Changes of diaphragmatic function and morphologic features in diabetic rats

AIM:To evaluate the changes of diaphragm contractile properties and morphologic features in experimental diabetic rats.METHODS:Diaphragm contractility was assessed by twitch kinetics,maximal tetanic force (P₀),tension-frequency relationship and fatigue index(FI)in vitro diaphragm strip preparations of 4th week diabetic Wistar rats,and compared with that of the control group.The structure of diaphragm was analysed in diabetic rats and control with Hematoxyline and eosin staining,Heidenhain staining and enzymatic histochemistry.RESULTS:The twitch (Pt),contraction time(CT),half relaxation time(RT_1/2)and FI decreased significantly in diabetic group as compared with the control group.There was no difference in Po between the two groups.The tension of diabetic diaphragm at frequencies of 25,50,75,100 and 125 Hz was significantly decreased compared with that of the normal rats.After diaphragm bundles were subjected to fatiguing stimulations and incubated with aminophylline at 250 mg/L for 30 minutes, the tension of diabetic diaphragm decreased remarkably at both high and low frequencies in comparison with that of the normal rats.In addition,the activity and optical density ofα-phosphoglycerol dehydrogenase in diaphragm from diabetic rats were significantly lower than that from control rats(P<0101).CONCLUSION:The reduction of contractile function,decreased diaphragm fatigue resistance,and damaged structure occurred in 4th week diabetic rat diaphragm.     

Effect of DADLE on lung injury in rats with acute global cerebral ischemia-reperfusion

AIM：To observe the effects of δ opioid receptor agonist DADLE on acute lung injury (ALI) induced by acute global cerebral ischemia-reperfusion in rats. METHODS：SD rats (n=30) were randomly divided into sham group, model (I/R) group and DADLE treatment group. Global cerebral ischemia-reperfusion model was established by a modified 2-vessel occlusion plus hypotension. DADLE (5 mg/kg) treatment was performed via the left jugular injection before reperfusion. After 120-min reperfusion, the pathological changes of the lung tissues were observed under light microscope and electronic microscope. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) level were detected. The partial pressure of arterial oxygen (PaO2) was also measured. RESULTS：In I/R group, widened alveolar septum, capillary dilatation and congestion, endovascular and perivascular cells in the lung with neutrophil infiltration, and significantly reduced type II epithelial cell surface microvilli, alveolar lumen cavity and trachea with serous exudate were observed. SOD activity decreased, but the MDA level increased. Compared with I/R group, the SOD activity increased and MDA level decreased in DADLE treatment group, with significantly reduced lung congestion, the degree of lung injury, and the infiltration of neutrophils. Compared with I/R group, the PaO2 and oxygenation index in DADLE treatment group were increased. CONCLUSION：Various degrees of pulmonary injury were observed in acute global cerebral ischemia reperfusion model. DADLE might have a protective effect on lung tissues of ALI in rats.     

Effects of bilirubin on oxygen free radicals and caspase-3 in acute lung injury rats

AIM:To investigate the mechanisms by which bilirubin inhibits acute lung injury (ALI). METHODS:30 female Wistar rats were divided into normal group, ALI group and bilirubin treatment group. ALI was induced by intravenous injection of LPS. The contents of OH^-, H₂O₂ and O₂^· in the lung as well as the expression of caspase-3 in the lungs were investigated. RESULTS:(1) The contents of OH^-, H₂O₂ and O₂^· in the lung homogenate and the expression of caspase-3 in the lungs in ALI group increased compared with those in normal group (P<0.05). (2) The contents of OH^-, H₂O₂ and O₂^· in the lung homogenate and the expression of caspase-3 in the lungs in bilirubin treatment group increased compared with those in normal group, but decreased compared with those in ALI models (P<0.05). CONCLUSION:(1) Bilirubin was shown to be able to ameliorate apoptosis in ALI rats. (2) The increase in the contents of OH^-, H₂O₂, O₂^· in ALI group indicated the development of oxidative lung injury, which was ameliorated by bilirubin. (3) Expression of caspase-3 confirmed that the model made by LPS was associated with apoptosis, which was reduced by bilirubin.     

Immunohistochemical observation and imaging analysis of iNOS and vasoactive intestinal peptide in experimental diabetic rat lung

AIM: The pathological changes in inducible nitric oxide synthase (iNOS) and vasoactive intestinal peptide (VIP) in diabetic rat lung were investigated.METHODS: Using immunohistochemical method and imaging analysis, the changes in iNOS and VIP were observed in normal and diabetic rat lung. RESULTS: In diabetic rats, the iNOS positive or weak positive staining was localized in epithelial cells of bronchi, epithelial cells of alveolar ducts, alveolar sacs, and arteries, but negative in endothelial cells of vein and capillary. Image analysis showed the area, the integral absorbance(IA) and relative contents of iNOS were significant lower than that of control (P＜0.01, P＜0.01 and P＜0.01). In diabetic rats, the ciliated cells of bronchi epithelium showed VIP positive reaction, while the bronchial smooth muscle, surounding tracheobronchial submucosal glands, the tunica adventitia of pulmonary arteries, and alveolar septum were stained weak positive or negative for VIP. Image analysis showed the area, the IA and relative contents of VIP were significantly lower than those of control (P＜0.01, P＜0.01 and P＜0.01).CONCLUSION: The pathological changes in diabetic rat lungs are not only involving autonomic neuropathy but also NANC nerves. Nitric oxide and VIP, which are NANC neurotransmitter, may play an important role in pathogenesis of pathological change in diabetic lung.     

Alteration of p38 MAPK activity in lung tissue in diabetic rats

AIM: To observe the pathologic changes in lung and the role of p38 MAPKinase signal pathways in pulmonary alteration in diabetic rats. METHODS: Diabetic rats were induced by intraperitoneally injected streptozotozin (STZ). After 4 weeks, we observed the pathologic changes in lungs, tested protein kinase C (PKC) activities by isotope in lungs of model rats, tested transforming growth factor (TGF-β₁) by Western blotting and immunohistochemical analysis, and determined the expression of p38 MAPKinase mRNA using in situ hybridization.RESULTS: After STZ administration for 4 weeks, we observed thickened pulmonary capillary basal lamina and increased number of fibre in Diabetes mellitus (DM) rats. TGF-β₁ levels, PKC and p38 MAPK activities were also found increased. CONCLUSION: The increased activities of TGF-β₁ and p38 MAPK suggeste that TGF-β₁ may play an important role in diabetic lung, and hyperglycemia-PKC-p38 MAPK signal pathways may be involved in the pathogenesis of diabetes.     

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.     

Liver protection by Sini decoction in hemorrhagic shock and its mechanism relating to oxygen free radical and nitric oxide

AIM:The work was designed to explore protective effects of a traditional Chinese medicine-sini decoction (SD) on liver in hemorrhagic shock and its mechanism relating to oxygen free radical and nitric oxide.METHODS:Anesthetized Wistar rats were subjected to a hemorrhagic shock protocol for 60 min followed by intravenous injection with normal sodium chloride solution or SD solution. Superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) in liver were examined. The inducible nitric oxide synthase (iNOS) was determined immunohistochemically. RT-polymerase chain reaction (RT-PCR)was used to assay the mRNA, which were corresponding to eNOS (endothelial nitric oxide synthase) and iNOS.RESULTS:The activity of SOD decreased, while the concentration of MDA increased in liver during hemorrhagic shock. SD enhanced SOD activity and inhibited a increase in MDA level in liver (P＜0.01). The NO concentrations in liver in SD group increased at three hours after resuscitation (P<0.01). In addition, it was found that the expression of iNOS was upregulated in sodium chloride-treated group, while SD upregulated the expression of eNOS.CONCLUSION:SD reduces the liver injury caused by oxygen free radicals during hemorrhagic shock. The increasing NO concentration by SD is through upregulation of endothelial NOS expression.     

Changes of free radicals and antioxidative system of prostate tissue in the male rats with short-term diabetes

AIM:To study the dynamic changes of free radicals and antioxidative system of prostate in rats with short-term diabetes.METHODS:48 Wistar male rats were divided into 6 groups at random (8 rats in each group). One group were injected(ip) with sodium citrate buffer to be the control group, the other 5 groups were injected (ip) with steptozocin(STZ) 60mg/kg body weight which were called diabetes group. Rats were killed after 1, 3, 5, 7 and 14 days; superoxide dismutase(SOD), malondialdehyde(MDA), glutathione(GST), glutathione s-transferase(GST), glutathione peroxidase(GSH-px), nitric oxide (NO) and nitric oxide synthase(NOS) of prostate were examined.RESULTS:The levels of MDA, GSH-px and NO in prostate homogenate of diabetes group were higher evidently as compared with control levels. The activities of SOD, GST, NOS and the levels of GSH in prostate homogenate were increased obviously after injection of STZ with control levels, then returned near to control values.CONCLUSION:The evidences mentioned above indicate that high-level of free radicals and decrease in antioxidative system rendere the prostate cells to be in oxidative stress.     

Changes of plasma levels of soluble VEGFR2 and SOD in hypertensive patients and hypertensive diabetic patients

AIM: To explore the changes of plasma levels of soluble vascular endothelial growth factor receptor 2 (sVEGFR2) and superoxide dismutase (SOD) in hypertensive patients and hypertensive diabetic patients. METHODS: In this cross-sectional study, 88 cases were enrolled, which were divided into hypertensive group (n=31), hypertensive diabetic group (n=31) and control group (n=26). Blood pressure was obtained from each participant with mercury sphygmomanometer. The levels of sVEGFR2 and SOD were measured by ELISA. Meanwhile, the levels of plasma glucose, glycosylated hemoglobin A1c (GHbA1c) and lipid profile were detected. RESULTS: The levels of total cholesterol (TC) and body mass index (BMI) were significantly higher in hypertensive group than those in control group (P<0.05). The levels of TC, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), BMI, waist circumference were significantly higher in hypertensive diabetic group than those in control group (P<0.05). The plasma levels of sVEGFR2 and SOD in both hypertensive diabetic group and hypertensive group were significantly decreased compared with control group (P<0.05), while the mean plasma levels of sVEGFR2 and SOD in hypertensive diabetic group were significantly decreased compared to the hypertensive group (P<0.05). A significantly positive correlation between sVEGFR2 and SOD in the whole study population (P<0.05) was observed. CONCLUSION: The plasma level of sVEGFR2 is decreased in both hypertensive and hypertensive diabetic patients, and more significantly decreased in hypertensive diabetic patients. Decreased SOD level may be associated with to the reduction of sVEGFR2.     

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.     

Protective effect of spermine on rats with acute cerebral ischemia/reperfusion injury

AIM: To study the effects and the possible mechanisms of exogenous spermine on the rats with acute transient focal cerebral ischemia/reperfusion (I/R) injury.METHODS: The rat model of focal cerebral ischemia/reperfusion was established by middle cerebral artery occlusion (2 h) and reperfusion (2 h). Healthy adult SD rats were divided into 5 groups;sham group,I/R group and spermine(4,20 and 40 mmol/L)groups.The degree of cerebral injury was evaluated by neurological deficit score, infracted volume, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The morphological changes of the brain were observed by HE staining and electron microscopy. RESULTS: Compared with I/R group, the neurological deficit score, infracted volume and the content of MDA were decreased, the SOD activity was increased and the ultrastructural changes were improved in spermine-treated groups. CONCLUSION: Exogenous spermine has a protective effect against acute focal cerebral ischemia/reperfusion injury. The mechanisms may be related to scavenging free radical by spermine.     

Protective effects of extract of Ginkgo biloba on diaphragm of diabetic rats

AIM: To investigate the effects of extract of Ginkgo biloba (EGb) on diaphragm from diabetic rats. METHODS: Sprague-Dauley rats were divided into three groups: normal control, diabetic group and EGb treatment group. The morphologic changes of diaphragm tissues were studied by light and electron microscopy, the activities of succinate dehydrogenase (SDH), superoxide dismutase (SOD), nitric oxide synthase (NOS) and contents of malondialdehyde (MDA), nitric oxide (NO₂^-/NO₃-) in the diaphragm mitochondria were assayed by spectophotometer, respectively. RESULTS: The activities of SOD, SDH decreased in diabetic diaphragm mitochondria, but the activitiy of NOS, the contents of NO₂^-/NO₃^-, MDA increased compared with control group. The activities of SOD, SDH were increased as well as NOS were decreased and the contents of NO₂^-/NO₃^-, MDA decreased in EGb treatment group compared with the diabetic group. CONCLUSION: EGb may protects the diaphragm mitochondria of diabetic rats by enhancing the function of respiratory chain, anti-oxidation and decreasing NO level.     

Study on the variation of trace elements and oxidative stress in diaphragm of the diabetic rats

AIM:To investigate the relationship between trace elements and oxidative stress in diabetic diaphragm. METHODS: Contents of copper(Cu), manganese(Mn), zinc(Zn), iron(Fe),cadmium(Cd),chromium(Cr),cobalt(Co),molybdenum(Mo) and lithium(Li) in the alloxan-induced diabetic rat diaphragm muscles were measured by atomic absorption spectrophotometer, andtheir superoxide dismutase(SOD) activities and malondiadehyde(MDA) contents were detected. RESULTS: The contents of Cu, Zn, Cr and Li were lower and the Fe and Cd were higher in diabetic rat diaphragm muscles than that of the control group, but there were no significant differences in Mn, Co and Mo, between control and diabetic group. SOD activities decreased significantly and MDA contents increased significantly in diaphragm of diabetic rats as compared with the control group.In addition,in diabetic rats diaphragm, there was a positive correlation between Zn and SOD, and negative correlation between Zn and MDA, and Cd and SOD. CONCLUSION: Changes of the contents of Cu, Zn, Fe, Cd, Cr, Li and the increase of oxidative stress in diaphragm were found in the early diabetes, and Zn, Cd play an importment role in oxidative stress of diabetic diaphragm.     

Protective effect of Ginkgo biloba extract on testicular tissue of diabetic rats

AIM: To investigate the protective effect of Ginkgo biloba extract (GBE) on diabetic testis and explore its possible mechanism. METHODS: Testicular structure of streptozotocin-induced diabetic rats was observed under light microscopy (LM) and transmission electron microscopy (TEM). Content of malondialdehyde (MDA), NO₂^-/NO₃^- and activity of superoxide dismutase (SOD), nitric oxide synthase (NOS) were determined in testicular homogenate. RESULTS: In diabetic rats, it was manifestated as deformation of seminiferous tubule, atrophy and shedding of germinal epithelium under LM, while expansion of smooth endoplasmic reticulum, formation of fatty vacuoles and decrease of lysosome obviously in the cytoplasm of sertoli cell under TEM, the injury of testicular tissue was improved by GBE. Compared with diabetic rats, activity of SOD increased while activity of tNOS and iNOS, content of MDA and NO₂^-/NO₃^- decreased in GBE-treated rats. CONCLUSION: GBE could effectively prevent the development of diabetic testis and the effect may be partly achieved by resisting lipid peroxidation,restraining the activity of testicular tissue iNOS and reducing the pathological alterations of NO.     

Effects of simvastatin on expression of Bcl-2 and Bax in myocardium of rats with renal ischemia-reperfusion injury

AIM: To observe the effect of simvastatin on myocardial tissue after renal ischemia-reperfusion injury and its mechanism. METHODS: A rat model of renal ischemia-reperfusion injury was prepared by clamping the bilateral renal arteries for 45 min. The rats (n=36) were randomly divided into sham operation group, renal ischemia-reperfusion (I/R) group and simvastatin group with 12 rats in each group. The content of serum creatinine (SCr), blood urea nitrogen (BUN) and myocardial tissue malondialdehyde (MDA), the myocardial activity of lactate dehydrogenase (LDH), creatine kinase (CK) and superoxide dismutase (SOD), and the myocardial protein expression of Bcl-2 and Bax were detected. RESULTS: Compared with sham operation group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in I/R group were significantly increased (P<0.05), and the activity of SOD was significantly decreased (P<0.05). Compared with I/R group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in simvastatin group were significantly decreased (P<0.05), while SOD activity was enhanced (P<0.05). The protein expression of Bcl-2 and Bax in sham operation group was less than that in I/R group (P<0.05), and the protein level of Bax in simvastatin group was significantly lower than that in I/R group (P<0.05), while the protein level of Bcl-2 was increased (P<0.05). CONCLUSION: Simvastatin has a protective effect on the myocardium of the rats with renal ischemia-reperfusion injury, and the protective mechanism may be related to the elimination of free radicals by simvastatin, increase in the protein expression of Bcl-2 and decrease in the protein expression of Bax.     

Effect of gingko biloba extract on liver from experimental type 2 diabetic rats

AIM:To study the protective effect of the ginkgo biloba (EGB) extract on liver from experimental type 2 diabetic rats and to explore its possible mechanism. METHODS:Thirty-nine male Sprague-Dawley rats were divided randomly into four groups: normal control group, high-fat group, diabetic group and EGB-treated group. After fed with high-fat diet for 4 weeks, the later two groups were injected with streptozotocin intraperitoneally to induce type 2 diabetes mellitus. EGB-treated group was injected intraperitoneally with EGB at a dose of 8 mg·kg^-1·d^-1, and the other three groups were treated with normal saline of the same volume. After 8 weeks, the morphologic change of hepatic tissue was observed under transmission electron microscope (TEM) and light microscope (LM), respectively. In addition, the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), total nitric oxide synthase (TNOS), inducable nitric oxide synthase (iNOS) and the content of malondialdehyde (MDA), nitric oxide (NO) in liver homogenate were detected biochemically. RESULTS:Obvious liver fatty degeneration, apparent decrease of glycogen granules in cytoplasm of hepatocytes under light microscope and hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of hepatic stellate cells and collagen under TEM were observed in diabetic group. The activity of SOD, CAT, GSH-PX decreased but the activity of tNOS, iNOS and the content of MDA, NO^-₂/NO^-₃ increased in diabetic group compared with normal control group. The pathological change was relieved in EGB-treated group. The activity of SOD, CAT, GSH-PX increased, the activity of tNOS, iNOS and the content of MDA, NO^-₂/NO^-₃ decreased in the liver of rats in EGB-treated group compared with diabetic group. CONCLUSION:EGB exerts a beneficial effect on liver in experimental type 2 diabetic rats. Anti-lipid peroxidation and suppression of NO production may be involved in this process.     

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.     

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.