Preconditioning with 3-nitropropionic acid induces rat cerebral ischemic tolerance and increases expression of glucose transporter 1 and glucose transporter 3

AIM: To explore the mechanism of 3-nitropropionic acid (3-NPA) preconditioning that induces cerebral ischemic tolerance in rats by affecting the expression of brain-type glucose transporters (GLUT1 and GLUT3) at mRNA and protein levels in cerebral tissues.METHODS: The male SD rats were used in the experiments and divided randomly into sham operation group (sham group, n=4), control group of 3-NPA preconditioning (3-NPA group, n=4), cerebral ischemia group (M group, n=16) and 3-NPA preconditioning group (IPC group, n=16). M group and IPC group were further divided into 4 subgroups according to the different reperfusion time(4 h, 12 h, 24 h and 48 h). All rats were killed at the corresponding time points. The cerebral tissues in the ischemic side (left) and coronal intermediate 1/3 of cortex were collected. The protein levels and mRNA expression of GLUT1 and GLUT3 were determined by Western blotting and RT-PCR. RESULTS: Compared with M group, the ischemic reperfusion and 3-NPA preconditioning induced the upregulation of GLUT1 and GLUT3 at protein levels with significant differences (F=5.848, P<0.05 and F=6.295, P<0.05, respectively), especially after ischemia-reperfusion for 48 h. The mRNA expression of GLUT1 in IPC group began to increase at 4 h, peaked at 48 h after reperfusion, with significant difference as compared to M group at the corresponding reperfusion time points in each group or sham group. In contrast, the mRNA expression of GLUT3 in IPC group increased at 24 h, and was the highest at 48 h as compared to cerebral ischemia group at the corresponding reperfusion time points or sham group.CONCLUSION: 3-NPA preconditioning increases the expression of GLUT1 and GLUT3 at protein and mRNA levels to maintain the energy supply in brain tissues, indicating a cerebral protective mechanism.     

Effects of EGCG on GLUT2/G6PD/GS expression and blood glucose level in type 2 diabetic rats

AIM To investigate whether epigallocatechin gallate （EGCG） improves blood glucose in type 2 diabetic rats through glucose transporter 2 （GLUT2）-glucose-6-phosphate dehydrogenase （G6PD）-glycogen synthase （GS） pathway. METHODS Type 2 diabetes mellitus （T2DM） model was established in male Sprague-Dawley （SD） rats by feeding with high-fat diet and injection of streptozotocin （STZ）. The rats were divided into 5 groups （n=10）： control （Con） group, T2DM model （M） group, metformin （Met; 200 mg/kg, ig） group, T2DM+low-dose （50 mg/kg, ig） EGCG （EL） group, and T2DM+high-dose （100 mg/kg, ig） EGCG （EH） group. Diabetic rats were given drugs for 8 weeks. After 8 weeks of administration, the rats were killed, and the blood and liver tissues were collected. The levels of fasting blood glucose （FBG）, fasting serum insulin （FINS） and serum glycosylated hemoglobin were measured by biochemical tests. Liver glycogen were test by periodic acid-Schiff （PAS） staining. The mRNA expression of G6PD in the liver was detected by real-time PCR. The protein levels of GS and GLUT2 were determined by Western blot and immunohistochemistry. RESULTS T2DM rat model was established successfully. Compared with Con group, the levels of FBG, FINS and serum glycosylated hemoglobin in M group were increased significantly （P<0.05）, while the insulin sensitivity index （ISI）, the liver glycogen, the G6PD mRNA expression, and the protein levels of GS and GLUT2 were decreased significantly （P<0.05）. Compared with M group, the levels of FBG and serum glycosylated hemoglobin in Met group and EH group were decreased significantly （P<0.05）, while the ISI, the liver glycogen, the G6PD mRNA expression, and the protein levels of GS and GLUT2 were increased significantly （P<0.05）. CONCLUSION EGCG reduces the blood glucose level in T2DM rats, which may be related to the regulation of GLUT2-G6PD-GS signaling pathway.     

Effect of acupuncture on expression of EGF and bFGF in brain tissues of rats with traumatic brain injury

AIM: To observe the effect of acupuncture on the expression of epidermal growth factor (EGF) and basic fibrolblast growth factor (bFGF) in the brain tissues of rats with traumatic brain injury. METHODS: Thirty SD rats were randomized into sham-operated group, model group and acupuncture group. The model of traumatic brain injury was established by free drop impact. Acupuncture was performed to the rats in acupuncture group once every day and 7 days altogether. Brain histotomy was conducted after the treatment. Immunohistochemical method was adopted to test the protein expression of EGF and bFGF. RESULTS: Compared to sham-operated group, the expression of EGF in the brain tissues of model group decreased (P<0.01), and the expression of bFGF increased (P<0.01). Compared to model group, the expression of EGF and bFGF in acupuncture group increased obviously (P<0.01). CONCLUSION: Acupuncture significantly increases the expression of EGF and bFGF, and improves the repair of injured brain tissues. This might be one of the mechanisms by which acupuncture can treat traumatic brain injury and improve the nervous function.     

Prevention and treatment of restenosis after PTA operation in rabbit artery by 3,3-diindolylmethane

AIM: To evaluate the safety and efficacy of 3,3-diindolylmethane (DIM) on neointimal proliferation of rabbit artery after balloon angioplasty. METHODS: Restenosis models of carotid artery after balloon injury was established in rabbits. 30 rabbits were randomly divided into 4 groups: sham-operated group, model group, low-dose DIM group and high-dose DIM group. DIM was given to the rabbits in low-dose treatment group (2 mg·kg-1·d-1) and high-dose treatment group (8 mg·kg-1·d-1) once a day from 3 d before operation to 4 weeks after operation. The two treatment groups were administered with intraperitoneal injection of emulsified DIM, while the other two groups with saline at the same volume. All rabbits were killed after 28 d and carotid arteries were removed. With HE staining, automatic image analysis and immunohistochemistry, artery morphology was observed and the thickness of arterial intima and media was measured. Platelet derived growth factor (PDGF) and transfer growth factor β1 (TGF-β1) were examined. The expression of 〖STBX〗bcl-2〖STBZ〗 gene was detected by in situ hybridization (ISH) with computer-assisted picture analysis system. RESULTS: No significant difference was found between low-dose DIM group and model group in intimal thickness, media thickness, luminal area and the expression of PDGF and TGF-β1 in low-dose DIM group (P>0.05). The intimal thickness was decreased in high-dose DIM group compared with model group and low-dose DIM group (P<0.01). The luminal area was significantly larger in high-dose DIM group than that in model group and low-dose DIM group (P<0.01). The expression of PDGF and TGF-β1 in low-dose DIM group was significantly reduced compared with model group and low-dose DIM group (P<0.01). CONCLUSION: The inhibition and treatment of DIM on arterial restenosis are safe and effective. The effect might be achieved by preventing neointimal proliferation and the expression of PDGF and TGF, enhancing apoptosis in the vascular smooth muscle cells.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.     

Effects of bFGF on brain edema,nerve function injury and autophagy related protein in rats with traumatic brain injury

AIM:To study the effects of basic fibroblast growth factor (bFGF) on brain edema, nerve function damage and autophagy related proteins in rats with head injury. METHODS:The rat model of craniocerebral injury (CI) was constructed. The rats were divided into control group, CI group, and low-, middle-and high-dose bFGF groups (n=10). The CI model was established in CI group, while the rats in control group were not given epidural impact. The rats in low-dose, middle-dose and high-dose bFGF groups were given bFGF at 2, 4 and 6 μg, respectively, by intraperitoneal injection after 30 min. The neurological function in the rats was evaluated by improved neurological function scoring. The rat brain tissues were taken, and the water content was detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in the brain tissue were measured by ELISA. The malondialdehyde (MDA) content was analyzed by thiobarbituric acid method. The activity of superoxide dismutase (SOD) was examined by WST-8 assay. The glutathine peroxidase (GSH-Px) activity was detected by colorimetric method. The protein levels of autophagy related proteins LC3-Ⅱ and beclin-1 in the brain tissues were determined by Western blot. RESULTS:The neurological function score was increased significantly of the rats in CI group. The rat model of craniocerebral injury was successfully constructed. Neurological function scores in the rats in low-dose, middle-dose and high-dose bFGF groups were reduced, the water content of the brain tissue was also reduced (P<0.05). The levels of TNF-α, IL-6 and IL-1 β were decreased in the brain tissues (P<0.05), the content of MDA was declined (P<0.05), the activities of SOD and GSH-Px were increased (P<0.05), the protein levels of LC3-Ⅱ and beclin-1 were decreased, compared with the untreated rats in CI group (P<0.05). CONCLUSION:bFGF improves the nerve function of the rats with craniocerebral injury, reduces the water content of the brain tissue, reduces the expression of autophagic protein LC3-Ⅱ and beclin-1.The mechanism is related to the inhibition of inflammatory reaction and oxidative damage.     

Effect of cimicifugoside H-1 on amino acid neurotransmitters in striatum of rats with cerebral ischemia

AIM: To study the neuroprotective effect of cimicifugoside H-1 and to explore the mechanism involved by determining the variation of amino acid neurotransmitters in extracellular fluid in the striatum of rats with cerebral ischemia. METHODS: The rats were randomly divided into sham-operated, cerebral ischemia, high-, middle- and low-dose cimicifugoside H-1, and ginkgo groups. Focal cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) with sutures. Normal saline was intraperitoneally injected into the rats in sham-operated group and cerebral ischemia group, while ginkgo and different doses of cimicifugoside H-1 were injected into the rats in ginkgo group and high-, middle- and low-dose cimicifugoside H-1 groups, respectively, once a day for 7 d. The striatal fluids were gained in vivo by brain microdialysis. The contents of aspartic acid, glutamic acid, glycine and γ-aminobutyric acid were tested by high-performance liquid chromatography electrochemical detector system. RESULTS: Compared with sham-operated group, the contents of excitatory amino acids (aspartic acid and glutamic acid) were significantly increased 2 h after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid were significantly decreased 2 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid did not show significant decrease 2 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. Compared with sham-operated group, the contents of inhibitory amino acid (γ-aminobutyric acid and glycine) were significantly decreased 3 h after cerebral ischemia in cerebral ischemia group (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine were significantly increased 3 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine did not show significant decrease 3 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. CONCLUSION: Cimicifugoside H-1 restrains the excessive releases of excitatory amino acids and increases the contents of inhibitory amino acids during cerebral ischemia. It doesn't only penetrate through the blood brain barrier, but also regulates the disorder of excitatory amino acid during cerebral ischemia, thus showing the protective function to cerebral neuron during cerebral ischemia.     

Protective effects of physcion against cerebral injury induced by ischemia-reperfusion in rats

AIM: To explore the effect of physcion (P) on the level of IL-1β and expression of ICAM-1 and caspase-3 during cerebral ischemia-reperfusion injury. METHODS: The 91 healthy adult SD rats were selected, and were randomly divided into normal group, sham-operated group, cerebral ischemia-reperfusion group (model), low-dose physcion (PLD) and high-dose physcion (PHD) treatment group. The level of IL-1β was detected by radioimmunoassay. The expression of ICAM-1 and caspase-3 was detected by immunohistochemistry. The changes of tissue pathology were also investigated. RESULTS: The level of IL-1β reached the peak at 6 h after ischemia-reperfusion (IR). The protein expression of ICAM-1 and caspase-3 reached the peak at 24 h after IR. The level of IL-1β and the protein expression of ICAM-1 and caspase-3 in PHD group decreased obviously compared with those in model group (P<0.05 or P<0.01), infiltration and adhesiveness of neutrophils were less serious at the same time. CONCLUSION: Physcion decreases the level of IL-1β and the protein expression of ICAM-1 and caspase-3 to protect brain tissue from cerebral ischemia-reperfusion injury.     

Expression of GLUT4 in endometrium of rats with polycystic ovary syndrome

AIM:To explore the expression of glucose transporter 4 (GLUT4) in the endometrium of rats with polycystic ovarian syndrom (PCOS) and evaluate the relationship between GLUT4 expression and insulin resistance (IR). METHODS:54 female SD rats of 85 days were randomized to control group (n=20), PCOS model group (n=17) and metformin treatment group (n=17). The rats in the latter two groups were induced by Poretsky’s method for PCOS model, followed by placebo or metformin, respectively. After 14 days of treatment, the rats were sacrificed and the expression of GLUT4 in endometrium was detected by Elivision^TM Plus two steps immunohistochemical staining. RESULTS:The expression of GLUT4 and insulin receptor(INS-R) proteins of endometrial glandulan epitheliu in PCOS rats were significantly lower (P<0.01,P<0.05) than those in control group, however, the expression of insulin(INS) protein in PCOS rats was higher than that in control group (P<0.01). The expression of GLUT4 in the treatment group increased (P<0.01), but was still lower than that in control group (P<0.01). However, compared with PCOS group, the expression of INS protein was decreased (P<0.05), but was still higher than that in control group (P<0.05). There was no GLUT4 expression in interstitial cells in endometrium, and the changes of the expressions of INS and INS-R proteins in those cells were similar with those in glandulan epitheliu. CONCLUSION:The decrease in GLUT4 expression of endometrium in PCOS rats is related with endometrial insulin resistance.     

Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

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.     

Expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage in neonatal A2AR knockout mice

AIM： To study the expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage (HIBD) in the neonatal adenosine A2A receptor knockout (A2AR-/-) mice. METHODS：Base on the modified Rice method, the model of HIBD was established. The total 64 C57/BL6 neonatal mice (7 days old) of A2AR-/-(KO) and corresponding wild type (A2AR+/+, WT) were randomized into sham-operated group and model group. The mice in model group were divided into 3 subgroups： 1 d after HIBD, 3 d after HIBD and 7 d after HIBD (n=8 for each group). The cortex and hippocampal CA1 region were used as the study areas. The neuronal apoptosis was detected using TUNEL assay combined with Nissl staining. The expression of p-p38 MAPK and activated caspase-3 was determined by the method of immunohistochemistry. The KO mice and WT mice were also taken from sham-operated group (SKO and SWT, n=10) and model group (MKO and MWT, n=30) 1 d after HIBD to assess the early neurological behavior. RESULTS：The apoptotic neurons, activated caspase-3 and p-p38 MAPK increased after HIBD and peaked at 1 d after HIBD in the cortex and the hippocampal CA1 region. The apoptotic neurons and the expression of activated caspase-3 in KO mice were significantly higher than those in WT mice at the same time point after HIBD. The expression level of p-p38 MAPK in KO mice were significantly higher than that in WT mice at 1 d and 3 d after HIBD. The expression of activated caspase-3 was positively correlated with the expression of p-p38 MAPK in neonatal mice after HIBD (in the cortex：r=0.957, P<0.01; in the hippocampal CA1 region： r=0.939, P<0.01). CONCLUSION：p-p38 MAPK might be involved in the aggravated neuron apoptosis and brain damage induced by A2AR knockout after neonatal HIBO.     

Effect of Qushi-Dizhuo decoction on urate transporters in hyperuricemia rats

AIM:To investigate the effects of Qushi-Dizhuo decoction (QSDZ) on the expression of uric acid transporters in rats with hyperuricemia. METHODS:Male Sprague-Dawley rats (n=60) were randomly divided into blank control group, model group, allopurinol group, and low-, middle- and high-dose QSDZ groups, with 10 rats in each. Except the blank control group, rats in the other groups were treated with adenine and potassium oxonate to prepare hyperuricemia rat model by intragastric administration once a day. Meanwhile, the rats in treatment groups were intragastric admi-nistrated with the corresponding preparation, after 28 days of continuous administration, the rats were sacrificed. Serum uric acid (UA) and creatinine (Cr) levels and xanthine oxidase (XOD) activity were measured. The morphological changes of renal tissues were observed by HE staining. The protein and mRNA expression of urate transporter1 (URAT1), glucose transporter 9 (GLUT9) and organic anion transporter 1 (OAT1) were detected by Western blot and real-time PCR. ATP-binding cassette subfamily Gmember 2 (ABCG2) and organic cation transporter 1 (OCT1) were also determined. RESULTS:Compared with the blank control group, the content of UA and Cr and the activity of XOD were significantly increased (P<0.05), the URAT1 and GLUT9 protein levels were significantly up-regulated, and the OAT1, ABCG2 and OCT1 protein levels were significantly down-regulated in model group (P<0.05). Compared with model group, the levels of serum UA and Cr and actioity of DXD were significantly decreased in QSDZ middle,high-dose group (P<0.05). The protein levels of URAT1 and GLUT9 were decreased, while the protein levels of OAT1, ABCG2 and OCT1 were increased in middle- and high-dose groups compared with model group (P<0.05). The mRNA expression of URAT1, GLUT9, OAT1, ABCG2, OCT1 was consistent with its protein expression trend. CONCLUSION:QSDZ decoction may through inhibiting the activity of XOD and regulating the expression of transporter to show its anti-hyperuricemia effect.     

Effects of Maxing-Shigan decoction on airway remodeling and expression of MMP-9 and TIMP-1 in lung tissues of asthmatic mice

AIM:To investigate the effects of Maxing-Shigan decoction on airway remodeling and expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissues of asthmatic mice, and to explore its possible mechanism in treatment of asthma. METHODS:The BALB/c mice were divided into blank control group, model group, low-dose Maxing-Shigan decoction group, middle-dose Maxing-Shigan decoction group, high-dose Maxing-Shigan decoction group and positive control group. The mice were sensitized and challenged with ovalbumin to establish asthma model. The mice in blank control group and model group were given saline by oral administration before 30 min of suscitation. The mice in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups were given Maxing-Shigan decoction at 5.0 g/kg, 10.0 g/kg and 20.0 g/kg, respectively, by oral administration before 30 min of suscitation. The mice in positive control group was given dexamethasone at 0.005 g/kg by oral administration before 30 min of suscitation. After consecutive administration for 7 d, the variations of airway responsiveness, the percentage of the goblet cells, the collagen deposition, and the eosinophil (EOS) counts in bronchoalveolar lavage fluid (BALF) of each group were observed. The protein levels of MMP-9 and TIMP-1 in the lung tissues were determined by ELISA and Western blot. The mRNA expression of MMP-9 and TIMP-1 was detected by RT-qPCR. RESULTS:Compared with blank control group, the airway responsiveness, the goblet cell percentage, the collagen deposition, the EOS counts in BALF, the protein levels of MMP-9 and TIMP-1, and the mRNA expression of MMP-9 and TIMP-1 were significantly increased in model group (P<0.01). Compared with model group, all of the indexes were reversed in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups and positive control group (P<0.05 or P<0.01). CONCLUSION:Maxing-Shigan decoction improves airway remodeling in asthma model mice by down-regulating the expression of MMP-9 and TIMP-1.     

High-fat diet affects plasma membrane GLUT4 content in skeletal muscle from Sprague-Dawley rats

AIM: To explore the change of the amount of GLUT4 protein at the plasma membrane of the rat skeletal muscle after high-fat feeding. METHODS: The animals were divided into three groups (ten for each): group I: control; group II: high-fat feeding; group III: high-fat feeding + dietary treatment. The rat model of insulin resistance (IR) was made by feeding high-fat diet for eight weeks. And then insulin-resistant rats were fed with chow diet for 4 weeks. Fasting plasma glucose and fasting serum insulin levels were measured before and after dietary treatment, respectively. Insulin treatment was achieved by intraperitoneal injection of insulin (10 unit insulin per kg body weight) 15 minutes before killing the animals. The right hindlimb skeletal muscle was rapidly dissected. Then the expression of GLUT4 protein at the plasma membrane in all the animals was assessed with Western bloting. RESULTS: The GLUT4 content at the plasma membrane in high-fat-fed rat skeletal muscle was significantly lower (about 31%) than that in controls (P<0.01). Dietary treatment partly corrected fasting blood glucose [from(6.20±0.39)mmol/L to(5.78±0.74)mmol/L]and fasting serum insulin levels [from(17.19±1.93)mU/L to(11.68±1.28)mU/L] and increased the GLUT4 content at the plasma membrane by 1.14-fold in insulin-resistant rat skeletal muscle. CONCLUSION: High-fat feeding induces IR in Sprague-Dawley rats. The mechanism may be involved in decreased cell-surface level of GLUT4 through affecting intracellular insulin signaling and then decreasing GLUT4 trafficking.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Flavonoids from stem and leaf of Scutellaria baicalonsis Georgi inhibit PHF abnormality and regulatory mechanism of protein phosphatase in rats' brain induced by okadaic acid

AIM: To investigate the effect of flavonoids from stem and leaf of Scutellaria baicalonsis Georgi (SSF) on paired helical filament (PHF) abnormality and the regulatory mechanism of protein phosphatase (PP) in rats' brain induced by okadaic acid (OA). METHODS: Male Sprague-Dawley (SD) rats were microinjected with OA (200 ng/kg) by the lateral ventricle to establish a memory impairment model. Morris water maze was used to screen the memory impairment model. The successful model rats were continuous intragastric infusion (ig) SSF for 36 days. The relative protein expression of PHF, PP1, PP2A-Cα, PP2A-Cβ, PP2CA and PP2CB in the rat cerebral cortex and hippocampus were detected by Western blot. GinKgo biloba leaf flavonoids (GLF) were used as positive control drug. RESULTS: Compared with the sham-operated rats, the relative protein expression of PHF in the cerebral cortex and hippocampus and PP1 in cortex of model rats were significantly increased (P<0.01), and the protein expression of PP2A-Cα, PP2A-Cβ in the cerebral cortex and hippocampus and PP2CB in the hippocampus were decreased (P<0.05), while the relative protein expression of PP2CA and PP2CB in the cortex were significantly increased (P<0.01). SSF reversed the abnormality in the protein expression of PHF, PP2A-Cα and PP2A-Cβ in rat cortex and hippocampus and PP1 in rat cortex induced by OA (P<0.01), which had no significant effect on the relative protein expression of PP2CA and PP2CB. GLF also showed similar results to SSF. CONCLUSION: SSF significantly reduces the abnormal formation of PHF in rats' brain induced by OA, which may be related to the regulation of PP1, PP2A-Cα and PP2A-Cβ expression, but not with PP2CA and PP2CB expression.     

Effect of peurarin pretreatment on endothelial nitric oxide synthase in rat brain tissues at the early stage of cerebral ischemia in rats

AIM: To investigate the neuroprotective effect of puerarin on the expression of endothelial nitric oxide synthase (eNOS) in rat brain tissues at the early stage of cerebral ischemia.METHODS: Forty-five rats were randomized into 3 groups: 5 in sham-operated group (S group), 20 in cerebral ischemia group (M group) and 20 in puerarin pretreatment group (P group).The rats in M group and P group were further divided into 4 subgroups to apply cerebral ischemia for 0.5 h, 1 h, 2 h and 4 h,respectively.The rats were subject to middle cerebral artery occlusion (MCAO) except those in S group.Puerarin was administered with intraperitoneal injection (100 mg/kg, ip) in P group 10 min before MCAO.The equal volume of the vehicle was administered in M groups and S group at the same time.Neurological deficit scores were determined to evaluate the functional changes of the central nervous system.The pathological changes of the brain tissues were observed under microscope with neuron nissl body staining.The protein expression and distribution of eNOS in the brain tissues were evaluated by the methods of immunohistochemistry and Western blotting.RESULTS: Neurological deficit scores of the rats in all subgroups of P groups were significantly lower than those in the corresponding subgroups of M groups (P<0.05).The dissolution extent of neuron nissl body in P groups was lower than that in M groups.The protein expression of eNOS in the brain tissues in all subgroups of P groups was higher than that in the corresponding subgroups of M groups.CONCLUSION: Pretreatment with puerarin protects brain tissues from injury of cerebral ischemia at the early stage by up-regulating the protein expression of eNOS in the brain tissues.     

Protective effect of progesterone on PC12 cells injured by oxygen-glucose deprivation

AIM: To investigate the effects of progesterone on the cell viability and expression of glucose transporter type 3(GLUT3) in PC12 cells injured by oxygen-glucose deprivation (OGD) in attempt to prove the neuroprotection of progesterone (PROG) against the hypoxic-ischemic injury in cultured cells in vitro. METHODS: Well-differentiated PC12 cells induced by nerve growth factor were randomly divided into 3 groups. In normal group, the cells were cultured without OGD treatment. In OGD group, the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N₂ and 5% CO₂ for 30 min. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In PROG+OGD group, the cells were given the same treatments as those in OGD group except that the medium contained progesterone at concentration of 10 nmol/L. Cellular morphological changes were observed after OGD for 30 min. The cell viability was assessed by WST-8 assay. The degree of the cell damage was evaluated by determining lactate dehydrogenase (LDH) leakage. The expression of GLUT3 at mRNA and protein levels was examined by RT-PCR and Western blotting, respectively. RESULTS: Progesterone attenuated the cellular swelling, decreased the leakage of LDH and improved the viability of PC12 cells injured by OGD (P<0.01). The expression of GLUT3 at mRNA and protein levels in PC12 cells in PROG+OGD group was significantly higher than that in OGD group (P<0.05). CONCLUSION: Progesterone has protective effect on in vitro cultured PC12 cells injured by OGD. The mechanism may be related to the up-regulation of GLUT3 protein.