gel compared to 72-percent degradation of tretinoin in the 0.025% gel following eight-hour ultraviolet A light exposure. The small increase in tretinoin degradation products with micronized tretinoin (0.05%) aqueous gel remained below six percent of the labeled concentration compared to a marked increase in tretinoin degradation products with tretinoin 0.025% gel at two hours that increased to over 66-percent labeled concentration at eight hours. Conclusion: Micronized tretinoin (0.05%) aqueous gel showed less than 10-percent degradation when exposed to eight hours of ultraviolet A light, while tretinoin (0.025%) gel showed significant tretinoin degradation.     

N-acetyl-seryl-aspartyl-lysyl-proline prevents renal insufficiency and mesangial matrix expansion in diabetic db/db mice

We have previously reported that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is a tetrapeptide hydrolyzed by ACE, inhibits the transforming growth factor-beta (TGF-beta)-induced expression of extracellular matrix proteins via inhibition of the Smad signaling in human mesangial cells. To test in vivo the antifibrotic efficacy of Ac-SDKP, we examined whether long-term Ac-SDKP treatment can prevent renal insufficiency and glomerulosclerosis in diabetic db/db mice. Diabetic db/db mice or nondiabetic db/m mice were treated with Ac-SDKP for 8 weeks using osmotic minipumps. The treatment with Ac-SDKP increased plasma Ac-SDKP concentrations by approximately threefold in both groups but did not affect the blood glucose levels. Histologically, the increased glomerular surface area, mesangial matrix expansion, and overproduction of extracellular matrix proteins in db/db mice were significantly inhibited by Ac-SDKP. Furthermore, Ac-SDKP treatment normalized the increased plasma creatinine value in db/db mice, whereas the albuminuria in Ac-SDKP-treated db/db mice was somewhat decreased as compared with nontreated db/db mice, although the difference was not statistically significant. In addition, the nuclear translocation of Smad3 was inhibited by Ac-SDKP. These results demonstrate that long-term Ac-SDKP treatment ameliorates renal insufficiency and glomerulosclerosis in db/db mice via inhibition of TGF-beta/Smad pathway, suggesting that Ac-SDKP could be useful in the treatment of diabetic nephropathy.     

db/db小鼠胰岛的分离与特征分析

目的 分离db/db小鼠胰岛,并对其特征进行检测和分析。方法 采用胶原酶V对10只db/db小鼠胰岛分离进行逆行胰管灌注和消化,用Ficoll-1077和Ficoll-1119进行胰岛的不连续密度梯度纯化,对分离的胰岛进一步进行手工挑选,并用DTZ进行胰岛和纯度鉴定,用透射电镜观察胰岛内部分泌颗粒等情况。结果 经本方法分离可得到db/db小鼠胰岛数量为（122.4±6.6）个/只,当量为（483.6±82.3）IEQ/只,与ICR小鼠胰岛分离结果差异有统计学意义（P<0.05）;db/db小鼠胰岛DTZ染色后显淡红色;胰岛大小指数为（3.96±0.64）,显著大于ICR小鼠胰岛（P<0.05）;透射电镜下显示β细胞中的胰岛素分泌颗粒减少,分泌颗粒颜色较浅。结论 采用本方法可分离得到db/db小鼠胰岛,为后续开展基于胰岛功能和特征变化的2型糖尿病患者治疗研究提供一种参考。     

阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用

目的 观察肾素抑制剂阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用。 方法 8周龄db/db和db/m小鼠行单侧肾切除,16周进入实验,分为4组：db/m小鼠对照组（db/m组）、db/db糖尿病小鼠对照组（db/db组）、db/db糖尿病小鼠阿利吉仑3 mg&#8226;kg-1&#8226;d-1治疗组（db/db+A3组）和db/db糖尿病小鼠阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗组(db/db+A25组)。阿利吉仑溶于磷酸盐缓冲液（PBS,350 mg/L）皮下泵入（0.25 μl/h）给药,疗程４周。治疗前后检测体质量、血糖、糖化血红蛋白、尿蛋白量、血压水平;PAS染色观察肾脏组织学变化;ELISA法检测肾皮质转化生长因子β1（TGF-β1）和纤溶酶原激活抑制因子1（PAI-1）含量;间接免疫荧光检测肾小球Ⅳ型胶原（ColⅣ）和纤连蛋白（FN）表达;实时定量PCR检测TGF-β1、PAI-1、ColⅣ、FN和肾素mRNA表达;放射免疫法检测肾皮质肾素活性和血管紧张素Ⅱ（AngⅡ）水平。 结果 与db/m组小鼠比较,db/db组小鼠有大量蛋白尿,肾小球细胞外基质沉积增加,TGF-β1、PAI-1、ColⅣ和FN蛋白及mRNA表达增加,同时肾皮质肾素mRNA、肾素活性和AngⅡ水平增高（均P < 0.05）。阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗在没有影响血压情况下,显著减少db/db小鼠24 h尿蛋白量,减少肾小球细胞外基质沉积,减少TGF-β1、PAI-1、ColⅣ、FN蛋白和mRNA表达,同时降低肾皮质肾素活性和AngⅡ水平（均P < 0.05）。 结论 阿利吉仑对2型糖尿病db/db小鼠肾脏损伤有保护作用。     

db/db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model

The pathophysiology of diabetic wound healing and the identification of new agents to improve clinical outcomes continue to be areas of intense research. There currently exist more than 10 different murine models of diabetes. The degree to which wound healing is impaired in these different mouse models has never been directly compared. We determined whether differences in wound impairment exist between diabetic models in order to elucidate which model would be the best to evaluate new treatment strategies. Three well-accepted mouse models of diabetes were used in this study: db/db, Akita, and streptozocin (STZ)-induced C57BL/6J. Using an excisional model of wound healing, we demonstrated that db/db mice exhibit severe impairments in wound healing compared with STZ and Akita mice. Excisional wounds in db/db mice show a statistically significant delay in wound closure, decreased granulation tissue formation, decreased wound bed vascularity, and markedly diminished proliferation compared with STZ, Akita, and control mice. There was no difference in the rate of epithelialization of the full-thickness wounds between the diabetic or control mice. Our results suggest that splinted db/db mice may be the most appropriate model for studying diabetic wound-healing interventions as they demonstrate the most significant impairment in wound healing. This study utilized a novel model of wound healing developed in our laboratory that stents wounds open using silicone splints to minimize the effects of wound contraction. As such, it was not possible to directly compare the results of this study with other studies that did not use this wound model.     

Association of SH2-containing inositol phosphatase 2 with the insulin resistance of diabetic db/db mice

SH-2-containing inositol 5'-phosphatase 2 (SHIP-2) is a physiologically important lipid phosphatase that functions to hydrolyze phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P3 to PI(3,4)P2 in the negative regulation of insulin signaling. We investigated whether SHIP-2 is associated with the insulin resistance of diabetic db/db mice. The amount of SHIP-2 protein was elevated in quadriceps muscle and epididymal fat tissue, but not in the liver, of db/db mice relative to that in control db/+m mice. In accordance with the enhanced expression of SHIP-2, its localization at the membrane preparation was increased in the skeletal muscle and fat tissue of db/db mice. Insulin stimulation of PI 3-kinase activity was modestly decreased in skeletal muscle, fat tissue, and liver of db/db mice compared with that of db/+m mice. In addition to the modest decrease at the level of PI 3-kinase, the activity of Akt and protein kinase C (PKC)-zeta/lambda, which are downstream molecules of PI 3-kinase, was more severely reduced in the skeletal muscle and fat tissue, but not in liver of db/db mice. Treatment with the insulin-sensitizing agent rosiglitazone decreased the elevated expression of SHIP-2 in the skeletal muscle and fat tissue of db/db mice. Insulin-induced Akt activation and PKC-zeta/lambda phosphorylation were restored to the control level, although insulin-stimulated PI 3-kinase activation was minimally affected in the skeletal muscle and fat tissue of db/db mice. These results indicate that SHIP-2 is a novel molecule associated with insulin resistance in the skeletal muscle and fat tissue, and that insulin-induced activity of the downstream molecules of PI 3-kinase is decreased, at least in part, by the elevated expression of SHIP-2 in diabetic db/db mice.     

Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice

Diabetic nephropathy is a leading cause of end-stage renal failure and is a growing concern given the increasing incidence of type 2 diabetes. Diabetic nephropathy is associated with progressive kidney macrophage accumulation and experimental studies suggest that intercellular adhesion molecule (ICAM)-1 facilitates kidney macrophage recruitment during type 1 diabetes. To ascertain the importance of ICAM-1 in promoting type 2 diabetic nephropathy, the development of renal injury in ICAM-1 intact and deficient db/db mice with equivalent hyperglycemia and obesity between ages 2 and 8 mo was examined and compared with results with normal db/+ mice. Increases in albuminuria (11-fold), glomerular leukocytes (10-fold), and interstitial leukocytes (three-fold) consisting of predominantly CD68+ macrophages were identified at 8 mo in diabetic db/db mice compared with nondiabetic db/+ mice. In comparison to db/db mice, ICAM-1-deficient db/db mice had marked reductions in albuminuria at 6 mo (77% downward arrow) and 8 mo (85% downward arrow). There was also a significant decrease in glomerular (63% downward arrow) and interstitial (83% downward arrow) leukocytes in ICAM-1-deficient db/db mice, which were associated with reduced glomerular hypertrophy and hypercellularity and tubular damage. The development of renal fibrosis (expression of TGF-beta1, collagen IV, and interstitial alpha-smooth muscle actin) was also strikingly attenuated in the ICAM-1-deficient db/db mice. Additional in vitro studies showed that macrophage activation by high glucose or advanced glycation end products could promote ICAM-1 expression on tubular cells and macrophage production of active TGF-beta1. Thus, ICAM-1 appears to be a critical promoter of nephropathy in mouse type 2 diabetes by facilitating kidney macrophage recruitment.     

Dehydroepiandrosterone suppresses the elevated hepatic glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in C57BL/Ksj-db/db mice: comparison with troglitazone.

The effect of dehydroepiandrosterone (DHEA) on the hepatic and muscle glucose metabolizing enzymes and on blood glucose were investigated in insulin-resistant diabetic C57BL/KsJ-db/db mice and their heterozygote littermates (db/+m). The results were compared with those after troglitazone administration under the same conditions. Despite hyperinsulinemia, hepatic glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FBPase) activities are higher in db/db than in db/+m mice. Dietary administration of DHEA and that of troglitazone for 15 days to respective groups of five mice each significantly decreased blood glucose in db/db mice and hepatic G6Pase and FBPase activities in both db/db and db/+m mice. Hepatic G6Pase and FBPase activities showed a linear relationship with blood glucose in all groups of mice, suggesting that the activities of G6Pase and FBPase are closely related to blood glucose levels. Because androstenedione, a DHEA metabolite, barely affected either of these enzyme activities or blood glucose in db/db mice, the actions of DHEA, which are similar to those of troglitazone, are presumed to be caused by DHEA itself. DHEA is considered to be a modulating agent for the activities of hepatic gluconeogenic enzymes in db/db mice.     

Long-term treatment of glucagon-like peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-beta1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator-activated receptor alpha and GLP-1 receptor-positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.     

Renoprotective effect of transforming growth factor beta activator kinase 1 inhibitor in diabetic db/db mice and its mechanism

Objective To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OZ) in diabetic db/db mice and the mechanism. Methods Twenty-four male db/db mice were randomly divided into two groups: db/db mice (db/db, n=12) and db/db mice with 5Z-7-oxozeaenol treatment (db/db+OZ, n=12). Another group of wild type mice (n=12) was held as the control group. OZ 2 mg/kg was administrated by intraperitoneal injection every other day. At week 8 and 12 after 5Z-7-oxozeaenol treatment, blood glucose (BG), body weight (BW), kidney weight (KW) and urinary albumin excretion rate (UAER) were evaluated. Kidney pathological lesions were detected by light and electron microscopy. NF-κB p65, monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were detected by immunohistochemistry. Western blotting was used to detect p-TAK1, TAB1, p-p38MAPK and IL-1β expression, while ICAM-1 and MCP-1 mRNA levels were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were higher (P＜0.01) in db/db mice group, while BW, KW and UAER levels were significantly decreased in db/db+OZ group compared with that in db/db mice group (P＜0.05). In week 8 and 12 db/db mice, glomerular volume and extracellular matrix were increased, while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor. Immunohistochemistry showed that NF-κB p65, MCP-1 and TNF-α expression levels were apparently increased in db/db mice group compared with that in control group (P＜0.05) and were significantly inhibited by TAK1 inhibitor (P＜0.05). Western blotting showed that p-TAK1, TAB1, p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group (P＜0.05) and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Moreover, real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Conclusions TAK1 Inhibitor can down-regulate MAPK and NF-κB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.     

Expression and role of microRNA-155 in db/db mice

Objective To investigate the expression of microRNA-155 (miR-155) in serum and kidney of C57BLKS/db (db/db) mice and its role in the pathogenesis of diabetic kidney disease (DKD). Methods The db/db mice (n=24) were divided into 6, 8, and 10 weeks old groups (n=8) with age increasing according to the random number table, and C57BL/6 mice of the same age were used as control group. The expression of miR-155 in mouse serum and kidney tissue was determined using real-time quantitative PCR. The mRNA and protein expression of Ets-1, eNOS, AGTR1 in renal tissues was verified by real-time quantitative PCR, Western blotting and immunohistochemistry. Results Compared with the control group, the expression of miR-155 in serum of db/db mice at 6, 8 and 10 weeks of age were significantly increased (all P＜0.01), and the increase of miR-155 was most obvious at 10 weeks of age (P＜0.01). Meanwhile the expression of miR-155 in kidney tissues of 6, 8 and 10 weeks old db/db mice was significantly up-regulated (all P＜0.01), and the highest expression of miR-155 was at 10 weeks of age (P＜0.01). Immunohistochemistry showed that Ets-1, eNOS and AGTR1 were localized in glomerular endothelial cells. The results of real-time quantitative PCR showed that the mRNA expression of Ets-1, eNOS and AGTR1 were down-regulated in the kidney tissues of db/db mice at 6, 8 and 10 weeks of age compared to the control(all P＜0.05), and the level of down-regulation was the most obvious at 10 week. Western blotting results showed that there was no significant change in Ets-1, eNOS and AGTR1 in 6-week-old db/db mice compared to the control group; the eNOS protein expression was down-regulated at 8 weeks of age (P＜0.05); the expression of AGTR1 protein was down-regulated (P＜0.05), and the protein expression of Ets-1 and eNOS were significantly down-regulated at 10-week age (both P＜0.01). Conclusions The expression of miR-155 in serum and kidney tissues of db/db mice increases during the progression of DKD, while the expression of miR-155 target genes Ets-1, eNOS and AGTR1 decreases with the progression of DKD. MiR-155 may participate in the development and progression of DKD by inhibiting its target genes Ets-1, eNOS and AGTR1, affecting endothelial cell function.     

Muscle protein turnover in the perfused hindquarters of lean and genetically obese-diabetic (db/db) mice

The stability of the perfused mouse hindquarter was assessed for a period of 5 h, and the preparation shown to remain stable for metabolic studies over this time period. Muscle protein synthesis and degradation rates in lean and diabetic-obese (db/db) mice were measured using the in situ perfused hindquarter preparation. The rates of protein synthesis were 48% lower in the muscles of intact db/db mice than in the lean controls when expressed per gram TCA precipitable protein and 46% lower when expressed per gram dry weight. Adrenalectomy, which has been shown to restore the lean body mass of the db/db mice to normal, had the effect of returning protein synthesis rate in muscle of db/db mice to lean control values. Insulin at a dose of 1 mU/ml stimulated protein synthesis in lean mice only, showing that the process of protein synthesis in the db/db mice is also insensitive to insulin. Measurements of the rates of degradation of muscle protein showed no differences between lean and db/db mice. These findings suggest that the decreased lean body mass of db/db mice is the result of a defect in protein synthesis rather than due to altered degradation.