1.Animal model: 160 male Wistar rats, each 200-350g in weight, were bought from the Experimental Animal Department, Epidemic Disease Institute, China Preventive Medical Academy. After the animals were weighed, urethan...1.Animal model: 160 male Wistar rats, each 200-350g in weight, were bought from the Experimental Animal Department, Epidemic Disease Institute, China Preventive Medical Academy. After the animals were weighed, urethane 1.2g/kg of body weight was injected into the abdominal cavity for anesthesia, and then LPS 5mg/kg of body weight (LPS 055 B5 Sigma Co) was injected into the sublingual vein to establish ALI animal model. The grouping of the animal: 1) control group (NS): Normal saline (The 4th Pharmaceutics, Shijiazhuang) was injected into the sublingual vein, and 2 hours later the specimen was collected. 2) ALI group: 2 hours after injection of LPS, the specimen was collected. 3) Rhubarb group (R): Rhubarb extract 20g/kg (made in the Pharmacy of Traditional Chinese Medicine of this hospital, 1 gm crude drug/ml) was injected into the abdominal cavity; 1 hour later, LPS was injected; and 2 hours later, the specimen was collected. 4) Dexamethasone group (D): Dexamethasone 70mg/kg of body weight (made in Zhengdatianjing Pharmaceutics, Ltd. Co. Lianyun-gang) was injected into the abdominal cavity; 1 hour later, LPS was injected; and 2 hours later, the specimen was collected.
2.Histological examinations: In each group, the lung specimens were taken from 5 animals. The specimens were fixed in 10% formaldehyde, embedded in paraffin and sectioned. HE staining was used, and the sections were examined under the light microscope. The lung specimen embedded in paraffin, 1 mm3 in size, was taken to be fixed in 25% glutaraldehyde and then again fixed in 1% OsO4. Alcohol and acetone were used for dehydration. The specimens were then embedded in EPON81, and ultrathin sections were prepared. Uranium-lead double staining was used, and the ultrathin sections were examined under the Philips EM 400T transmission electron microscope.
3.Measurement of lung tissue wet weight/dry weight and the rate of cell count and protein content in the pulmonary alveolar lavage fluid: The thoracic cavity was opened, and the lung was taken out. Its wet weight was measured, then the lung was put in an oven 80℃ for 20 hours to obtain the constant weight of the lung, and finally the rate of wet/dry weight of the lung was calculated. In another group of rats, pulmonary alveolar lavage was carried after the thoracic cavity was opened. The total cell count and the cell classification were measured in the lavage fluid under the light microscope. The Lowry method was used for determination of the protein content. The lung permeability index was calculated (the protein in the pulmonary alveolar fluid/serum protein, LPI).
4.Pulmonary vascular permeability change:6 After the drug was administered in each animal group, Evans blue (50kg/kg) was injected into the femoral vein at the time when LPS was injected. After opening the thoracic cavity, the lung was taken out, its peripheral tissues were resected. The lung was immersed in the formamide solution (20 mg/100g of animal weight); the lung in the solution was incubated for 72 hours in an incubator at 45-50 ℃; after the pigment was completely extracted from the lung tissue, the tissue was taken out and solution was centrifuged and the supernatant fluid collected. Spectrophotometer 72 Model was used to make colorimetry at 620 nm. The Evans blue content was calculated from a standard curve and the value of the content was used for the determination of the change of pulmonary vascular permeability.
5.Measurement of NO: The reagent kit for the measurement of NO was bought from Bangding Company. Reagent 0.6ml was added into the serum or plasma 0.1ml, and they were mixed well. Double distilled water 0.4ml was added into the mixture, and then D reagent (4% sodium hydroxide) was added into the mixture. The final mixture was incubated on ice for 60 minutes. The mixture was centrifuged for 2 minutes at 12000 rpm. The double distilled water 0.4ml and reagent A 0.1ml (0.3% sulfonamide) were added into the supernatant solution 0.6ml. The mixture was incubated for 15 minutes on ice. Afterwards, reagent B 0.1ml was added, and the mixture was put in room temperature for one hour; colorimetry was performed at 545 nm wave length; and the OD value was read. Sodium nitrite was used as the standard to prepare the standard curve. According to the OD value of the specimen and the standard curve, the NO content was calculated.
6.Measurement of iNOS activity in the lung tissue: After the rat was decapitated, the left lung was taken out soon. The pre-cooled solvent 1:5 (w/v) was added, and at 4℃, a homogenate was formed in an ice bath. The homogenate was centrifuged at 20000g for 60 minutes, then the supernatant fluid was collected, that was the NOS extract. The Lowrys method was used to determine the protein concentration in the NOS extract, and 3H-arginine transformation experiment was used to determine the activity of NOS in the extract, the unit being expressed by number of pmol 3H-guanidine produced by one mg of protein in one minute.展开更多
Hepatic ischemia reperfusion injury(HIRI) is a clinical condition which may lead to cellular injury and organ dysfunction. The role of nitric oxide(NO) in HIRI is complicated and inconclusive. NO produced by endotheli...Hepatic ischemia reperfusion injury(HIRI) is a clinical condition which may lead to cellular injury and organ dysfunction. The role of nitric oxide(NO) in HIRI is complicated and inconclusive. NO produced by endothelial nitric oxide synthase(e NOS) activation plays a protective role during early HIRI. But e NOS overexpression and the resulting excessive NO bioavailability can aggravate liver injury. NO induced by inducible nitric oxide synthase(i NOS) may have either a protective or a deleterious effect during the early phase of HIRI, but it may protect the liver during late HIRI. Here, we reviewed the latest findings on the role of NO during HIRI:(1) NO exerts a protective effect against HIRI by increasing NO bioavailability, downregulating p53 gene expression, decreasing inflammatory chemokines, reducing ROS via inhibiting the mitochondrial respiratory chain, activating s GCGTP-c GMP signal pathway to reduce liver cell apoptosis, and regulating hepatic immune functions;(2) e NOS protects against HIRI by increasing NO levels, several e NOS/NO signal pathways(such as Akt-e NOS/NO, AMPK-e NOS/NO and HIF-1α-e NOS/NO) participating in the anti-HIRI process, and inhibiting over-expression of e NOS also protects against HIRI; and(3) the inhibition of i NOS prevents HIRI. Thus, the adverse effects of NO should be avoided, but its positive effect in the clinical treatment of diseases associated with HIRI should be recognized.展开更多
AIM To evaluate the protective effects of glutamine in a model of portal hypertension(PH) induced by partial portal vein ligation(PPVL).METHODS Male Wistar rats were housed in a controlled environment and were allowed...AIM To evaluate the protective effects of glutamine in a model of portal hypertension(PH) induced by partial portal vein ligation(PPVL).METHODS Male Wistar rats were housed in a controlled environment and were allowed access to food and water ad libitum. Twenty-four male Wistar rats were divided into four experimental groups:(1) control group(SO)-rats underwent exploratory laparotomy;(2) control + glutamine group(SO + G)-rats were subjected to laparotomy and were treated intraperitoneally with glutamine;(3) portal hypertension group(PPVL)-rats were subjected to PPVL; and(4) PPVL + glutamine group(PPVL + G)-rats were treated intraperitoneally with glutamine for seven days. Local injuries were determined by evaluating intestinal segments for oxidative stress using lipid peroxidation and the activities of glutathione peroxidase(GPx), endothelial nitric oxide synthase(e NOS) and inducible nitric oxide synthase(i NOS) after PPVL.RESULTS Lipid peroxidation of the membrane was increased in the animals subjected to PH(P < 0.01). However, the group that received glutamine for seven days after the PPVL procedure showed levels of lipid peroxidation similar to those of the control groups(P > 0.05). The activity of the antioxidant enzyme GTx was decreased in the gut of animals subjected to PH compared with that in the control group of animals not subjected to PH(P < 0.01). However, the group that received glutamine for seven days after the PPVL showed similar GTx activity to both the control groups not subjected to PH(P > 0.05). At least 10 random, non-overlapping images of each histological slide with 200 × magnification(44 pixel = 1 μm) were captured. The sum means of all áreas, of each group were calculated. The mean areas of e NOS staining for both of the control groups were similar. The PPVL group showed the largest area of staining for e NOS. The PPVL + G group had the second highest amount of staining, but the mean value was much lower than that of the PPVL group(P < 0.01). For i NOS, the control(SO) and control + G(SO + G) groups showed similar areas of staining. The PPVL group contained the largest area of i NOS staining, followed by the PPVL + G group; however, this area was significantly smaller than that of the group that underwent PH without glutamine(P < 0.01).CONCLUSION Treatment with glutamine prevents gut mucosal injury after PH in rats.展开更多
Multiple factors are involved in the etiology of cardiovascular disease(CVD). Pathological changes occur in a variety of cell types long before symptoms become apparent and diagnosis is made. Dysregulation of physiolo...Multiple factors are involved in the etiology of cardiovascular disease(CVD). Pathological changes occur in a variety of cell types long before symptoms become apparent and diagnosis is made. Dysregulation of physiological functions are associated with the activation of immune cells,leading to local and finally systemic inflammation that is characterized by production of high levels of reactive oxygen species(ROS). Patients suffering from inflammatory diseases often present with diminished levels of antioxidants either due to insufficient dietary intake or,and even more likely,due to increased demand in situations of overwhelming ROS production by activated immune effector cells like macrophages. Antioxidants are suggested to beneficially interfere with diseases-related oxidative stress,however the interplay of endogenous and exogenous antioxidants with the overall redox system is complex. Moreover,molecular mechanisms underlying oxidative stress in CVD are not fully elucidated. Metabolic dybalances are suggested to play a major role in disease onset and progression. Several central signalingpathways involved in the regulation of immunological,metabolic and endothelial function are regulated in a redox-sensitive manner. During cellular immune response,interferon γ-dependent pathways are activated such as tryptophan breakdown by the enzyme indoleamine 2,3-dioxygenase(IDO) in monocyte-derived macrophages,fibroblasts,endothelial and epithelial cells. Neopterin,a marker of oxidative stress and immune activation is produced by GTP-cyclohydrolase Ⅰ in macrophages and dendritic cells. Nitric oxide synthase(NOS) is induced in several cell types to generate nitric oxide(NO). NO,despite its low reactivity,is a potent antioxidant involved in the regulation of the vasomotor tone and of immunomodulatory signaling pathways. NO inhibits the expression and function of IDO. Function of NOS requires the cofactor tetrahydrobiopterin(BH4),which is produced in humans primarily by fibroblasts and endothelial cells. Highly toxic peroxynitrite(ONOO-) is formed solely in the presence of superoxide anion(O2-). Neopterin and kynurenine to tryptophan ratio(Kyn/Trp),as an estimate of IDO enzyme activity,are robust markers of immune activation in vitro and in vivo. Both these diagnostic parameters are able to predict cardiovascular and overall mortality in patients at risk. Likewise,a significant association exists between increase of neopterin concentrations and Kyn/Trp ratio values and the lowering of plasma levels of vitamin-C,-E and-B. Vitamin-B deficiency is usually accompanied by increased plasma homoycsteine. Additional determination of NO metabolites,BH4 and plasma antioxidants in patients with CVD and related clinical settings can be helpful to improve the understanding of redox-regulation in health and disease and might provide a rationale for potential antioxidant therapies in CVD.展开更多
文摘1.Animal model: 160 male Wistar rats, each 200-350g in weight, were bought from the Experimental Animal Department, Epidemic Disease Institute, China Preventive Medical Academy. After the animals were weighed, urethane 1.2g/kg of body weight was injected into the abdominal cavity for anesthesia, and then LPS 5mg/kg of body weight (LPS 055 B5 Sigma Co) was injected into the sublingual vein to establish ALI animal model. The grouping of the animal: 1) control group (NS): Normal saline (The 4th Pharmaceutics, Shijiazhuang) was injected into the sublingual vein, and 2 hours later the specimen was collected. 2) ALI group: 2 hours after injection of LPS, the specimen was collected. 3) Rhubarb group (R): Rhubarb extract 20g/kg (made in the Pharmacy of Traditional Chinese Medicine of this hospital, 1 gm crude drug/ml) was injected into the abdominal cavity; 1 hour later, LPS was injected; and 2 hours later, the specimen was collected. 4) Dexamethasone group (D): Dexamethasone 70mg/kg of body weight (made in Zhengdatianjing Pharmaceutics, Ltd. Co. Lianyun-gang) was injected into the abdominal cavity; 1 hour later, LPS was injected; and 2 hours later, the specimen was collected.
2.Histological examinations: In each group, the lung specimens were taken from 5 animals. The specimens were fixed in 10% formaldehyde, embedded in paraffin and sectioned. HE staining was used, and the sections were examined under the light microscope. The lung specimen embedded in paraffin, 1 mm3 in size, was taken to be fixed in 25% glutaraldehyde and then again fixed in 1% OsO4. Alcohol and acetone were used for dehydration. The specimens were then embedded in EPON81, and ultrathin sections were prepared. Uranium-lead double staining was used, and the ultrathin sections were examined under the Philips EM 400T transmission electron microscope.
3.Measurement of lung tissue wet weight/dry weight and the rate of cell count and protein content in the pulmonary alveolar lavage fluid: The thoracic cavity was opened, and the lung was taken out. Its wet weight was measured, then the lung was put in an oven 80℃ for 20 hours to obtain the constant weight of the lung, and finally the rate of wet/dry weight of the lung was calculated. In another group of rats, pulmonary alveolar lavage was carried after the thoracic cavity was opened. The total cell count and the cell classification were measured in the lavage fluid under the light microscope. The Lowry method was used for determination of the protein content. The lung permeability index was calculated (the protein in the pulmonary alveolar fluid/serum protein, LPI).
4.Pulmonary vascular permeability change:6 After the drug was administered in each animal group, Evans blue (50kg/kg) was injected into the femoral vein at the time when LPS was injected. After opening the thoracic cavity, the lung was taken out, its peripheral tissues were resected. The lung was immersed in the formamide solution (20 mg/100g of animal weight); the lung in the solution was incubated for 72 hours in an incubator at 45-50 ℃; after the pigment was completely extracted from the lung tissue, the tissue was taken out and solution was centrifuged and the supernatant fluid collected. Spectrophotometer 72 Model was used to make colorimetry at 620 nm. The Evans blue content was calculated from a standard curve and the value of the content was used for the determination of the change of pulmonary vascular permeability.
5.Measurement of NO: The reagent kit for the measurement of NO was bought from Bangding Company. Reagent 0.6ml was added into the serum or plasma 0.1ml, and they were mixed well. Double distilled water 0.4ml was added into the mixture, and then D reagent (4% sodium hydroxide) was added into the mixture. The final mixture was incubated on ice for 60 minutes. The mixture was centrifuged for 2 minutes at 12000 rpm. The double distilled water 0.4ml and reagent A 0.1ml (0.3% sulfonamide) were added into the supernatant solution 0.6ml. The mixture was incubated for 15 minutes on ice. Afterwards, reagent B 0.1ml was added, and the mixture was put in room temperature for one hour; colorimetry was performed at 545 nm wave length; and the OD value was read. Sodium nitrite was used as the standard to prepare the standard curve. According to the OD value of the specimen and the standard curve, the NO content was calculated.
6.Measurement of iNOS activity in the lung tissue: After the rat was decapitated, the left lung was taken out soon. The pre-cooled solvent 1:5 (w/v) was added, and at 4℃, a homogenate was formed in an ice bath. The homogenate was centrifuged at 20000g for 60 minutes, then the supernatant fluid was collected, that was the NOS extract. The Lowrys method was used to determine the protein concentration in the NOS extract, and 3H-arginine transformation experiment was used to determine the activity of NOS in the extract, the unit being expressed by number of pmol 3H-guanidine produced by one mg of protein in one minute.
基金Supported by National Natural Science Foundation of China,No.81260504,No.81660151 and No.81660751Science Foundation of Science Commission of Jiangxi Province,China,No.20161BBG70067School Teaching Reform Fund of Nanchang University,No.NCUJGLX-14-1-111
文摘Hepatic ischemia reperfusion injury(HIRI) is a clinical condition which may lead to cellular injury and organ dysfunction. The role of nitric oxide(NO) in HIRI is complicated and inconclusive. NO produced by endothelial nitric oxide synthase(e NOS) activation plays a protective role during early HIRI. But e NOS overexpression and the resulting excessive NO bioavailability can aggravate liver injury. NO induced by inducible nitric oxide synthase(i NOS) may have either a protective or a deleterious effect during the early phase of HIRI, but it may protect the liver during late HIRI. Here, we reviewed the latest findings on the role of NO during HIRI:(1) NO exerts a protective effect against HIRI by increasing NO bioavailability, downregulating p53 gene expression, decreasing inflammatory chemokines, reducing ROS via inhibiting the mitochondrial respiratory chain, activating s GCGTP-c GMP signal pathway to reduce liver cell apoptosis, and regulating hepatic immune functions;(2) e NOS protects against HIRI by increasing NO levels, several e NOS/NO signal pathways(such as Akt-e NOS/NO, AMPK-e NOS/NO and HIF-1α-e NOS/NO) participating in the anti-HIRI process, and inhibiting over-expression of e NOS also protects against HIRI; and(3) the inhibition of i NOS prevents HIRI. Thus, the adverse effects of NO should be avoided, but its positive effect in the clinical treatment of diseases associated with HIRI should be recognized.
文摘AIM To evaluate the protective effects of glutamine in a model of portal hypertension(PH) induced by partial portal vein ligation(PPVL).METHODS Male Wistar rats were housed in a controlled environment and were allowed access to food and water ad libitum. Twenty-four male Wistar rats were divided into four experimental groups:(1) control group(SO)-rats underwent exploratory laparotomy;(2) control + glutamine group(SO + G)-rats were subjected to laparotomy and were treated intraperitoneally with glutamine;(3) portal hypertension group(PPVL)-rats were subjected to PPVL; and(4) PPVL + glutamine group(PPVL + G)-rats were treated intraperitoneally with glutamine for seven days. Local injuries were determined by evaluating intestinal segments for oxidative stress using lipid peroxidation and the activities of glutathione peroxidase(GPx), endothelial nitric oxide synthase(e NOS) and inducible nitric oxide synthase(i NOS) after PPVL.RESULTS Lipid peroxidation of the membrane was increased in the animals subjected to PH(P < 0.01). However, the group that received glutamine for seven days after the PPVL procedure showed levels of lipid peroxidation similar to those of the control groups(P > 0.05). The activity of the antioxidant enzyme GTx was decreased in the gut of animals subjected to PH compared with that in the control group of animals not subjected to PH(P < 0.01). However, the group that received glutamine for seven days after the PPVL showed similar GTx activity to both the control groups not subjected to PH(P > 0.05). At least 10 random, non-overlapping images of each histological slide with 200 × magnification(44 pixel = 1 μm) were captured. The sum means of all áreas, of each group were calculated. The mean areas of e NOS staining for both of the control groups were similar. The PPVL group showed the largest area of staining for e NOS. The PPVL + G group had the second highest amount of staining, but the mean value was much lower than that of the PPVL group(P < 0.01). For i NOS, the control(SO) and control + G(SO + G) groups showed similar areas of staining. The PPVL group contained the largest area of i NOS staining, followed by the PPVL + G group; however, this area was significantly smaller than that of the group that underwent PH without glutamine(P < 0.01).CONCLUSION Treatment with glutamine prevents gut mucosal injury after PH in rats.
文摘Multiple factors are involved in the etiology of cardiovascular disease(CVD). Pathological changes occur in a variety of cell types long before symptoms become apparent and diagnosis is made. Dysregulation of physiological functions are associated with the activation of immune cells,leading to local and finally systemic inflammation that is characterized by production of high levels of reactive oxygen species(ROS). Patients suffering from inflammatory diseases often present with diminished levels of antioxidants either due to insufficient dietary intake or,and even more likely,due to increased demand in situations of overwhelming ROS production by activated immune effector cells like macrophages. Antioxidants are suggested to beneficially interfere with diseases-related oxidative stress,however the interplay of endogenous and exogenous antioxidants with the overall redox system is complex. Moreover,molecular mechanisms underlying oxidative stress in CVD are not fully elucidated. Metabolic dybalances are suggested to play a major role in disease onset and progression. Several central signalingpathways involved in the regulation of immunological,metabolic and endothelial function are regulated in a redox-sensitive manner. During cellular immune response,interferon γ-dependent pathways are activated such as tryptophan breakdown by the enzyme indoleamine 2,3-dioxygenase(IDO) in monocyte-derived macrophages,fibroblasts,endothelial and epithelial cells. Neopterin,a marker of oxidative stress and immune activation is produced by GTP-cyclohydrolase Ⅰ in macrophages and dendritic cells. Nitric oxide synthase(NOS) is induced in several cell types to generate nitric oxide(NO). NO,despite its low reactivity,is a potent antioxidant involved in the regulation of the vasomotor tone and of immunomodulatory signaling pathways. NO inhibits the expression and function of IDO. Function of NOS requires the cofactor tetrahydrobiopterin(BH4),which is produced in humans primarily by fibroblasts and endothelial cells. Highly toxic peroxynitrite(ONOO-) is formed solely in the presence of superoxide anion(O2-). Neopterin and kynurenine to tryptophan ratio(Kyn/Trp),as an estimate of IDO enzyme activity,are robust markers of immune activation in vitro and in vivo. Both these diagnostic parameters are able to predict cardiovascular and overall mortality in patients at risk. Likewise,a significant association exists between increase of neopterin concentrations and Kyn/Trp ratio values and the lowering of plasma levels of vitamin-C,-E and-B. Vitamin-B deficiency is usually accompanied by increased plasma homoycsteine. Additional determination of NO metabolites,BH4 and plasma antioxidants in patients with CVD and related clinical settings can be helpful to improve the understanding of redox-regulation in health and disease and might provide a rationale for potential antioxidant therapies in CVD.
