OBJECTIVETo investigate the effect of Xuezhikang (, XZK) on renal cell apoptosis in diabetic rats and the possible mechanism.

METHODSSixty-six rats were randomly divided into 3 groups: the normal, model and XZK groups. In each group, the rats were further randomly divided into 3-month and 6-month subgroups, respectively. Diabetes of rats was induced by a single intraperitoneal injection of 1% streptozocin at 60 mg/kg body weight. Rats in the XZK group received gastric perfusion of XZK (1200 mg/kg body weight) everyday for 3 or 6 months, while rats in the normal and model groups received equal volume of saline. Twenty-four hours' urine was collected for urinary albumin excretion (UAE) measurement. Periodic acid-Schiff (PAS) and Masson's trichrome staining were used for saccharides and collagen detection. Cell apoptosis of renal cortex was investigated by TdT-mediated dUTP nick end labeling (TUNEL) staining. Bax and Bcl-2 expressions were detected by immunohistochemistry and Western blot, respectively. Cytochrome C (Cyt C) and caspase-9 concentration were detected by Western blot.

RESULTSCompared with the model group, XZK treatment could significantly decrease the kidney hypertrophy index, 24 h UAE, renal cell apoptosis, cytoplasmic Cyt C level and active caspase-9 level, as well as suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio at 3 and 6 months (P<0.05 or P<0.01). Moreover, XZK treatment could alleviate the deposition of PAS-stained saccharides and Masson's trichromestained collagen to different extent.

CONCLUSIONSRenal cell apoptosis was observed in diabetic kidney, in which mitochondrial apoptotic pathway might be involved. XZK treatment could attenuate pathological changes in diabetic kidney and reduce renal cell apoptosis, probably via the suppression of Bax/Bcl-2 ratio, which lead to inhibition of Cyt C release and following caspase-9 activation.

Albuminuria ; blood ; complications ; Animals ; Apoptosis ; drug effects ; Blood Glucose ; metabolism ; Caspase 9 ; metabolism ; Cytochromes c ; metabolism ; Diabetes Mellitus, Experimental ; blood ; drug therapy ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypertrophy ; In Situ Nick-End Labeling ; Kidney ; drug effects ; pathology ; Kidney Glomerulus ; pathology ; Lipids ; blood ; Male ; Mesangial Cells ; drug effects ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats, Sprague-Dawley ; Streptozocin ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the deregulation of autophagy in diabetic peripheral neuropathy (DPN) and investigate whether Jinmaitong ( JMT) alleviates DPN by inducing autophagy.

METHODSDPN models were established by streptozotocin-induced diabetic rats and Schwann cells (SCs) cultured in high glucose medium. The pathological morphology was observed by the improved Bielschowsky's nerve fiber axonal staining and the Luxol fast blue-neutral red myelin staining. The ultrastructure was observed by the transmission electron microscopy. Beclin1 level was detected by immunohistochemistry and Western blot. The proliferation of cultured SCs was detected by methylthiazolyldiphenyl-tetrazolium bromide.

RESULTSDiabetic peripheral nerve tissues demonstrated pathological morphology and reduced autophagic structure, accompanied with down-regulation of Beclin1. JMT apparently alleviated the pathological morphology change and increased the autophagy [in vivo, Beclin1 integral optical density (IOD) value of the control group 86.6±17.7, DM 43.9±8.8, JMT 73.3 ±17.8, P<0.01 or P<0.05, in vitro Beclin1 IOD value of the glucose group 0.47±0.25 vs the control group 0.88±0.29, P<0.05]. Consequently, inhibition of autophagy by 3-methyladenine resulted in a time- and concentration-dependent decrease of the proliferation of SCs (P<0.05, P<0.01).

CONCLUSIONSDown-regulation of autophagy in SCs might contribute to the pathogenesis of DPN. JMT alleviates diabetic peripheral nerve injury at least in part by inducing autophagy.

Animals ; Autophagy ; drug effects ; Axons ; drug effects ; pathology ; Beclin-1 ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; pathology ; Diabetic Neuropathies ; complications ; drug therapy ; pathology ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Glucose ; pharmacology ; Immunohistochemistry ; Male ; Rats, Wistar ; Schwann Cells ; drug effects ; pathology ; Sciatic Nerve ; drug effects ; pathology ; ultrastructure ; Staining and Labeling

OBJECTIVETo observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats.

METHODSThirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues.

RESULTSCompared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure.

CONCLUSIONThese results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.

Animals ; Calcium-Binding Proteins ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetes Mellitus, Type 2 ; Diabetic Cardiomyopathies ; metabolism ; physiopathology ; Disease Progression ; Heart ; physiopathology ; Male ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Wistar ; Receptors, Calcium-Sensing ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Streptozocin

OBJECTIVETo investigate the effects of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) on wound healing and microRNA expression in diabetic rats.

METHODSEighteen male SD rats of clean grade were used to reproduce diabetes model. Four weeks later, a total of 64 full-thickness skin wounds were created on the back of 16 rats with established diabetes, with 4 wounds on each rat. Two symmetrical wounds on either side of the spine were created as a pair according to paired design. Then the wounds were divided into groups A and B according to the random number table and blind method (red and blue tags on the rhGM-CSF or the gel vehicle), with 32 wounds in each group. The ointment with red tag was applied on the wounds of group A and the blue one on group B. The application was conducted once a day, with a thickness of 3 mm, up to post injury day (PID) 14. Gross observation of wound healing was conducted on PID 3, 7, 14. The wound healing rate was determined on PID 3 and 7. On PID 3, 7, 14, tissues from 2, 4, and 8 wounds were harvested from each group respectively for the observation of the histopathological changes with HE staining, and also for analyzing the expression of proliferating cell nuclear antigen (PCNA) and CD31 with immunohistochemical staining (denoted as absorbance value). On PID 7, tissues from 6 wounds in each group were harvested for microarray gene chip to screen the differentially expressed microRNAs. Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway on the differentially expressed microRNAs were performed after the microRNA screening results were validated by real-time fluorescent quantitative RT-PCR. Data were processed with paired t test or two-sample t test.

RESULTS(1) On PID 3, the wound area was significantly decreased, and the wound granulation was significantly proliferated in both groups. On PID 7, the wound area was further decreased, and the wound area was almost filled by granulation in both groups; the conditions in group A were better. On PID 14, all the wounds in group A were almost healed, while a small area of raw wound with incrustation still remained in some wounds of group B. On PID 3 and 7, the wound healing rates of group A were (41 ± 5)% and (75 ± 4)%, significantly higher than those of group B [(31 ± 9)% and (71 ± 4)%, with t values respectively 10.13 and 8.06, P values below 0.001]. (2) On PID 3, the epidermal cells, endothelial cells, and Fbs in the wounds of 2 groups were sparse, with heavy infiltration of inflammatory cells. The above condition in the wounds was better in group A than in group B. On PID 7, the epidermal cells, endothelial cells, and Fbs were gradually well arranged in group A; infiltration of inflammatory cells decreased, and the condition was better than that of group B. On PID 14, the wounds of group A were completely covered by epidermis, while infiltration of inflammatory cells still remained in some wounds of group B. (3) On PID 3, 7, 14, the positive expressions of CD31 and PCNA in group A were respectively 0.275 ± 0.018, 0.345 ± 0.034, 0.305 ± 0.023; 0.406 ± 0.063, 0.223 ± 0.011, 0.045 ± 0.022. They were significantly higher than those of group B (0.222 ± 0.020, 0.229 ± 0.018, 0.197 ± 0.015; 0.324 ± 0.039, 0.162 ± 0.012, 0.018 ± 0.020, with t values from 2.281 to 9.652, P < 0.05 or P < 0.01). (4) According to the microRNAs detection and screening, as compared with group B, 18 microRNAs were up-regulated while 13 were down-regulated in the wounds of group A. (5) The results of real-time fluorescent quantitative RT-PCR had good consistency with the results of microRNAs detection. (6) Enrichment analysis of KEGG signaling pathway showed that among the 31 differentially expressed microRNAs, 4 took part in the MAPK signaling pathway, 3 took part in the Wnt signaling pathway, 1 took part in the TGF-β signaling pathway, 3 took part in the epidermal growth factor receptor signaling pathway, 2 took part in the cell cycle pathway, 5 took part in the axon guidance signaling pathway, 6 took part in the focal adhesion pathway, 3 took part in the regulation of actin cytoskeleton pathway, 1 took part in the extracellular cell matrix receptor pathway, 3 took part in the adherens junction pathway, and 1 took part in the cell adhesion molecules pathway. After disclosing the blind, it showed that the ointment with red tag was the rhGM-CSF gel and the blue one was gel vehicle.

CONCLUSIONSThe rhGM-CSF gel can promote wound healing in diabetic rats, producing significant differential microRNA expression in wounds, and they may be the target at gene post-transcriptional level of rhGM-CSF gel in promoting wound healing.

Animals ; Bacteria ; isolation & purification ; Burns ; drug therapy ; microbiology ; pathology ; Diabetes Mellitus, Experimental ; complications ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Male ; MicroRNAs ; genetics ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Recombinant Proteins ; Signal Transduction ; Wound Healing ; drug effects

We aimed to elucidate the effect of bilirubin on dyslipidemia and nephropathy in a diabetes mellitus (DM) type I animal model. Sprague-Dawley rats were separated into control, DM, and bilirubin-treated DM (Bil) groups. The Bil group was injected intraperitoneally with 60 mg/kg bilirubin 3 times per week and hepatoma cells were cultured with bilirubin at a concentration of 0.3 mg/dL. The Bil group showed lower serum creatinine levels 5 weeks after diabetes onset. Bilirubin treatment also decreased the amount of mesangial matrix, lowered the expression of renal collagen IV and transforming growth factor (TGF)-beta1, and reduced the level of apoptosis in the kidney, compared to the DM group. These changes were accompanied by decreased tissue levels of hydrogen superoxide and NADPH oxidase subunit proteins. Bilirubin decreased serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), free fatty acids, and triglycerides (TGs), as well as the TG content in the liver tissues. Bilirubin suppressed protein expression of LXRalpha, SREBP-1, SCD-1, and FAS, factors involved in TG synthesis that were elevated in the livers of DM rats and hepatoma cells under high-glucose conditions. In conclusion, bilirubin attenuates renal dysfunction and dyslipidemia in diabetes by suppressing LXRalpha and SREBP-1 expression and oxidative stress.
Animals ; Bilirubin/pharmacology/*therapeutic use ; Cell Line, Tumor ; Creatine/blood ; Diabetes Mellitus, Experimental/chemically induced/complications/*pathology ; Diabetic Nephropathies/*drug therapy/etiology ; Disease Models, Animal ; Kidney/pathology ; Lipoproteins, HDL/blood ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase/metabolism ; Orphan Nuclear Receptors/antagonists & inhibitors/genetics/metabolism ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Streptozocin/toxicity ; Triglycerides/analysis/*biosynthesis/blood

OBJECTIVETo investigate the expression of C-type natriuretic peptides (CNP) and natriuretic peptide receptor-B (NPR-B) receptor in diabetic rats renal cortex, and the regulation by Tongluo Recipe (TLR).

METHODSSixty male SD rats were divided into 3 groups: the normal control group, diabetic model group and diabetic TLR group. Each group was further divided into two subgroups of ten in each, according to 4-week or 12-week observation period. Streptozotocin (STZ)-induced diabetic rats were treated with TLR (1.0 g·kg(-1)·d(-1)) for 4 and 12 weeks, respectively. (1) The essential information was collected for comparing renal mass, serum creatinine and 24 h urine albumen on each group was calculated. (2) CNP mRNA and NPR-B mRNA were detected by realtime-polymerase chain reaction (PCR) on rats renal cortex. (3) Concentration of CNP on renal cortex or serum were analyzed by enzyme-linked immunosorbent assay (ELISA). (4) Pathological evaluation and NPR-B immunostaining for renal tissue were also performed.

RESULTS(1) CNP and NPR-B mRNA levels were detected in each treated or untreated group, with slight elevated in untreated diabetes rats administrated with STZ after 4-week and CNP mRNA level remarkable elevated at 39.21 times higher than normal control group after 12 weeks, but NPR-B mRNA level showed a remarkably down-regulation at 98.07% after 12 weeks. CNP mRNA of TLR-treated group was also elevated after 12-week treatment, but less than untreated group. (2) Concentrations of CNP in renal cortex were obviously increased in treated or untreated diabetes rats, within these groups the treatment of TLR was found more significantly on prompting CNP concentration. Comparing to normal group, serum concentrations of CNP were also increased in treated or untreated diabetic groups, but there was no difference between these diabetic groups. (3) Renal lesions like glomerular volume increased are observed mostly in the relative early stage after 4 weeks. Although TLR treated group had no significant difference in their glomerular volume, the degrees of injury of glomerulus were ameliorated, as well as the NPR-B immunostaining enhanced in glomerulus. Weakly positive immunostaining of NPR-B are observed in glomerulus of normal control, and negative in glomerulus of untreated diabetes rats administrated with STZ after 12 weeks, whereas TLR-treatment groups showed a little enhancement.

CONCLUSIONCNP and NPR-B showed different characteristic on renal cortex at different pathological period in diabetes rats, and TLR regulated their expression.

Animals ; Body Weight ; drug effects ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; genetics ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Hematuria ; complications ; genetics ; pathology ; Immunohistochemistry ; Kidney ; drug effects ; metabolism ; pathology ; Kidney Cortex ; drug effects ; metabolism ; pathology ; Kidney Glomerulus ; drug effects ; metabolism ; pathology ; Male ; Natriuretic Peptide, C-Type ; genetics ; metabolism ; Organ Size ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Atrial Natriuretic Factor ; genetics ; metabolism ; Staining and Labeling ; Streptozocin

OBJECTIVETo investigate the role of the non-canonical Wnt signaling pathway in development of non-alcoholic steatohepatitis (NASH) related to type 2 diabetes mellitus (T2DM) using a rat model.

METHODSTwenty-four male Sprague-Dawley rats were randomly divided into two equal groups: control group, fed a stand diet; T2DM-NASH model group, fed a high sucrose and fat diet for 4 weeks and intraperitoneally injected with streptozotocin (30 mg/kg). Twelve weeks after model establishment, all rats were sacrificed. Serum levels of glucose, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by biochemical analysis. Liver pathological changes were assessed microscopically by hematoxylin-eosin and oil red O staining. The liver expression of Wnt5a and NF-kB p65 were detected by immunohistochemistry and western blotting (protein), and quantitative real-time PCR (mRNA).

RESULTSThe T2DM-NASH model group showed significantly higher levels of glucose (control group: 6.25 +/- 1.28 vs. 31.21 +/- 0.86 mmol/L, t = -36.204, P less than 0.01), ALT (31.00 +/- 3.69 vs. 301.50 +/- 8.62 U/L, t = -99.94, P less than 0.01), and AST (77.58 +/- 1.83 vs. 344.75 +/- 1.82 U/L, t = -358.85, P less than 0.01). The T2DM-NASH group also showed remarkable signs of steatosis and inflammation in hepatic tissues. The T2DM-NASH group had significantly higher integral optical density (IOD) detection of Wnt5a (control group: 1.15E4 +/- 577.45 vs. 4.04E5 +/- 2.42E4, t = -56.24, P less than 0.01) and NF-kB p65 (1.28E4 +/- 1.59E3 vs. 4.21E5 +/- 1.68E4, t = -83.895, P less than 0.01), as well as protein levels detected by western blot (Wnt5a: 4.21 +/- 0.34 vs. 1.00 +/- 0.25, t = 17.030, P less than 0.01; NF-kB p65: 4.93 +/- 0.76 vs. 1 +/- 0.13, t = 11.438, P less than 0.01). The hepatic mRNA levels followed the same trend (Wnt5a: 9.53 +/- 0.64 vs. 1.04 +/- 0.35, t = 20.165, P less than 0.01; NF-kB p65: 0.60 +/- 0.13 vs. 0.74 +/- 0.10, t = -1.802, P = 0.125). In the T2DM-NASH group, hepatic Wnt5a protein expression was positively correlated with ALT (r = 0.64, P less than 0.05), AST (r = 0.59, P less than 0.05), and NF-kB p65 protein expression (r = 0.58, P less than 0.05).

CONCLUSIONWnt5a may activate NF-kB to stimulate an inflammatory response leading to development of NASH related to T2DM.

Animals ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; metabolism ; Liver ; pathology ; Male ; Non-alcoholic Fatty Liver Disease ; etiology ; pathology ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; metabolism ; Wnt Proteins ; metabolism ; Wnt Signaling Pathway ; Wnt-5a Protein

To evaluate the effect of chlorogenic acid (CGA), a polyphenol abundant in coffee, on retinal vascular leakage in the rat model of diabetic retinopathy, Sprague-Dawley rats were divided into four groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with 10 and 20 mg/kg chlorogenic acid intraperitoneally daily for 14 days, respectively. Blood-retinal barrier (BRB) breakdown was evaluated using FITC-dextran. Vascular endothelial growth factor (VEGF) distribution and expression level was evaluated with immunohistochemistry and Western blot analysis. Expression of tight junction proteins, occludin and claudin-5, and zonula occludens protein, ZO-1 was also evaluated with immunohistochemistry and Western blot analysis. BRB breakdown and increased vascular leakage was found in diabetic rats, with increased VEGF expression and down-regulation of occludin, claudin-5, and ZO-1. CGA treatment effectively preserved the expression of occludin, and decreased VEGF levels, leading to less BRB breakdown and less vascular leakage. CGA may have a preventive role in BRB breakdown in diabetic retinopathy by preserving tight junction protein levels and low VEGF levels.
Animals ; Blood-Retinal Barrier/*drug effects ; Chlorogenic Acid/metabolism/*pharmacology ; Claudin-5/metabolism ; Dextrans/chemistry ; Diabetes Mellitus, Experimental/complications/metabolism/*pathology ; Diabetic Retinopathy/etiology/prevention & control ; Down-Regulation ; Fluorescein-5-isothiocyanate/chemistry ; Male ; Occludin/metabolism ; Rats ; Rats, Sprague-Dawley ; Retina/*metabolism ; Tight Junction Proteins/metabolism ; Vascular Endothelial Growth Factor A/metabolism ; Zonula Occludens-1 Protein/metabolism

OBJECTIVETo evaluate the anti-apoptotic effect of aldehyde dehydrogenase 2 (ALDH2) on myocardial ischemia/reperfusion (I/R) injury in diabetic rats.

METHODSNormal male SD rats were divided into normal, diabetes and ethanol (the agonist of ALDH2) + diabetes groups. In the latter two groups, diabetes was induced by an intraperitoneal injection of 55 mg/kg STZ. Four weeks after the modeling, myocardial I/R was mimicked ex vivo, and lactate dehydrogenase (LDH) content in the coronary flow was determined. The activities of caspase-3 and ALDH2 were evaluated, and the expressions of Bcl-2 and Bax mRNA in the left anterior myocardium were detected using RT-PCR.

RESULTSIn diabetic group, LDH release and caspase-3 activity were increased, while ALDH2 activity and Bcl-2/Bax mRNA expression were decreased as compared to those in normal control group. Compared with the diabetic group, ALDH2 agonist ethanol significantly reduced LDH release and caspase-3 activity, increased ALDH2 activity and Bcl-2/Bax mRNA expression.

CONCLUSIONIn diabetic rats, enhanced ALDH2 expression can offer mycardial protection possibly in relation to suppress cell apoptosis.

Aldehyde Dehydrogenase ; metabolism ; Aldehyde Dehydrogenase, Mitochondrial ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Diabetes Mellitus, Experimental ; complications ; enzymology ; Ethanol ; pharmacology ; Male ; Mitochondrial Proteins ; agonists ; metabolism ; Myocardial Ischemia ; enzymology ; etiology ; Myocardial Reperfusion Injury ; enzymology ; pathology ; prevention & control ; Myocardium ; enzymology ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the expressions of 78-kDa glucose-regulated protein (GRP78) and Caspase-12 and their relationship with apoptosis in renal cortex of diabetic rats.

METHODSUninephrectomized Wistar rats were used to induce diabetes by intraperitoneal injection of Streptozotocin (STZ 65 mg/kg). After 8 weeks, the expression and distribution of GRP78, Caspase-12, proliferating cell nuclear antigen (PCNA) were examined by immunohistochemistry. Flow cytometry was used to detect the levels of protein of GRP78 and Caspase-12. Apoptosis was evaluated by means of terminal deoxynucleotidyl transferase-mediated d-UDP nick-end labeling (TUNEL) and Flow cytometry. Serum creatinine, blood urea nitrogen and 24-hour urine protein excretion were checked.

RESULTSCompared with those in normal control group, the numbers of apoptosis and the expression of GRP78, Caspase-12 in glomerular and tubular cells were much higher in the diabetic kidneys at 8 weeks. There was no significant difference between group A and group B.

CONCLUSIONActivation of endoplasmic reticulum stress may play an important role in the development of diabetic nephropathy.

Animals ; Apoptosis ; Caspase 12 ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetic Nephropathies ; pathology ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Kidney Cortex ; metabolism ; pathology ; Male ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Wistar