Effects of hydrogen sulfide on wound healing of skin ulcer in diabetic rats

AIM: To study the effect of hydrogen sulfide on wound healing of skin ulcer in diabetic rats.METHODS: Male SD rats were randomly divided into 3 groups, including non-diabetic control(NDC) group, untreated diabetic control(UDC) group, and treated diabetic administration(TDA) group. Diabetic rats were induced by intraperitoneal injection of streptozotocin(STZ). After 1 week, wound healing model was prepared by making a round incision(2.0 cm in diameter) on the dorsal skin in full thickness. The rats from TDA group received 2% sodium bisulfide ointment on the skin ulcer wound, and the animals from UDC and NDC groups received control cream. After 21 d of treatment with sodium bisulfide, blood samples were collected for biochemical analysis, including prothrombin time(PT), thrombin time(TT), and fibrinogen(FIB) in plasma, as well as the activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA) in the serum. White blood cells(WBC) and lymphocytes were also counted. Granulation tissues from the wound were processed for histological examination and Western blot analysis was used to detect heme oxygenase-1(HO-1) and tumor necrosis factor α(TNF-α) expression.RESULTS: Compared with UDC group, sodium bisulfide treatment accelerated wound healing of skin ulcer(P<0.01), and increased the activity of SOD in serum(P<0.01) in the diabetic rats. The declined number of WBC and lymphocytes, prolonged PT and TT, and decreased FIB levels in rats treated with sodium bisulfied were also confirmed. Pathological section showed that there were inflammatory cell infiltration, and irregular and loose fibril alignment in the granulation tissue of rats from the UDC group, but there were regular fibril alignment and increased angiogenesis in the granulation tissue of rats from the TDA group(P<0.05). Furthermore, sodium bisulfide treatment raised HO-1 protein expression, and decreased TNF-α protein expression in the diabetic rats.CONCLUSION: Hydrogen sulfide accelerates the wound healing of skin ulcer in the rats with diabetes. The beneficial effect of H₂S may be associated with formation of granulation, anti-inflammation, and antioxidation.     

Effect of JNK expression in dorsal root ganglia on histological change of foot skin in diabetic rats

AIM: To observe the change of skin histology in diabetic rats and to investigate the possible me-chanism of c-Jun N-terminal kinase (JNK) protein in the dorsal root ganglion (DRG) during the process. METHODS: Diabetic animal model was established in the male SD rats by intraperitoneal injection of streptozotocin. Plantar skin specimens of the rats were collected from control group, DM 2-week group (DM2), DM 4-week group (DM4), and DM 8-week group (DM8). Immunohistochemical staining and HE staining were used to observe the change of PGP 9.5 immunoreactive nerve terminals and the structures of the skin tissues. The protein expression of PGP 9.5 in the plantar skin tissues, and JNK and p-JNK protein in the DRG within lumbar 5, 6 (L5, 6), and sacral 1 (S1) spinal cord segments were detected by Western blotting. RESULTS: PGP 9.5 immunoreactive nerve terminals of the plantar skin of the rats mainly distributed in the basal layer of the epidermis and papillary dermis. Compared with control group, PGP 9.5 positive nerve terminals in DM4 group showed reduced density and sparse distribution. PGP 9.5 positive nerve terminals in DM8 group showed significantly reduced distribution, thinner nerve diameter, shorter length and distorted shape. Histological changes of the thinner epidermal tissue, reduced epidermal cell layers, uneven cell distribution and arrangement in DM4 group, and significantly reduced epidermal cell layers, swollen and blurred cells, increasing cell gap, lack of stratified epidermis arrangement for part of epidermis, atropal and degenerated dermal collagen fiber, significantly decreased subcutaneous fat in DM8 group were observed. The results of Western blotting showed that the protein expression of PGP 9.5 in the plantar skin tissue of DM rats was progressively decreased along with the disease, while the protein level of p-JNK in L5, 6-DRG or S1-DRG showed a gradual increasing trend. PGP 9.5 immunoreactive positive nerve terminal density of plantar skin in DM rats had a negative correlation with the protein level of p-JNK in L5, 6-DRG and S1-DRG (P<0.01), but showed a significant positive correlation with the plantar skin thickness (P<0.01). CONCLUSION: The protein level of p-JNK within L5, 6-DRG or S1-DRG in DM rats shows a progressive enhancement. At the same time, there is a significant change in the skin tissue density and structure. The changes of skin tissue and nerve morphology in DM rat may be related to the activation of JNK/SAPK pathway in L5, 6-DRG or S1-DRG cells. Blocking or inhibiting JNK/SAPK pathway may delay the diabetic peripheral neuropathy and reduce the risk of skin lesions.     

Thiazolidinedione improves Alzheimer-like changes in the hippocampus of type 2 diabetic rats by Wnt pathway

AIM: The abnormal level of insulin and glycemia in type 2 diabetes mellitus(T2DM) are important risk factors of Alzheimer's disease (AD). To explore the mechanism that thiazolidinedione (TZD) decreases the incidence of AD in T2DM, we use TZD on T2DM rats for an intervention and detect the change of Wnt pathway before and after the intervention.METHODS: To establish a T2DM model, the rats were fed with high glucose, high fat and high protein for 8 weeks, and then injected with STZ. TZD was administered intragastrically for 2 and 4 weeks and the rats were divided into TZD2W and TZD4W groups, respectively. Plasma insulin level was measured by RIA method, and the plasma glucose was detected by glucose-oxidase method. Total tau level, the phosphorylation level of tau at individual phosphorylation sites and the level of amyloid β precursor protein(APP), β-catenin, glycogen synthase kinase-3β (GSK-3β) and PPARγ in rat hippocampus were analyzed by Western blotting. The activity of GSK-3β in the hippocampus of rats was determined using γ- -ATP and the specific peptide substrate. The level of APP was also determined by immunochemistry. The insulin resistant (IR) degree was valued by HOMA-IR.RESULTS: Glycemia level in T2DM and TZD2W groups was obviously higher than that in control group. No significant difference of glycemia level between TZD4W and control group was observed. Plasma insulin levels in all groups were evidently higher than that in control group. The IR degree in T2DM and TZD2W groups increased significantly as compared to control group, but no obvious difference between TZD4W and control group was observed. The level of phosphorylated tau protein at site Ser199/202 and Ser422, and APP level in hippocampus of T2DM rats were found to be notably raised as compared to control group, but the level of phosphorylated tau protein at those sites and APP level were decreased gradually. No change of the PPARγ level was found in the hippocampus in T2DM and control group, but a notable increase was observed after TZD intervention. There was a decrease in β-catenin level in hippocampus of T2DM rats, which increased after TZD intervention for 2 and 4 weeks. There was a rise of GSK-3β activity in T2DM rats, which decreased after intervention.CONCLUSION: These findings suggest that TZD may improve the AD-like changes in the hippocampus of T2DM rats by up-regulation of Wnt pathway, which acts before the insulin signal transduction in the contribution of AD-like changes in T2DM rats.     

Protective effects of riboflavin on diabetic nephropathy in STZ-induced diabetic rats

AIM: To explore the protective effects of riboflavin on the kidney in streptozotocin (STZ)-induced diabetic rats. METHODS: Male Sprague-Dawley rats were randomly divided into 3 groups: normal control group, diabetic model group and riboflavin-treated group. Diabetes was induced by a single injection of STZ (dissolved in 0.01 mol/L citrate buffer, pH 4.5, 65 mg/kg, ip) in rats. The biochemical methods were used to measure the contents of urine protein and malondialdehyde in the kidney, and the activities of superoxide dismutase (SOD) and catalase (CAT) in serum and renal tissues. Furthermore, the protein expression of TGF-β1 and plasminogen activator inhibitor-1(PAI-1) in renal cortex was detected by Western blotting. The morphological changes of renal tissue were observed under microscope.RESULTS: Compared to the diabetic model group, riboflavin significantly increased the activities of SOD and CAT (P<0.01) in the serum and renal tissues, and decreased the contents of urine protein and MDA (P<0.01) in the renal tissues in riboflavin-treated group. The levels of TGF-β1 and PAI-1 in the renal cortex were dramatically decreased in the treated diabetic rats compared to the diabetic model rats (P<0.01).CONCLUSION: Riboflavin inhibits the protein expression of TGF-β1 and PAI-1 in renal tissue of STZ-induced diabetic rats. Riboflavin may alleviate the pathologic changes and play an important protective role in diabetic kidneys.     

Accelerated cardiac remodeling of post-infarction was associated with changes of gene expression profile in untreated streptozotocin-induced diabetic rats

AIM: To study the time-dependent effects of diabetes mellitus (DM) on the development of cardiac remodeling in untreated streptozotocin (STZ)-induced rats with acute myocardial infarction (MI). METHODS: The left anterior descending coronary arteries were ligated 10 weeks after DM induction without any therapy. Transmission electron microscopy, echocardiography, heart weight to tibial length ratios, histological examination, microarray analysis, and real time-PCR were utilized to monitor the changes up to 56 d. RESULTS: After MI, the diabetic rats experienced lower survival rate compared to non-diabetic animals. The pathophysiologic changes indicated that DM accelerated the cardiac remodeling post-infarction. In primary examination, 164 genes related to cardiac remodeling were found to be candidates for hierarchical analysis, such as leucine-rich PPR-motif containing (interleukin-6 signaling pathway), procollagen type I and III, fibronectin-1, RT1, and TIMP-1, etc. The gene expression profile at 14 d in diabetic rats were comparably similar to both 14 d and 28 d in non-diabetic rats, while such changes at 28 d and 56 d in diabetic rats was also similar to the ones at 56 d in non-diabetic rats. CONCLUSION: The accelerated cardiac remodeling of post-infarction in STZ-induced untreated diabetic rats seems be associated with the different profile of gene expressions.     

Changes of corneal ultrastructure in early diabetic rats

AIM:To investigate the changes of ultrastructure in early diabetic rat cornea and the pathogenesis of diabetic keratopathy.METHODS: 20 Sprague-Daxley rats were sacrificed 6, 8, 10 and 12 weeks after induction of diabetes mellitus by streptoxotocin. 20 untreated rats at the same age were used as normal controls and were sacrificed at the same intervals. The ultrastructures of cornea were observed with transmission electronic microscopy and scanning electron microscopy.RESULTS:During the experimental period, the corneal ultrustructure of diabetic rats showed that epithelial and endothelial cells were swollen, the mitochondrions in the cytoplasm were swollen and increased. The collagen fibers appeared in disarrangement 10 weeks after streptoxotocin treatment. The endothelial of cornea was damaged from the periphery to the center gradually.CONCLUSION:The ultrastructural changes of cornea leads to dysfunction in streptoxotocin-induced diabetic rats, which may be related to the abnormal metabolism in hyperglycemia condition.     

Protective effect of curcumin on myocardium in diabetic rats

AIM: To study the effects of curcumin (Cur) on diabetic cardiomyopathy (DCM) in rats. METHODS: Male Wistar rats (n=75) were divided into control group and diabetes model group, in which the rats were fed with high-fat diet and then intraperitoneally injected with streptozotocin (STZ, 40 mg/kg). Fasting blood glucose was measured 72 h and 1 week after STZ injection. The diabetic rats were diagnosed when sustained fasting blood glucose levels ≥ 11.6 mmol/L. The diabetic rats were randomly divided into DCM group, DCM+Cur 100 mg/kg group and DCM+Cur 200 mg/kg group. After treatment for 16 weeks, glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) level were measured, and the level of cardiac troponin I (cTnI) in the serum was determined by enzyme-linked immunosorbent assay. The protein expression of protein kinase C (PKC) was detected by Western blotting. RESULTS: Curcumin significantly decreased the blood glucose level, increased the body weight, inhibited MDA content and up-regulated the GSH-Px activity in the diabetic rats. Furthermore, curcumin treatment inhibited the diabetes-induced protein expression of PKC. CONCLUSION: Curcumin may have a protective effect on diabetic cardiomyopathy by attenuating oxidative stress.     

An enzyme histochemical study of diaphragm in diabetic rats

AIM: The aim of this research is to study the earlier enzyme activity changes of the diaphragm in diabetic rats. METHODS: An enzyme histochemical method was used to observe the changes in the enzyme activities of dehydrogenases,hydrolases and oxidases in 4th week diabetic rat diaphragm. RESULTS: The activites of enzymes including SDH(Succinate dehydrogenase),MDH(Malate dehydrogenase), GDH(Glutamate dehydrogenase), ICDH(Isocitrate dehydrogenase), NADHD(NADH diaphorase), G-6-PD(Glucose-6-phosphate dehydrogenase), ACP(Acid phosphatase) and ANAE(Acid α-naphtyl acid esterase) were increased in diabetic diaphragm compared with the control. LDH (Lactate dehydrogenase)and CCO(Cytochrome oxidase) activities were decreased, whereas NADPHD(NADPH diaphorase) showed no changes in diabetic rats. Eleven kinds of enzyme were analysed with image analysis.Optical density (A) of SDH, MDH, GDH, ICDH, NADHD, G-6-PD, ACP and ANAE in diaphragm of diabetic rats were significantly higher than that of control rats (P<0.01). A value of LDH and CCO in diaphragm from diabetic rats were significantly lower than that of control rats (P<0.01). A value of NADPHD in diaphragm from diabetic rats showed no apparent alteration compared with the control rats(P>0.05). CONCLUSION: Increase in the aerobic capacity, decrease in the glycolytic capacity, and disturbance of lipid and energy metabolism were found in diaphragm of 4th week diabetic rats.     

Effect of lentinan on myocardial impairment in diabetic rats

AIM:To study the protective effect of lentinan against myocardial impairment in diabetic rats.METHODS:Morphology of myocardium from streptozocin induced diabetic rats treated with lentinan was observed under light microscopy(LM) and transmission electron microscopy(TEM). Activity of superoxide dismutase(SOD), nitric oxide synthase (NOS) and contents of malondialdehyde (MDA) and nitric oxide (NO) were detected biochemically in myocardial homogenate.RESULTS:Vacuolar degeneration, local lysis of myocardium and interstitial proliferation under LM and expansion of mitochondria, shortening of mitochondrial crest, lysis of myofibril and proliferation of interstitial collogenous fiber under TEM were observed. The activity of SOD decreased and the activity of NOS, the contents of NO, MDA increased, but the morphological change became slight in LNT-treatment group. Activity of SOD increased while activity of NOS and contents of MDA, NO decreased in LNT-treated rats compared with diabetic rats.CONCLUSION:LNT protectes diabetic myocardium, and the anti-lipid peroxidation and decreasing of NO level may be involved in it.     

Role of autophagy in intervertebral disc degeneration in diabetic rats

AIM:To explore the levels of apoptosis and autophagy in the nucleus pulposus tissues of intervertebral discs in diabetic rats. METHODS:Sixteen weeks after injection of streptozocin (STZ), the lumbar intervertebral discs were obtained from the rats. The histological changes were observed by hematoxylin-eosin (HE) staining and alcian blue staining. The apoptosis of the nucleus pulposus cells was measured by the methods of terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), immunohistochemistry, and Western blotting. The level of autophagy in the nucleus pulposus cells was detected by Western blotting and immunohistochemistry. RESULTS:Compared with normal group, HE and alcian blue staining suggested that the intervertebral discs of the diabetic rats became degenerate. The expression of caspase-3 and the apoptotic rate were increased in intervertebral disc nucleus pulposus of the diabetic rats. The results of immunohistochemistry and Western blotting showed that the expression levels of LC3Ⅱ/LC3Ⅰand beclin-1 in the diabetic rats were higher than those in normal group. CONCLUSION: The STZ-induced diabetes accelerates degeneration of the intervertebral discs. In addition, the apoptosis and autophagy are increased in the intervertebral discs of diabetic rats.     

Expression of angiogenic factors in myocardial tissue of diabetic rats

AIM：To observe the expression of angiogenesis factors in the myocardial tissue of streptozotocin-induced diabetic rats. METHODS：The diabetic rat model was induced by intraperitoneal injection of streptozotocin. After 12 weeks, the cardiac function was measured by MPA cardiac function analysis system. The myocardial collagen volume fraction (CVF) was assessed by Masson staining. The capillary vessels was quantified as the ratio of capillary to myocyte (C/M) using CD31 immunostaining. The expression levels of vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1, endostatin and Ang-2 were observed by Western blotting. RESULTS：Compared with normal control group, the left ventricular end-diastolic pressure (LVEDP) was evidently increased (P<0.01), but left ventricular pressure rise maximum rate (+dp／dtmax), left ventricular pressure decrease maximum rate (-dp／dtmax) and the ratio of capillary/myocyte (C/M) were significantly decreased (P<0.05). The CVF and the expression level of endostatin were significantly increased, whereas the expression levels of VEGF and Ang-1 evidently decreased (both P<0.05) in diabetic rats. However, no marked difference in the expression of Ang-2 between the 2 groups was observed (P>0.05). CONCLUSION：Imbalances between the angiogenic factors (VEGF and Ang-1) and anti-angiogenic factors (endostatin) may play an important role in the pathogenesis of diabetic cardiomyopathy.     

Role of oxidative stress in gastric motility of diabetic rats

AIM: To investigate the changes of oxidative stress in the stomach tissues and their roles in gastric motility and interstitial cells of Cajal (ICC) in diabetic rats. METHODS: Thirty-eight SD rats (8-week-old, male) were intraperitoneally injected with streptozotocin (STZ). Diabetes was successfully induced in 36 of them. The diabetes rats were randomly divided into untreated diabetes group and treated diabetes group. Eighteen healthy SD rats (8-week-old, male) served as controls. The body weight and the levels of blood glucose and glycosylated hemoglobin were measured. At the end of week 1 and week 10, 9 rats were sacrificed in each group. The gastric emptying rate and the levels of malondialdehyde (MDA),superoxide dismutase (SOD), tumor necrosis factor α (TNF-α), tyrosine kinase receptor c-Kit and stem cell factor (SCF) in gastric smooth muscle were analyzed. The apoptosis of ICC in gastric tissues was detected by the methods of immunocytochemistry and TUNEL. RESULTS: Compared with control group, gastric motility and SOD activity in untreated diabetes group were significantly weakened, the levels of MDA and TNF-α increased, the levels of c-Kit and SCF decreased, and apoptosis of ICC enhanced. In treated diabetes group, the oxidative stress level was attenuated, antioxidant capacity was enhanced, the levels of c-Kit and SCF were significantly increased, and the ICC apoptosis was reduced. Gastric motility was significantly improved after antioxidant therapy. CONCLUSION: Hyperglycemia affects the expression of antioxidant enzymes in the stomachs of diabetic rats. Oxidative stress is caused by hyperglycemia and is an important factor in the etiology of gastric motility dysfunction in diabetic rats, which may be correlated with the augmentation of ICC apoptosis resulting from oxidative stress-induced c-Kit/SCF damage.     

Microarray-based miRNAs profiling in lower limb arteriosclerosis of diabetic rats

AIM: To establish the profiling of microRNAs (miRNAs) in the lower extremity arterial tissue between diabetic rats with lower limb arteriosclerosis (DAS) and diabetic rats with normal lower limb (DN), and to explore the possible molecular mechanisms involved in aberrant miRNA expression in DAS. METHODS: The rat models of DAS and DN were successfully established. The respective lower extremity arterial tissue was isolated. The total miRNAs were purified for a hybridization detection by miRNA microarray. The results of chip scanning and data were analyzed and verified by RT-qPCR. RESULTS: Ten miRNAs related to DAS, including rno-miR-206-3p, rno-miR-133a-5p, rno-miR-133b-3p, rno-miR-133a-3p, rno-miR-325-5p, rno-miR-675-3p, rno-miR-411-5p, rno-miR-329-3p, rno-miR-335 and rno-miR-126a-3p, were determined. All 10 abnormally expressed miRNAs were up-regulated. The validating results of RT-qPCR confirmed 9 of the miRNAs in line with chip expression. Just rno-miR-335 showed the opposite between PCR detection and microarray result. CONCLUSION: A group of miRNAs in diabetic rats suffering from lower limb arteriosclerosis plays an important role in the vascular atherosclerosis process. The abnormal expression of miRNAs is likely to affect the vascular atherosclerosis process.     

Effect of Ginkgo biloba extract on retinopathy in diabetic rats

AIM: To investigate the effect of Ginkgo biloba extract(GBE) on diabetic retinopathy(DR) and its possible mechanism in rats. METHODS: Goto-Kakizaki(GK) rats were used as a DR model, and were treated with different doses of GBE. Normal Wistar rats were used as the control. Blood glucose and retina barrier injury were analyzed, respectively. Ganglion cell apoptosis was detected by TUNEL staining. Moreover, the protein expression of nuclear factor E2-related factor 2(Nrf2), ERK, Bcl-2 and P53, and ERK phosphorylation were examined by Western blot.RESULTS: GBE reduced blood glucose in the DR rats, attenuated retina barrier injury, and decreased the apoptosis of ganglion cells. Furthermore, the expression of Nrf2 and Bcl-2, and phosphorylation of ERK were increased after GBE treatment, whereas P53 expression was decreased. CONCLUSION: GBE protects ganglion cells against apoptosis in DR rats, which may be through activation of Nrf2/ERK pathway and regulating Bcl-2 and P53 expression.     

Protective effects of sulforaphane on retinal neurons in diabetic rats

AIM: To clarify whether sulforaphane (SF) has protective effects on retina neuronal cells in diabetic rats and to identify the related mechanisms involved in this process. METHODS: The diabetic rat model was induced by single intraperitoneal injection of streptozotocin (STZ). The protective effects of SF were evaluated by measuring the generation of reactive oxygen species (ROS), detecting apoptosis of retina neuronal cells with TUNEL staining and counting the survival retinal ganglion cells (RGCs). The nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) and the protein expression of heme oxygenase-1 (HO-1) were examined by immunofluorescence analysis and Western blot. RESULTS: SF treatment significantly attenuated ROS generation, decreased the apoptosis of retina neuronal cells and increased the numbers of survival RGCs in the diabetic rats. Meanwhile, SF significantly increased the nuclear accumulation of Nrf2 and the protein level of HO-1 in the retinas of diabetic rats. However, HO-1 inhibitor, protoporphyrin IX zinc (Ⅱ) diminished the inhibitory effects of SF on RGCs apoptosis. CONCLUSION: SF partially exerts the beneficial neuroprotective effects via the activation of the Nrf2/HO-1 antioxidant pathway, therefore alleviating retinal oxidative stress and decreasing the apoptosis of retina neuronal cells.     

Renoprotective effect of L-carnitine on streptozotocin-induced diabetic nephropathy in rats

AIM: To investigate whether L-carnitine (LC) treatment confers renoprotection in a rat model of streptozotocin (STZ)-induced diabetic nephropathy (DN). METHODS: Diabetic animal model was established by intraperitoneal injection of STZ (65 mg/kg) in Sprague-Dawley rats. Diabetic rats were treated with LC (50 mg·kg^-1·d^-1 or 200 mg·kg^-1·d^-1 intravenously) daily for 12 weeks. The effects of LC on STZ-induced DN were evaluated by assessing renal function, urinary protein excretion, histopathological changes, macrophage infiltration, the expression of proinflammatory and prosclerotic cytokines, and the expression of nuclear factor-κB (NF-κB) and apoptosis-related gene. RESULTS: LC administration significantly decreased glomerulosclerosis, preserved the number of podocytes, and reduced macrophage infiltration. These changes were accompanied by improvements in urinary protein excretion and renal dysfunction. LC treatment suppressed the expression of proinflammatory and prosclerotic cytokines, and these changes were paralleled by significant attenuation of NF-κB and apoptosis-related gene expression. CONCLUSION: LC has a renoprotective effect against STZ-induced DN in rats.