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

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

Actins ; metabolism ; Animals ; Collagen Type III ; metabolism ; Collagen Type IV ; metabolism ; Desmin ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetic Nephropathies ; metabolism ; pathology ; HSP47 Heat-Shock Proteins ; metabolism ; Kidney Glomerulus ; metabolism ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Vimentin ; 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

OBJECTIVETo analyze the relationship between cutaneous glycometabolic disorders and cutaneous neuropathy in diabetic rats, and to look for the mechanism of neuropathy and impaired wound healing.

METHODSEighty male SD rats were randomly divided into the normal control group (NC, n = 20), diabetic group (D, n = 20), aminoguanidine-interfered group (AI, n = 20), and insulin-interfered group (II, n = 20) by drawing lots. Diabetes was reproduced in rats of D, AI, and II groups with intraperitoneal injection of streptozotocin (STZ). Then, rats in AI group were fed with 100 mg×kg(-1)×d(-1) aminoguanidine, while rats in II group were subcutaneously injected with insulin for satisfactory control of blood glucose. Changes in mechanical and heat pain thresholds of pad of hind limb were measured at post injection week (PIW) 2, 4, 8. Skin specimens were collected during PIW 2-8 from pads for determination of contents of glucose, advanced glycation end product (AGE), substance P (SP), calcitonin gene-related peptide (CGRP), and observation of distribution and ultrastructure of skin nerve fibers. Data were processed with t test.

RESULTSThe mechanical and heat pain thresholds in D group at PIW 2 [(6.3 ± 1.5) g, (6.0 ± 0.9) s, respectively ] were obviously lower than those in NC group [(13.0 ± 3.2) g, (10.3 ± 1.2) s, with t value respectively 2.71, 3.42, P values all below 0.05]. Contents of glucose and AGE in skin tissue in D group were significantly increased when compared with those in NC group, especially at PIW 8 [(2.85 ± 0.33) mg/g, (31.7 ± 3.2) U/mg of hydroxyproline vs. (0.82 ± 0.22) mg/g, (22.2 ± 1.9) U/mg of hydroxyproline, with t value respectively 1.65, 6.47, P values all below 0.01]. The myelinated nerve fibers were edematous and degenerated, with axons compressed, while the unmyelinated nerve fibers were vacuolated, with microfilament and microtubule disorderly arranged. Content of SP in skin tissue in D group was lower as compared with that in NC group, especially at PIW 2 [(16.8 ± 3.4) pg/g vs. (28.5 ± 5.0) pg/g, t = 2.42, P < 0.01]. There was no obvious difference in content of CGRP between NC and D groups, and also in content of glucose in skin between D and AI groups. Compared with those in D group, content of AGE in AI group at PIW 8 was decreased markedly [(27.2 ± 1.4) U/mg of hydroxyproline, t = 3.38, P < 0.05]; contents of glucose and AGE in II group at PIW 8 were significantly decreased [(1.42 ± 0.38) mg/g, (23.6 ± 1.3) U/mg of hydroxyproline, with t value respectively 1.74, 8.17, P < 0.05 or P < 0.01]. Compared with that in D group, contents of SP in AI and II groups were increased, with a delay in time of trough value. Content of CGRP showed no obvious difference among D, AI, and II groups.

CONCLUSIONSHigh glucose and accumulation of AGE are key mediators of cutaneous neuropathy and impaired wound healing in diabetes mellitus, which confirms that diabetic wound takes an atypical footing during wound repairing. Aminoguanidine and insulin can reduce contents of glucose and AGE in diabetic skin tissue, and ameliorate diabetic cutaneous neuropathy.

Animals ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Glucose ; metabolism ; Glycation End Products, Advanced ; metabolism ; Male ; Peripheral Nervous System Diseases ; etiology ; Rats ; Rats, Sprague-Dawley ; Skin ; metabolism ; pathology ; Skin Diseases ; etiology ; Wound Healing

OBJECTIVETo observe the effect of heart protection on diabetic cardiomyopathy in rats by tripterysium glucosides.

METHODThe rat diabetic cardiomyopathy rats model are made by streptozotocin, then divided into tripterysium glucosides group (n=8) and model group (n=8). In addition, the control group is established (n=8). Glucosides group was orally administrated tripterysium glucosides (18 mg x kg(-1)), the control groups was orally administrated same volume NS for 3 months. Blood sugar, heart function and cardiac index were detected after 3 months. Immunohistochemical techniques were used to detect NF-kappaB and ICAM-1 expression. Ultrastructure of cardiac muscle cell were observed by electronmicroscope.

RESULTCompared with model group, cardiac index was decreased after tripterysium glucosides administration, and LVSP, LVEDP, + dp/dtmax, -dp/dtmax, were improved, and the expression of nuclear Factor-kappaB (NF-kappaB) and intercellular adhension molecule-1 (ICAM-1) was inhibited. Ultrastructure of cardiac muscle cell such as mitochondrion and cardiac muscle fibers was atttenuated.

CONCLUSIONTripterysium glucosides could protect rat diabetic cardiomyopathy rats heart. These function may be related to inflammatory reaction inhibition and immunosuppression of tripterysium glucosides.

Animals ; Blood Glucose ; metabolism ; Cardiomyopathies ; etiology ; metabolism ; pathology ; physiopathology ; Diabetes Mellitus, Experimental ; complications ; Gene Expression Regulation ; drug effects ; Glucosides ; administration & dosage ; pharmacology ; therapeutic use ; Heart ; drug effects ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Myocardium ; ultrastructure ; NF-kappa B ; metabolism ; Rats

OBJECTIVE: To evaluate the effect of hepatic insulin gene therapy on diabetic enteric neuropathy. METHODS: Mice were randomly allocated into 3 groups: a normal control group, a diabetic group, and a diabetic gene therapy group. Diabetes were induced by penial vein injection of streptozocin (STZ). The gene therapy group received hepatic insulin gene therapy while the other 2 groups only received an empty virus expressing green fluorescent protein. Random blood glucose, body weight growth, gastric emptying, total bowel length, absolute and relative bowel transit, electric field stimulation of colon smooth muscle, colon nuclei staining and counting were measured. RESULTS: We successully established a mouse model of diabetic enteric neuropathy which manifests as: 8 weeks of continuous hyperglycemia,increased total bowel length, decreased relative bowel transit, impaired colon smooth muscle relaxation and loss of inhibitory neurons in colon. Through gene therapy, the above indexes were normalized or ameliorated, suggesting hepatic insulin gene therapy is capable of preventing diabetic enteric neuropathy. CONCLUSION Hepatic insulin gene therapy can prevent STZ induced diabetic enteric neuropathy.
Adenoviridae ; Animals ; Diabetes Mellitus, Experimental ; complications ; therapy ; Diabetic Neuropathies ; therapy ; Enteric Nervous System ; metabolism ; pathology ; Gastrointestinal Diseases ; etiology ; therapy ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Hepatocytes ; metabolism ; Insulin ; genetics ; metabolism ; Mice ; Proinsulin ; genetics

OBJECTIVETo study the possible pathogenic mechanism of liver injury in type 2 diabetes mellitus (T2DM) and the intervening effect of puerarin on it.

METHODSMice with T2DM (KKAy) were randomly divided into two groups, the model group and the puerarin group. And the C57BL/J mice of the same age were set up as normal controls. They were sacrificed at 28 weeks old for observing serum fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) by automatic biochemistry; liver cell apoptosis by flow cytometry; pathomorphology by electron microscope; and mRNA expressions of bcl-2 and bax genes by RT-PCR; as well as the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), Na(+)-K(+)-ATPase; and content of malondialdehyde (MDA) in liver tissue by spectrophotometer.

RESULTSIn KKAy mice, blood levels of FBG, TG, TC, ALT, AST and liver cell apoptosis rate were higher; the bax mRNA expression was higher and bcl-2 mRNA was lower markedly; the activities of SOD, GSH-Px, Na(+)-K(+)-ATPase in liver tissue were lower, and MDA content was higher than those in the normal control significantly (all P <0.01). Besides, mitochondria swelling and damage were found in liver tissue. While in the puerarin group after treatment, all the above-mentioned changes were alleviated to some extent.

CONCLUSIONSObvious liver injury emerges in KKAy mice. Puerarin shows a protective effect on the T2DM caused oxidative damage by way of up-regulating bcl-2 to inhibit oxidative stress, and improving the energy metabolic dysfunction in liver of mice.

Animals ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; pathology ; Diabetes Mellitus, Type 2 ; complications ; drug therapy ; pathology ; Isoflavones ; therapeutic use ; Liver ; metabolism ; physiopathology ; Liver Diseases ; drug therapy ; etiology ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria, Liver ; pathology ; Oxidative Stress ; Phytotherapy ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2

This study examined the potential roles of astragalus and angiotensin II type 2 receptor (AT2) in rats with streptozotocin (STZ)-induced diabetic cardiomyopathy. Of 52 female 4-week-old Wistar rats treated with high glucose and lipid diet to induce insulin resistance, 7 treated with sodium citrate buffer (pH=4.5) served as controls (con1) and the other 45 were treated by intraperitoneal injection (ip) of STZ to induce type 2 diabetes. After 20 weeks, the maximal velocity decrease of pressure per second in left ventricle within the period of isovolumic relaxation (-dp/dtmax) was detected by inserting cannula through right carotid artery. Of the 45 rats, 24 with -dp/dtmax < or = 700 mmHg/s (1 mmHg=0.133 kPa) developing diabetic cardiomyopathy were grouped as follows: 7 treated with double distilled H2O (ip) were included in control group 2 (con2); other 8 treated with AT2 agonist (CGP42112A, ip) were included in experimental group1 (exp); 9 treated with astragalus (po) constituted experimental group 2 (exp2). All injections lasted 4 weeks (qd) and the heart weight (HW) was recorded. Cardiomyocyte apoptosis index (CAI), mRNA of AT2 and Bcl-2 as well as AT2 and Bcl-2 protein values in cardiomyocytes were also measured. Our results showed that -dp/dtmax in exp1, exp2 and con2 were much lower than those in con1 (P<0.01). CAI and AT2 in both mRNA and protein in con1 were lower than those in the other three groups (P<0.01). The three parameters above were higher in exp1 but less in exp2 than those in con2, respectively (P<0.01). The three parameters and HW in exp1 were much higher than those in exp2 (P<0.01). Changes of Bcl-2 were opposite to those of AT2. Our results suggested that high expression of AT2 might accelerate the apoptosis of cardiomyocytes in diabetic rats and play an important role in precipitating diabetic cardiomyopathy and astragalus protects diabetic rats from developing cardiomyopathy by downregulating AT2.
Animals ; Apoptosis ; drug effects ; Astragalus membranaceus ; chemistry ; Cardiomyopathies ; etiology ; pathology ; prevention & control ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Myocytes, Cardiac ; pathology ; Phytotherapy ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 2 ; biosynthesis ; genetics

OBJECTIVETo study the effect of Sanhuang Jiangtang recipe (SJR) on renin-angiotensin system in local myocardium in diabetic rats.

METHODSRats were made into diabetes model by intraperitoneally administering of streptozocin, and medicated through gastrogavage with SJR (50 g/kg), gliclazide (20 mg/kg), captopril (15 mg/kg) and nitrendipine (30 mg/kg) respectively, for successive 8 weeks, started from 2 weeks after modeling. Levels of fasting blood sugar (FBS), serum insulin (Ins), heart/body weight ratio (H/BW), myocardial angiotensin II (Ang II), angiotensin converting enzyme (ACE) and aldosterone (ALD) were determined. And the mRNA expression of type I angiotensin receptor (AT1R) in myocardium were detected by RT-PCR assay.

RESULTSAs compared with those in the normal rats, levels of FBS, H/BW, Ang II, ACE, ALD and AT1R mRNA expression were higher (all P < 0.05) and level of serum Ins was lower (P < 0.01) in the model rats. SJR, gliclazide, captopril and nitrendipine could slightly reduce the blood sugar level in model rats, but with no increase of serum Ins. All the four drugs could reduce H/BW, Ang II, ACE and AT1R mRNA expression. SJR and captopril could also decrease the ALD content in myocardium.

CONCLUSIONCardiac hypertrophy has been induced in 10 weeks after diabetic modeling. Activation of local myocardial RAS is related to the genesis of diabetic cardiomyopathy. SJR, gliclazide, captopril and nitrendipine could antagonize the genesis of diabetic cardiomyopathy, the mechanism is related to the inhibition of RAS activation in local myocardium.

Animals ; Cardiomegaly ; etiology ; prevention & control ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypoglycemic Agents ; pharmacology ; therapeutic use ; Male ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Renin-Angiotensin System ; drug effects