Expression of urotensin II and its receptor in nephridial tissues of rats with acute renal damage

AIM: To observe the mRNA expression of urotensin II (UII) and its receptor (G protein-coupled receptor 14,GPR14) in nephridial tissues of rats with acute renal damage. METHODS: Male Wistar rats were divided into 2 groups: the rats in control group (n=10) were administered with normal saline by gavage; the rats in model group (n=30) were administered with Caulis Aristolochiae manshuriensis (CAM) by gavage for 25 d to induce acute renal damage. Every 5 rats in model group were sacrificed on the 3rd, 7th, 15th and 25th days during CAM treatment and all rats in the 2 groups were killed 10 d after withdraw of CAM. The kidneys were collected for pathological observation and the UII and GPR14 mRNA examination.RESULTS: The degeneration, necrosis and disintegration in tubules were observed as major pathological changes in the rat kidneys after 3 d of CAM administration. The pathological changes were aggravated following the duration of in CAM administration, and were remained and even worsen when CAM was withdrawn for 10 d. Compared with control group, the mRNA expression of UII was significantly elevated (P<0.05) at the time point of CAM administration for 15 d,even obviously increased (P<0.01) at the time point of CAM administration for 25 d, and remained at the highest levels to the end of the observation. The mRNA expression of GPR14 was significantly increased (P<0.05) at the time point of CAM administration for 7 d, became higher (P<0.01) on the 15th day, and gradually increased as the experimental time went on. CONCLUSION: The mRNA levels of UII and its receptor are significantly elevated in CAM-induced renal lesion in rats, suggesting that UII plays a pathological role in the development of acute renal damage.     

Effect of urantide on liver function and histomorphology in atherosclerotic rats

AIM:To investigate the effect of urantide on the liver function and histomorphology in the rats with atherosclerosis (AS).METHODS:The AS Wistar rat model was induced by intraperitoneal injection of vitamin D₃ (VD₃) and feeding with high-fat diet. The rats were randomly divided into normal control group, AS model group, positive medicine group and urantide group. The liver function indexes of the rats were measured by biochemical test, and the pathological changes of the aorta and liver of the rats were observed by hematoxylin-eosin (HE) staining. The mRNA expression of urotensin Ⅱ (UⅡ) and GPR14 at mRNA and protein levels in rat livers was determined by RT-qPCR and Western blot. RESULTS:The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (γ-GT), lactate dehydrogenase (LDH), total bilirubin (TBIL), indirect bilirubin (IBIL) and alkaline phosphatase (ALP) in AS model group were significantly increased compared with normal control group (P<0.05). The above indexes in urantide group were remarkably decreased compared with AS model group (P<0.05). No change of the levels of direct bilirubin (DBIL), total protein (TP), globulin (GLB) and albumin (ALB) in each group was observed. Urantide postponed hepatocyte fatty degeneration and repaired hepatocyte injury in the AS rats. Compared with normal control group, the mRNA and protein levels of UⅡ and GPR14 in the liver were significantly increased in AS model group (P<0.05). With the prolongation of dosing time, the mRNA and protein levels of UⅡ and GPR14 in the liver were significantly decreased in urantide group compared with AS model group (P<0.05). CONCLUSION:Urantide significantly attenuates the liver damage caused by liver fatty degeneration in AS rats.     

Hypoxic tubulointerstitial fibrosis and swimming exercise change expression of urotensin II and its receptor GPR14 in rats

AIM: To observe the expression of urotensin II (UII) and its receptor GPR14 in rats with hypoxic tubulointerstitial fibrosis, and to explore the changes after swimming exercise. METHODS: The animal model of hypoxic renal interstitial fibrosis was established by exposing the rats to isobaric hypoxic chamber for 7 weeks (8 h/d, 7 d/week). Forty-five male SD rats were randomly divided into normal control group (control), hypoxic 7-week group (hypoxia) and hypoxic 3-week and swimming without loads 4-week group (swimming). Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by chemical colorimetry, and UII was detected by ELISA. The content of hydroxyproline (Hyp) in the renal homogenate was assayed. The mRNA expression of UII and GPR14 were detected by RT-PCR. The protein level of UII was determined by the method of immunohistochemistry. Meanwhile, the renal specimens were prepared to observe renal interstitial fibrosis by van Gieson(VG) staining. RESULTS: The content of Scr and BUN in hypoxia group was lower than that in control group by 18.5% and 14.1%,respectively, while there was no significant difference between swimming group and hypoxia group. The content of Hyp in hypoxia group was 42.9% higher than that in control group, while swimming group was 26.1% lower than that in hypoxia group. The plasma content of UII in hypoxia group was 380.8% higher than that in control group, while swimming group was 42.6% lower than that in hypoxia group. The mRNA expression of UII in the kidneys was obviously up-regulated by 104.5% in hypoxia group compared with control group, while it was markedly down-regulated by 33.2% in swimming group compared with hypoxia group. The mRNA expression of GPR14 in the kidneys was significantly up-regulated by 35.4% in hypoxia group compared with control group. However, no significant difference between hypoxia group and swimming group was observed. The UII protein level in the kidneys of hypoxia rats was distinctly higher than that in control group and lower in swimming group than that in hypoxia group. VG staining revealed that the renal interstitial fibrosis was found in hypoxia group, which was significantly alleviated by swimming exercise. CONCLUSION: The levels of UII and GPR14 increase in the kidneys of the rats with hypoxic tubulointerstitial fibrosis. Moderate swimming exercise alleviates the tubulointerstitial fibrosis induced by hypoxia and decreases the expression of UII.     

Effect of renal transporter Glut9 on hyperuricemia in rats induced by fructose

AIM: To explore the effect of renal transporter glucose transporter 9 (Glu9) on hyperuricemia in the rats induced by fructose.METHODS: SD male rats (n=30) were randomly divided into normal group, model group and benzbromarone group, according to the weight. The rats in normal group was given water, while the rats in model group and benzbromarone group were given 10% fructose solution to establish hyperuricemia model. At the same time, the rats in normal group and model group were given a gavage of distilled water, while the rats in benzbromarone group were given benzbromarone at the dose of 20 mg/kg. The rats were sacrificed on the 40th day. The serum uric acid (SUA) and urinary uric acid (UUA) were detected to calculate the clearance rate of uric acid (CUA) in the kidney. The activity of hepatic xanthine oxidase (XOD) was also measured. The expression of renal Glut9 at mRNA and protein levels was determined by RT-qPCR and immunohistochemical staining. RESULTS: From the 20th day to the 40th day, the SUA in model group was significantly higher than that in normal group, but the UUA and CUA had no difference. On the 20th day, the SUA in benzbromarone group was markedly decreased as compared with model group, but UUA and CUA had no significant difference. On the 40th day, the hepatic XOD activity in model group was significantly elevated, and no difference of XOD between model group and the benzbromarone group was observed. Compared with normal group, the protein expression of Glut9 in the renal tissues of model group were markedly increased, and that in benzbromarone group was significantly lower than that in model group. However, no difference of the Glut9 mRNA expression was observed among groups. CONCLUSION: Fructose drinking induces hyperuricemia in rats, which is probably related to the up-regulation of renal Glut9 expression at protein level, and the increase in the reabsorption of uric acid in the kidneys.     

Mechanisms of hyperglycemia induced by immunosuppressant FK506

AIM：To investigate the effect of immunosuppressant FK506 on serum glucose in rats and to explore its mechanism. METHODS：Sprague-Dawley rats (n=12) were randomly divided into drug group and normal group. The rats in drug group were intraperitoneally injected with FK506 at dose of 1 mg·kg-1·d-1 and the rats in normal group received saline (1 mL·kg-1·d-1, ip) for 14 d. The fasting weight and fasting glucose were regularly measured every 2 d. Visceral fat was isolated from the rats at the end of experiment. The mRNA expression of adiponectin, leptin, visfatin, resistin, retinol-binding protein 4 (RBP4) and peroxisome proliferator-activated receptors γ (PPAR-γ) was determined by real-time fluorescence quantitative PCR. The protein expression of PPAR-γ and adiponectin was measured by Western blotting. RESULTS：Compared with normal group, the concentration of fasting blood glucose in model group was significantly increased from the 10th day (P<0.05). At day 14, the fasting blood glucose of the model group increased from (5.10±062) mmol/L to (7.73 ± 0.73) mmol/L. No significant change of blood glucose in normal group between the 10th day and the 14th day ［from (4.66 ± 0.32) mmol/L to (5.80±0.10) mmol/L］ was observed. Compared with normal group, the mRNA expression of PPAR-γ, adiponectin and leptin in the adipose tissue of model group was significantly decreased (P<001), whereas the expression of visfatin, resistin and RBP4 was significantly increased (P<005). Compared with normal group, the expression of PPAR-γ and adiponectin in model group was decreased (P<001). CONCLUSION：FK506 may decrease the expression of PPAR-γ to change the expression of adipocytokines and induce hyperglycemia in rats.     

Antidepressant-like effect of N-palmitoylethanolamide by prefrontal cortex PPARα pathway in a rat model

AIM: To investigate the role of cortical peroxisome proliferator-activated receptor α (PPARα) in the regulation of depression-like behavior in the rats by N-palmitoylethanolamide (PEA). METHODS: A rat model of chronic unpredictable mild stress (CUMS) was established. The rats (n=70) were randomly divided into normal control group, CUMS model group, CUMS+ fluoxetine (10 mg/kg) group, CUMS+ PEA (2.5, 5 and 10 mg/kg) groups and CUMS+ PEA (10 mg/kg)+ MK886 (3 mg/kg) group. On the 8th day during CUMS, the drugs were continuously admi-nistered for 28 d. The body weight and the related behavioral changes in the open-field test and sucrose consumption test were monitored every week. On the 36th day, some of the brain tissues from the rats were fixed in 4% formalin solution for histomorphological and immunohistochemical observations to determine the number and morphological changes of prefrontal cortex (PFC) neurons and the protein expression of synaptophysin (SYP). Other brain tissues were quickly removed, PFC was separated and weighed, and Western blot and RT-PCR were used to detect the expression of PPARα at protein and mRNA levels in the PFC of rats. RESULTS: Compared with CUMS model group, PEA increased the body weight gain, the sucrose preference rate, and the locomotion time and distance in the open-field test, and shortened the immobility time in the open-field test. PEA increased the weight of PFC, the percentage of PFC/brain weight and the number of neurons in PFC, and improved the morphological changs of the neurons. PEA also up-regulated the protein expression of SYP in PFC, and down-regulated the expression of PPARα at mRNA and protein levels in the PFC of CUMS model rats (P<0.05). In addition, compared with PEA (10 mg/kg) group, MK886 significantly reduced the body weight gain of the rats, the percentage of sucrose preference and the locomotion distance in the open-field test, and increased the immobility time in the open-field test on the 35th day during CUMS. The number of neurons SYP expression in PFC tissues were decreased, and the expression of PPARα at protein and mRNA levels was increased in MK886 group. CONCLUSION: PEA may antagonize the depression-like behavior of rats by regulating the PPARα pathway in PFC, improving synaptic plasticity of PFC and protecting the neurons.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Effects of orexin-A on food intake and spontaneous physical activity in obesity-resistant rats and high-fat diet-induced obese rats

AIM: To investigate the differences of food intake and spontaneous physical activity (SPA) among obesity-resistant (OR) rats, diet-induced obese (DIO) rats and normal Sprague-Dawley (SD) rats and the role of orexin-A in these processes. METHODS: The rostral lateral hypothalamic area (rLHa) catheter was implanted into the OIO, OR, and SD rats. Orexin A at doses of 0, 31.25, 62.5, 125 and 250 pmol was injected through the catheter. The SPA and food intake were measured and recorded for 2 h after injection. The mRNA expression of prepro-orexin, orexin-A receptor (OX1R) and orexin-B receptor (OX2R) in the rLHa and hypothalamus of OR, DIO and SD rats was detected by real-time PCR. The protein expression of OX1R and OX2R in the hypothalamus and rLHa of the rats was measured by radioimmunoassay. RESULTS: A small-dose injection of orexin-A into rLHa significantly increased the food intake in all the rats. Orexin A-induced SPA had significant differences, showing that the OR and SD rats had the higher motion than the DIO rats. The mRNA and protein levels of OX1R and OX2R in the rLHa of OR rats were significantly higher than those in DIO and SD rats. CONCLUSION: Hypothalamic orexin-A participates in the regulation of energy metabolism in obese and normal rats, in which the regulatory effect on OR rats is the best.     

Effects of astragalan on neurotransmitters and expression of c-fos mRNA in hippocampus after ischemic brain injury in rats

AIM: To explore the effects of astragalan (AG) on the neurotransmitters,acetylcholine (ACh), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), and the expression of c-fos mRNA in hippocampus after ischemic brain injury in rats. METHODS: Male Wistar rats (180~220 g) were randomly divided into 10 groups (n=10): sham-operated group (SOG), 3 model groups (MG 1 d, 3 d, 7 d) and 3 low- or high-dose AG treatment groups (L/H-AGTG 1 d, 3 d, 7 d), respectively. The middle cerebral artery of the rats in MG group and AGTG group were blocked by operation to induced brain injury. The cerebral blood vessels of the animals were blocked on day 1, day 2 and day 7, respectively, after the L/H-AGTG were treated with AG (5 mg/kg and 15 mg/kg, ip). The content of ACh,5-HT and NE was determined using their respective ELISA kits, and the expression of c-fos mRNA in the hippocampus homogenate was semiquantitative analyzed by RT-PCR after neurologic impairment (NIP) was scored. RESULTS: AG attenuated the injury in hippocampus by cerebral ischemia in a dose-dependent manner. The content of ACh, 5-HT and NE in L-AGTG 7 d,H-AGTG 3 d and 7 d groups was significantly higher than that in MG group, but was lower in SOG group (P<0.05 or P<0.01). The mRNA expression of c-fos in SOG group was lower than that in MG group (P<0.05 or P<0.01), indicating that reinforcement expression of c-fos mRNA by cerebral ischemia and the expression of downstream genes may be beneficial for protecting the neurons. The mRNA expression of c-fos in H-AGTG 3 d/7 d groups was higher than that in MG group (P<0.05). CONCLUSION: AG attenuates the damage of neurons and improves the functions of hippocampus under the condition of cerebral ischemia/reperfusion by increasing the content of ACh, NA and 5-HT, and the mRNA expression of c-fos in hippocampus.     

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases during wound healing in diabetic rats

AIM: To investigate the imbalance between the expression of metalloproteinases (MMPs) and that of tissue inhibitors of metalloproteinase (TIMPs) during wound healing in diabetic rats. METHODS: Diabetic rats were induced with streptozotocin. All rats were maintained for 6 weeks. A full-thickness excisional wound was created on the back of each rat. Every group was randomly divided into 3 subgroups of 7 rats: 3 d group, 7 d group, 14 d group and animals were killed at 3rd, 7th and 14th day. Routine pathological examination, Masson′s trichrome staining and immunohistochemistry were made to calculate the score of epidermal and dermal regeneration, granulation tissue thickness, angiogenesis, matrix density, and infiltrated cells at different time points. RT-PCR and Western blotting were used to detect the expression of mRNA and protein of MMP-9 and TIMP-1 in the skin at those time points. RESULTS: Six weeks after streptozotocin treatment, Three days after injury, the wound healing rate of normal rats was faster than that of diabetic rats. From 3rd to 14th day, there were a lot of fibroblast and macrophage in normal skin, while few such cells were observed in diabetic skin. The other histological scores in normal skin were higher than those in diabetic rats at 7th and 14th day. Both MMP-9 and TIMP-1 had minimally detectable levels before wounding but exhibited rapid, significantly large increases within 3 d after wounding. Subsequently, they showed a rapid decline by 14 d. The relative values of expression of MMP-9 mRNA and protein in diabetic group were higher than those in normal group at different time points. However, the values of TIMP-1 mRNA and protein in diabetic group were significantly lower than those in control group. Significant difference was observed between two groups with the ratio of MMP-9/TIMP-1, higher in diabetic group than that in normal group. CONCLUSION: Abnormal reepithelialization, angiogenesis, inflammatory cell infiltration, collagen fibers generation, granulation tissue deposition, seem to be the basic histopathology that delays wound healing. The imbalance between MMPs and TIMPs in diabetic skin tissue before and after injury may be one of the important reasons of these alterations of histopathology.     

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

AIM: To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae (MP)-infected rat lung tissues and to explore its mechanism. METHODS: SD rats (n=40) were randomly divided into 4 groups:blank control group, model group, Jiedu-Qingfei group and positive control group, with 10 rats in each group. The rats in experimental groups were slowly dripped with 1×10⁹ CFU/L MP solution into their nostrils for 4 d. One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation, and the other rats were given gavage therapy. The rats in blank control group and model group were intragastrically given the same volume of normal saline, the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks, and the rats in psoitive control group were given dexmethasone sodium phosphate (0.5 mg·kg^-1·d^-1). After the experiment, the rats were killed. The serum and bronchoalveolar lavage fluid (BALF) were collected for detecting the levels of interleukin-12 (IL-12), IL-13 and TNF-α by ELISA. The right lung tissues were used for pathological observation and HE staining, while the left lung tissues were used to detect the expression of NF-κB p50, I-κBα and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels. RESULTS: The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive, indicating that the rat model of MP infection was successfully established. On the 1st day of the treatment, the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group (P<0.05). After treatment, the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group. The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection (P<0.05), while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group (P<0.05). The levels of IL-13 and TNF-α in the serum and BALF of MP-infected rats were increased significantly, while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group (P<0.05). The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly (P<0.01). After treatment, the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group (P<0.01). The mRNA expression level of I-κBα in model group was significantly lower than that in control group. After treatment, the mRNA expression of I-κBα in Jiedu-Qingfei group was significantly higher than that in model group (P<0.05). The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group. After treatment, the protein expression of NF-κB p50 and p38 MAPK was decreased significantly. The protein level of I-κBα in model group was significantly lower than that in blank control group, and after treatment with Jiedu-Qingfei mixture, the protein expression level of I-κBα was increased significantly (P<0.05). CONCLUSION: Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.     

Roles of nuclear factor-κB in the development of rat pancreatic fibrosis mediated by angiotensin II

AIM: To determine the effects of NF-κB on the development of rat pancreatic fibrosis mediated by angiotensin II. METHODS: Spraque-Dawley rats (200-300g) were randomly divided into normal group, control group and losartan-treatment group. Pancreatic fibrosis was induced by injection of 2% TNBS into biliopancreatic duct. Rats in losartan-treatment group and control group were respectively treated with losartan (10 mg·kg^-1·d^-1) by gavage and the same volume of saline vehicle. The expression, distribution, and activation of NF-κB were studied by Western blot, immunohistochemistry and TransAM^TM. Toluidine blue staining and transmission electron microscopy were also used to observe the number, distribution and degranulation of mast cells. In addition, RT-PCR was performed to detect the intrapancreatic ICAM-1 mRNA expression. RESULTS: The expression and activity of intrapancreatic NF-κB p65 protein were significantly increased on day 3 after operation, reaching peak on day 7 [(0.406±0.086)mg/g total protein].. Mast cell activation was observed and ICAM-1 mRNA levels on day 3 and 7 were up-regulated in control group. Losartan treatment inhibited NF-κB expression and activation, reduced mast cell infiltration and degranulation and decreased ICAM-1 mRNA expression compared with control rats. CONCLUSION: It might be associated with the expression and activation of NF-κB that angiotensin II mediates inflammation and fibrosis in the early stage of pancreatic fibrosis.     

Effects of pirenzepine on expression of MMP-2 and TIMP-2 in stroma of sclera in chick myopic eyes

AIM:To observe the effect of intravitreal injection of pirenzepine on form-deprivation myopia in chicks. METHODS:Forty-one day old chicks were randomly divided into 4 groups:normal control group, form deprivation group, vehicle control group and pirenzepine injection group. The right eyes of all chicks were used as experimental eyes. The deprived eyes in vehicle control group and pirenzepine injection group received daily intravitreal injection of vehicle control solution(0.01 mol/L PBS) and pirenzepine(1%in PBS), respectively. Optical examinations such as refraction, axial length and equatorial diameter were made at the end of the 5th day. The mRNA and protein levels of matrix metalloproteinase 2(MMP-2) and tissue inhibitor of metalloproteinase-2(TIMP-2), and the activity of MMP-2 were detected by RT-PCR, Western blotting and zymography analysis,respectively. RESULTS:Refraction, axial length and equatorial diameter of the eyes in pirenzepine injection group were significantly lower than those in form deprivation group and vehicle control group(P<0.05), but those were higher(P<0.05) and the eyes were relatively myopic as compared with normal control group. The mRNA expression, protein le vels and activity of MMP-2 in pirenzepine group were significantly higher than those in normal control group(P<0.01), and were significantly lower than those in form deprivation group and vehicle control group(P<0.01, P<0.01). The mRNA and protein levels of TIMP-2 in pirenzepine group were significantly lower than those in normal control group(P<0.01), and was significantly higher than those in form deprivation group and vehicle control group(P<0.01, P<0.01). CONCLUSION:Intravitreal injection of pirenzepine may partly prevent form-deprivation myopia by modulating the expression of MMP-2 and TIMP-2 in the fibrous layer of sclera.     

Astragalus injection inhibits expression of Apaf-1 in rat hippocampus after cerebral ischemia and reperfusion

AIM: To investigate the effect of Astragalus injection on the expression of apoptotic protease-activating factor 1 (Apaf-1) in the hippocampus of global cerebral ische-mia-reperfusion rats. METHODS: Male SD rats were randomly divided into 4 groups with 30 each: sham operation group, cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group, and cerebral ischemia-reperfusion+vehicle group. The global cerebral ischemia-reperfusion model of the rats was established by 4-vessel occlusion. The rats in cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group and cerebral ischemia-reperfusion+vehicle group were further divided into 7 subsets, according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. After reperfusion, the brains were removed at the corresponding time points. The protein expression of Apaf-1 in hippocampal neurons was detected by immunohistochemistry and Western blotting. The mRNA expression of Apaf-1 was observed by RT-PCR. RESULTS: Compared with sham operation group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h increased obviously in cerebral ischemia-reperfusion group (P<0.05). Compared with cerebral ischemia-reperfusion group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h decreased obviously in cerebral ischemia-reperfusion+Astragalus injection group (P<0.05). However, those in cerebral ischemia-reperfusion+vehicle group had no obvious change (P>0.05). CONCLUSION: Astragalus injection inhibits the expression of Apaf-1 at mRNA and protein levels in hippocampus of global cerebral ischemia-reperfusion rats, thus inhibiting the apoptosis of hippocampal neurons.     

Influence of Nao Xue Kang tablet on dynamic expression of PAR-1 after acute intracerebral hemorrhage in rats

AIM: To study the dynamic expression of protease-activated receptors-1 (PAR-1) after intracerebral hemorrhage (ICH) and the influence of Nao Xue Kang Tablet (NXKT) on it's expression. METHODS: 72 Wistar rats were divided into normal group, ICH model groups (ICH, 6 h, 24 h, 3 d, 7 d), Nao Xue Kang groups (NXKT, 6 h, 24 h, 3 d,7 d). ICH models were produced with the induction of collagenase typeⅦ-S, except normal group. Immunohistochemical method was used to detect PAR-1 protein and RT-PCR technique was used to detect PAR-1 mRNA in brain tissues around the haematoma at different time points of different groups. RESULTS: PAR-1 protein and mRNA were mildly positive in normal group. In ICH model groups, intensity of PAR-1 expression started to increase at 6 h, and further increased at 24 h. PAR-1 expression reached the peak at 3 d and began to descend. At 7 d the decent was obvious. At 6 h, 24 h, 3 d, and 7 d time points, the PAR-1 protein positive cell number and PAR-1 mRNA absorbance ratio in ICH model and NXKT groups were significantly higher than those in normal group (P<0.05 or P<0.01). The PAR-1 protein positive cell number and PAR-1 mRNA absorbance ratio in NXKT group were significantly lower than in ICH model group (P<0.05 or P<0.01). CONCLUSION: After ICH, PAR-1 is continuously activated because of the stimulation of thrombin. Action of thrombin after ICH may be mediated by PAR-1; NXKT may inhibit the activation of PAR-1, so the praxiology is improved. This may be one of the main mechanisms that NXKT could facilitate the recovery of nervous function.     

Effects of imidapril on blood gas,oxidative stress and inflammatory factors in rats with acute lung injury induced by ammonium chloride

AIM: In this study, the rat lung injury model was induced by ammonium chloride for studying the effect of imidapril on blood gas, serum TNF-α, IL-6 and MDA concentrations, and AngⅡ and CD54 protein expression in rat lung tissue. METHODS: Male rats were randomly divided into 3 groups: control group, lung injury model group and drug group. The rats in control group were given saline (2 mL/kg), while the rats in lung injury model group were given 6% ammonium chloride (2 mL/kg). In drug group, imidapril (3 mg·kg^-1·d^-1) was given to the rats once daily for 1 week by intragastric gavage after given 6% ammonium chloride. On the 7th day, the rats were anesthetized with 2% so-dium pentobarbital. Abdominal aorta blood, venous blood and lung tissue were collected. The blood gas indexes and serum TNF-α, IL-6 and MDA concentrations were determined. The lung tissues were fixed and sliced, and the expression of AngⅡ and CD54 proteins was detected by immunohistochemistry. RESULTS: The PaCO₂ increased in lung injury model group compared with control group and drug group (P < 0.05).The expression of AngⅡ and CD54, and the concentrations of TNF-α, IL-6 and MDA also increased significantly (P < 0.01) in model group. Pulmonary edema, inflammation, alveolus congestion, hemorrhage and hyperplasia in model group were obvious compared with control group and drug group. CONCLUSION: Imidapril improves blood gas indexes, and reduces lipid peroxidation and inflammatory responses in the rats with lung injury induced by ammonium chloride.     

Effect of traditional Chinese medicine Muniziqi on gonad axis in rats with chronic stress premature ovarian failure

AIM: To investigate the effects of traditional Chinese medicine Muniziqi on chronic premature ovarian failure (POF) by observing the histomorphological changes of pituitary, hypothalamus and ovary in rats with chronic POF. METHODS: Mature Sprague-Dawley rats (female, n=90) were randomly divided into normal group (n=10), and stress model group (n=80). After the model was established, the rats with POF were screened. The model rats were divided into POF group, and POF with high-, medium- and low-dose Muniziqi groups. HE staining and Masson staining were used. The morphological changes of pituitary, hypothalamic and ovarian tissues were observed, and the ovarian and uterus indexes were calculated. The serum levels of estradiol (E₂), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the rats were measured by ELISA. RESULTS: The traditional Chinese medicine Muniziqi had certain effects on the morphological changes and hormone levels of pituitary, hypothalamic and ovarian tissues in the rats with chronic POF. Compared with POF group, each drug intervention group had different degrees of improvement. Compared with normal group, the E₂ level in POF group was significantly decreased (P<0.05), and the levels of LH and FSH were significantly increased (P<0.05). CONCLUSION: Chronic stress results in the occurrence of POF. The traditional Chinese medicine Muniqizi has the effects on prevention and treatment of POF and improvement of histomorphological changes and hormone levels of the gonaol axis.     

Analgesic effect of angiotensin angiotensin Ⅱ type 2 receptor antagonist EMA401 on neuropathic pain in rats and its mechanism

AIM: To explore whether angiotensin Ⅱ type 2 receptor antagonist EMA401 decreases neuropathic pain and the expression of growth-associated protein-43 (GAP-43), protein kinase C (PKC) and calmodulin (CaM) in dorsal root ganglia (DRG) during chronic constriction injury (CCI) in rats. METHODS: SD rats were used to establish CCI model and randomly divided into 4 groups. The rats in model group were given equal volume of normal saline by intragastric administration. The rats in low dose (LD) group were given 5 mg/kg EMA401 by intragastric administration. The rats in middle dose (MD) group were given 10 mg/kg EMA401 by intragastric administration. The rats high dose (HD) group were given 20 mg/kg EMA401 by intragastric administration. The rats in sham operation group received equal volume of normal saline by intragastric administration. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured before operation and 7 d, 14 d and 28 d after CCI. After behavioral test, DRG of lumbar spinal was obtained from each group, and was used to determine Ca²⁺ concentration by o-cresolphthalein complexone microplating method, and the expression of GAP-43, PKC and CaM at mRNA and protein levels by Western blotting and RT-PCR. RESULTS: Compared with model group, EMA401 significantly increased the TWL and MWT (P<0.05). Meanwhile, EMA401 significantly reduced Ca²⁺ concentration and the expression of GAP-43, PKC and CaM at mRNA and protein levels in the DRG (P<0.05). CONCLUSION: EMA401 may attenuate neuropathic pain of CCI by inhibiting Ca²⁺ concentration and the expression of GAP-43, PKC and CaM.