Effects of EGb761 on synaptophysin expression in hippocampus of vascular dementia rats

AIM: To investigate the effects of Ginkgo biloba extract 761 (Egb761) on synaptophysin (SYN) expression in hippocampus of vascular dementia (VD) rats.METHODS: VD rat models, established by repeatedly cerebral ischemia/reperfusion, were randomly divided into two groups: model group and EGb761 treated group (both n=30), and another 30 condition-matched rats were selected as the sham-operated controls. Spatial learning and memory abilities of rats were assessed by Morris water maze (MWM) task, and SYN expression in hippocampal formation of rats in different groups was detected by immunohistochemical technique and image analysis.RESULTS: The MWM escape latency (EL) in model group was highly longer than that in the sham-operated group (P<0.01), while the EL of EGb761-treated group was significantly shorter than that in model group, but still longer than that in the sham-operated group at 1 m, 2 m and 4 m after VD modeling operation (P<0.05 or P<0.01). Immunohistochemical analysis showed that the SYN immunoreactive expression in hippocampal formation in model group greatly decreased and mean optical density of SYN expression highly increased compared with both sham-operated group and EGb761-treated group at three time points (P<0.01).CONCLUSION: EGb761 can increase the expression of SYN in hippocampus, which may be one of important mechanisms of EGb761 in improving learning and memory dysfunction of VD rats.     

Histopathological observation of cerebral cortex and hippocampus in the mouse with synthetic vascular dementia

AIM:To observe pathomorphological changes in cerebral cortex and hippocampus in the mouse with synthetic vascular dementia.METHODS:The synthetic vascular dementia model was produced in the mouse. Animals were killed 7 d, 15 d, and 30 d after the operation, brain tissues were removed and embedded in paraffin. Section of 8μm thickness were stained with hematoxylin-eosin(HE)and Nissl methods, and observed with light microscope.RESULTS:The cerebral cortex in the mouse became thinner on the seventh day, karyopyknosis in partial nervous cells was formed, the number of local neurons was reduced, sieve structure was observed, and glial cells pro liferated, with the similar results 15 d and 30 d afteroperation.Model mouseπs hippocampal cells in CA₁ area were reduced and almost disappeared 30 d after operation.At the same time, glial cells were abundantly proliferated, tu bercles were formed.Cells in CA₂, CA₃ area were also reduced and hippocampal sclerosis occurred.CONCLUSION:Delayed necrosis of hippocampal pyramidal cells may be the pathological basis of ischemia cerebral vascular dementia.     

Effect of protein S-nitrosylation on cAMP response element-binding protein activity in RSC-364 cells

AIM: To investigate the effect of protein S-nitrosylation on cAMP response element-binding protein(CREB) activity in RSC-364 cells. METHODS: ① RSC-364 cells cytoplasmic extracts were incubated with 100 μmol/L GSH (control chemical), 100 μmol/L GSNO (a donor of NO) in the presence or absence of 10 mmol/L DTT (inhibitor of S-nitrosylation) for 15 min at room temperature. The S-nitrosylated proteins were determined by biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. ② RSC-364 cell nuclear extracts were subjected to S-nitrosylation and electrophoretic mobility shift assay (EMSA) to analyze the CREB DNA binding activity after 1 h stimulation of rIL-1β (10 μg/L). RESULTS: GSNO obviously increased the expression of nitrosylated proteins and inhibited the CREB activity, which was reversed by DTT. CONCLUSION: S-nitrosylation may inhibit the CREB activity in RSC-364 cells.     

Phosphodiesterase 4 inhibitor rolipram prevents depression- and anxiety-like behaviors in rats exposed to chronic restraint stress

AIM: To discuss the impact of phosphodiesterase 4 (PDE4) inhibitor rolipram on chronic restraint stress-induced depression- and anxiety-like behaviors in rats. METHODS: (1)Forty SD rats were randomly divided into 4 weight-matched groups: unstressed animals injected with vehicle of lithium chloride (LiCl) and rolipram, restraint-stressed animals injected daily with vehicle prior to stress, restraint stress plus 100 mg/kg LiCl group and restraint stress plus 1 mg/kg rolipram group. The open field test was conducted 24 h before the first stress and drug administration,and then the rats received drugs daily 1 h prior to restraint stress (6 h/d) for 25 d. Daily body weight recording, forced swimming test, elevated plus-maze and open field test were conducted to determine the changes of depression- and anxiety-like behaviors. The expression of phosphorylated cAMP response element-binding protein (p-CREB), brain-derived Reurotrophic factor (BDNF), p-Ser21-glycogen synthase kinase (GSK) 3α, p-Ser9-GSK3β, p-Tyr279-GSK3α, p-Tyr216-GSK3β, total GSK3α and total GSK3β was measured by Western blotting. (2)Thirty SD rats were randomly divided into 6 groups and the cannula was surgically placed above the CA1 region in the hippocampus. Seven days after the surgery, the restraint stress was conducted for 21 d after microinjection of protein kinase A (PKA) antagonist H89 and intraperitoneal injection of LiCl and rolipram everyday. The expression of PDE4D, PKA, p-CREB and p-Ser9-GSK3β was measured by Western blotting. RESULTS: (1)No difference of the locomotor activity among all groups before stress was observed. After repeated stress, the body weight,and the crossing, rearing and grooming in open field test were lower than those in control group, and LiCl and rolipram reversed these effects significantly. In addition, in comparison with control group, the immobility in forced swimming test was increased, the climbing in forced swimmming test and the open-arm exploration in elevated plus-maze were decreased and the expression of p-CREB, BDNF, p-Ser21-GSK3α and p-Ser9-GSK3β was down-regulated. Stress induced depression- and anxiety-like behaviors, and rolipram reversed these changes. The LiCl showed similar effects as rolipram except for the expression of p-CREB and BDNF. No significant difference of the expression of p-Tyr279-GSK3α, p-Tyr216-GSK3β, total GSK3α and total GSK3β among all groups was found. (2)The expression of PDE4D was increased, the expression of PKA, p-CREB and p-Ser9-GSK3β was decreased in the hippocampus induce by restraint stress. However, the effect of rolipram on the expression of PKA, p-CREB and p-Ser9-GSK3β was blocked by PKA inhibitor H89. CONCLUSION: Rolipram significantly reduces the depression- and anxiety-like behaviors, possibly through CREB/BDNF signaling and inhibitory serine-phosphorylation of GSK3-mediated signaling. Importantly, the CREB/BDNF signaling also plays a key role in the down-regulation of serine-phosphorylation of GSK3.     

Effect of Shaofu-Zhuyu decoction on MAPK/ERK signaling pathway in rats with endometriosis

AIM: To observe the effects of Shaofu-Zhuyu decoction (SFZY) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway in the rats with endometriosis (EM), and to explore the mechanism of SFZY for treatment of EM.METHODS: Healthy female SD rats were used to establish the EM model. The rats were randomly divided into blank control group, model group, positive control group, and low dose, middle dose and high dose of SFZY groups. The pathological changes of the endometriotic tissue were observed by HE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and IL-8 in the uterine tissue were detected by ELISA. The mRNA expression of ERK, vascular endothelial growh factor (VEGF) and matrix metalloprotein-9 (MMP-9) was detected by RT-qPCR. The protein expression of nuclear factor-κB (NF-κB), MAPK and MAPK-ERK kinase (MEK) was determined by Western blot.RESULTS: Compared with model group, the levels of TNF-α, IL-6 and IL-8 in the uterine tissue of the rats in middle dose and high dose of SFZY groups were significantly decreased (P<0.05), the mRNA expression of ERK, VEGF and MMP-9 was significantly reduced, and the protein expression of NF-κB, MEK and MAPK was decreased significantly in the rat endometriotic tissues (P<0.05).CONCLUSION: SFZY may play a key role in the treatment of EM by regulating MAPK/ERK signaling pathway.     

Effect of ligustrazine on protein kinase C signaling pathway in human peripheral blood lymphocytes

AIM:To investigate whether Ligustrazine(LTZ) has an effect on the changes of protein kinase C(PKC) signaling pathway induced by inflammatory mediators involved in asthma in normal human peripheral blood lymphocytes (PBL).METHODS:10 mL peripheral venous blood was obtained from each of 63 health humans and treated as follows. The activities of PKC from cytosolic and membrane fractions in PBL were measured by -ATP-catalyzing assay, after PBL had been isolated and performed by following processes: (1) First: three groups treated with 5 g/L LTZ(n=6) or 5 μmol/L Ro31-8220 (n=6); Paired untreated PBL served as control of this group, as well as the negative controls of the following groups(n=6); (2)Second : three groups treated with 100 nmol/L Methacholine (Mch, n=5), 5 g/L LTZ+100nmol/L Mch(n=5)or 5 μmol/L Ro31-8220(a PKC inhibitor)+100 nmol/L Mch(n=5); (3)Third: three groups treated with 100 nmol/L histamine, 5 g/L LTZ+100 nmol/L histamine(n=5) or 5 μmol/L Ro31-8220+100nmol/L histamine(n=5); (4)Fourth: three groups treated respectively with 100nmol/L PMA(a PKC activator, n=5), 5 g/L LTZ+100nmol/L PMA(n=5) or 5 μmol/L Ro31-8220+100nmol/L PMA(n=5).RESULTS:(1)LTZ had no effect on the activities of PKC in inactive PBL in normal humans; (2) Methacholine or histamine resulted in an increase in membrane PKC activity of normal human PBL, which was partly suppressed by LTZ (all P＜0.05); (3) PMA caused an increase in membrane PKC activity of normal human PBL, which was partly decreased by LTZ (all P＜0.05).CONCLUSION:LTZ has an inhibitory effect on activation of PKC signaling pathway in PBL in normal humans induced by some inflammatory mediators involved in asthma, which may be one of the mechanisms that LTZ plays a role in the prevention and therapy of asthma.     

Effect of chronic intermittent hypoxia on AMPK pathway in young rats

AIM: To investigate the effect of chronic intermittent hypoxia on AMP-activated protein kinase(AMPK) pathway in the brain of young rats. METHODS: Part one: SD mice(3~4 weeks old) were randomly divided into 4 groups(n=8): simulated air control group for 2 weeks(2AC), chronic intermittent hypoxia group for 2 weeks(2IH), simulated air control group for 4 weeks(4AC) and chronic intermittent hypoxia group for 4 weeks(4IH). Part two: SD mice(3~4 weeks old) were randomly divided into 2 groups(n=8): chronic intermittent hypoxia group for 4 weeks(4IH) and chronic intermittent hypoxia group treated with AMPK inhibitor for 4 weeks(4IHI). After modeling, the eight-arm maze test was performed. TUNEL method was used to detect the neuronal apoptosis in the hippocampal and prefrontal cortical tissues. The mRNA expression of adenosine A2a receptor was examined by RT-qPCR, and the protein levels of phosphorylated AMPK(p-AMPK) and mammalian target of rapamycin(p-mTOR) were determined by Western blot. RESULTS: Compared with control group, the numbers of reference memory error(RME), working memory error(WME) and total error(TE) in 2IH group and 4IH group significantly increased(P<0.01). Compared with 2IH group, the numbers of errors in 4IH group also increased significantly(P<0.01). Compared with 4IH group, the values in 4IHI group significantly decreased. Compared with control group, the neuronal apoptosis of hippocampus and prefrontal cortex in 2IH group and 4IH group increased, and that in 4IH group was more evident(P<0.05). In 4IHI group, the neuronal apoptosis decreased. The mRNA expression of adenosine A2a receptor in the hippocampal and cortical tissues in 2IH group and 4IH group was higher than that in control group. The protein level of p-AMPK was higher, and p-mTOR was lower in 2IH group and 4IH group, and those in 4IH group were more evident(P<0.05). Compared with 4IH group, the protein level of p-AMPK was lower, and p-mTOR was higher in 4IHI group. CONCLUSION: Chronic intermittent hypoxia induces neuronal apoptosis, resulting in impairment of learning and memory in a time-dependent manner by upregulating adenosine A2a receptor, activating AMPK activity, and inhibiting mTOR phosphorylation in rats.     

Effects of CCK-8 and its receptor antagonists on expression of CREB and pCREB in prefrontal cortex and hippocampus of morphine withdrawal rats

AIM：To observe the effects of cholecystokinin octapeptide (CCK-8) and its receptor antagonists on cAMP response element binding protein (CREB) and phosphorylated CREB (pCREB） expression in frontal cortex and hippocampus of morphine withdrawal rats, which aim to explore the post-receptor mechanism through which CCK-8 regulates morphine withdrawal. METHODS：After the morphine dependence and naloxone-precipitated withdrawal animal models were established, the effects of CCK-8, L-364718 (CCK1 receptor antagonist) and LY-288513 (CCK2 receptor antagonist) pretreatment on CREB and pCREB expression in frontal cortex and hippocampus were observed by Western blotting and immunohistochemistry. RESULTS：In rat frontal cortex neuron, CREB was expressed in both cytoplasm and nucleus, but pCREB was only highly expressed in the nucleus. In the pyramidal cell layer of hippocampal CA1 region, CREB showed high expression in the cytoplasm and low expression in the nucleus, while pCREB was only expressed in the nu-cleus. No obvious change of CREB was observed after either chronic morphine treatment or naloxone withdrawal. The pCREB expression was increased after chronic morphine treatment and further increased after naloxone withdrawal. Compared with the withdrawal group, chronic pretreatment with CCK-8, L-364718 and LY-288513 had no effect on CREB expression in the frontal cortex, but obviously decreased the pCREB expression. In the hippocampus, pretreatment with L-364718 and LY-288513 decreased CREB and pCREB expression, but only the pCREB expression was decreased after CCK-8 treatment. CONCLUSION：CCK-8 and CCK receptor antagonists may alleviate morphine withdrawal symptoms by regulating CREB, with specificity in different brain regions.     

Expression of CRF and PKC proteins in hippocampus of rats with cerebral ischemia and reperfusion

AIM: To observe the expression of CRF and PKC in rats with cerebral ischemia.METHODS: Using immunohistochemistry technique we measured the expression quantitatively of CRF and PKC proteins in the hippocampus in rats induced by MCAO at 2 h,6 h and 24 h after reperfusion,contrast to CRF antagonist.RESULTS: (1) CRF: there were lots of positive and deeper dyeing neurons in hippocampus in model group and normal saline group rats,while there were a few positive and lighter dyeing neurons in sham group and CRF antagonist group.The positive expression areas of CRF protein in hippocampus in model group and normal saline group were significantly bigger than those in sham group and CRF-antagonist group(P<0.01),respectively.(2) PKC：there were a great number of denser positive granules in hippocampus in model group and normal saline group rats,while there were a few of scattered positive granules in sham group and CRF antagonist group.The positive expression areas of CRF protein in hippocampus in model group and normal saline group were significantly bigger than that in sham group and CRF-antagonist group (P<0.01),respectively.CONCLUSION: The high expression of CRF and PKC induced by cerebral ischemia may be one important factors that resulted in the delayed neuronal death in hippocampus.The CRF protein activated PKC expression,indicating an important pathology mechanism of nerve tissue damage induced by CRF.     

Effect of ischemia or hypoxia on vascular endothelial growth factor production in rat myocardium and its significance

AIM:To determine the relationship between ischemia, hypoxia and the production of vascular endothelial growth factor in rat myocardium and its basic mechanism. METHODS:(1) 28 Wistar rats were randomly divided into 4 groups: group A, normal control;group B, 1 day's acute myocardial infarction;group C, 3 day's acute myocardial infarction;group D, 7 day's acute myocardial infarction. (2) Rat cardiac myocytes cultured were primarily divided into some groups, hypoxia incubated 24 hours; PMA groups, hypoxia incubated 24 hours with PKC activator (PMA), A 0 ng/mL; B 10 ng/mL; C 100 ng/mL; D 1 000 ng/mL; Chelerythrine groups, hypoxia incubated 24 hours with PKC inhibitor (chelerythrine), A 0 nmol/L; B 10 nmol/L. (3) By computer scanned and quantitated, vascular endothelial growth factor (VEGF) protein was detected with immunohistochemical technique. RESULTS:The longer time of ischemia and hypoxia was, the higher the VEGF production.The relat ionship was found between the time of ischemia or hypoxia and the production of VEGF.The product ion of VEGF protein was further promoted by PMA with different concentrat ion, decreased by chelerythrine.CONCLUSION: Ischemia or hypoxia strongly stimulated the production of VEGF in myocardium, which played an important role in autoprotecting of ischemic or hypoxic myocardium. Hypoxia-induced PKC activation is one kind of basic mechanisms in this course.     

Effects of cerebral ischemia and postconditioning on signaling molecules PERK and GRP78 in hippocampus tissue of tree shrew during endoplasmic reticulum stress

AIM: To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage. METHODS: The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia. The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cerebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot. RESULTS: The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly. The expression of PERK at mRNA and protein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P<0.05), while postconditioning downregulated the expressions of PERK at ischemia and postconditioning 4 h (IP4 h) gruop and IP24 h group (P<0.05). The expression of GRP78 at mRNA and protein levels was not changed at 4 h, 24 h and 72 h after ischemia, while postconditioning upregulated the expressions of GRP78 at IP24 h group (P<0.05). CONCLUSION: The focal thrombotic cerebral ischemia activates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and protein expression of PERK in the PERK/eIF2α signal transduction pathway. The postconditioning treatment alleviates endoplasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.     

The effect of buyanghuanwu decoction on expression of AMPA receptor GluR1 subunit in mRNA and protein levels in rat hippocampus with vascular dementia

AIM: To observe the effect of buyanghuanwu decoction, a Chinese medicine, on the expression of AMPA receptor GluR1 subunit in mRNA and protein levels in rat hippocampus with vascular dementia (VD). METHODS: One hundred and forty-four rats were randomly divided into 4 groups: sham-operation group, VD model group, nimodipine group and buyanghuanwu decoction treatment group. The rat model of VD was built up by the method of 4 vessel occlusion. The VD rats were intragastrically treated with buyanghuanwu decoction suspension (pharmacognostic 50 g·kg-1·d-1) and nimodipine suspension (20 mg·kg-1·d-1) for 30 d. The learning and memory abilities were evaluated by Morris water maze testing. The change of GluR1 protein in hippocampal neurons in each group of rats was measured with immunohistochemistry and Western blotting techniques. The expression of GluR1 mRNA in hippocampus was determined by real-time fluorescence quantitative PCR. RESULTS: Compared to sham-operation group, the average escaping latency period (s) of Water maze tests in VD rats prolonged significantly and cross-platform time (numbers/min) shortened distinctly (P<0.05). The VD rats treated with buyanghuanwu decoction significantly improved the above-mentioned learning and memory performances (P<0.05); no significant difference of above-mentioned learning and memory performances among the rats in sham-operation group, nimodipine group and buyanghuanwu decoction treatment group was observed (P>0.05). Compared to the rats in sham-operation group, the mRNA and protein levels of GluR1 were apparently decreased in VD rats (P<0.05). The mRNA and protein levels of GluR1 in the neurons of hippocampus in buyanghuanwu decoction treated VD rats were higher than those in the model animals (P<0.05), and no difference was discovered in the rats among sham operation group, buyanghuanwu decoction treatment group and nimodipine group (P>0.05). CONCLUSION: Buyanghuanwu decoction improves the learning and memory abilities in VD rats. The therapeutic mechanism is associated with lessening the neuron injury on CA1 field in hippocampus and restoring the mRNA and protein expression of GluR1.     

Role of Cdk5-CRMP pathway in sevoflurane-induced dendritic developmental disorder of neurons in prefrontal cortex of neonatal rats

AIM: To investigate the effect of sevoflurane (Sevo) on the dendritic development in prefrontal cortex (PFC) of neonatal rats and the role of cyclin dependent kinase 5 (Cdk5)- collapsin response mediator protein (CRMP) pathway in it. METHODS: Eighty-eight postnatal day 7 Sprague Dawley (SD) rats were randomly divided into 4 groups (n=22): Air+NS group, Air+roscovitine (Ros) group, Sevo+NS group and Sevo+Ros group. The rats in Air+NS group and Air+Ros group were exposed to the air for 4 h, while the rats in the other 2 groups were exposed to 2.8% sevoflurane for 4 h. The rats received intraperitoneal injection of 150 μL normal saline 15 min before exposure in the Air+NS group and Sevo+NS group, while the rats in the Air+Ros group and Sevo+Ros group received intraperitoneal injection of 150 μL roscovitine (in DMSO solution, 10 mg/kg) 15 min before exposure. At the end of exposure, the cortices of the rat brain were collected and the protein levels of P35, P25, Cdk5, CRMP1, CRMP2, CRMP4 and p-CRMP2 Ser522 in PFC were detected by Western blot. On the postnatal day 30, the rat brains were sectioned for Golgi-Cox staining and morphological analysis of dendrites in the PFC neurons. Open-field test and contextual fear conditioning test were performed on postnatal days 25~27 and 31~32, respectively. RESULTS: Compared with Air+NS group, the expression of P35 in the Sevo+NS group was significantly decreased, and the expression of P25 was dramatically increased (P <0.05), whereas roscovitine partly reversed the changes above induced by sevoflurane (P <0.05). The expression of Cdk5 was not significantly different among all groups. Compared with the Air+NS group, the expression of CRMP1, 2, and 4 in the Sevo+NS group were decreased, and the protein level of p-CRMP2 Ser522/CRMP2 was increased (P <0.05), whereas roscovitine partly reversed the changes above induced by sevoflurane (P <0.05), except for the expression of CRMP2. Compared with Air+NS group, the total dendrite length, secondary dendritic length and interactions on 60 and 80 μm shells in the Sevo+NS group were decreased (P <0.01), whereas roscovitine partly reversed the changes above induced by sevoflurane (P <0.05). Compared with Air+NS group, the percentage of freezing time in the Sevo+NS group was decreased (P <0.01), whereas roscovitine partly reversed the changes induced by sevoflurane (P <0.05). No significant difference among groups in the open-field test was observed. CONCLUSION: Sevoflurane exposure disturbed dendritic development of neurons in PFC, learning and memory ability of neonatal rats, which may be mediated by Cdk5-CRMP pathway.     

Effects of Chaihu-Shugan decoction on ROCK/JNK signaling pathway and atherosclerosis in spontaneously hypertensive rats

AIM To observe the effect of Chaihu-Shugan decoction (CHSGD) on atherosclerosis in spontaneously hypertensive rats (SHR) and its possible mechanism. METHODS The male SHR (n=50) were randomly divided into model group (gavage of normal saline), compound kendir leaves (CKL) group (gavage of 0.5 g/kg CKL), and low-, medium- and high-dose CHSGD (CHSGD-L, CHSGD-M and CHSGD-H) groups (gavage of 2.5, 5 and 10 g/kg CHSGD, respectively), and another 10 male Wistar rats of the same origin were selected as normal control （NC) group (gavage of normal saline). The blood pressure was measured by intelligent noninvasive sphygmomanometer. The levels of blood lipids were measured by automatic biochemical analyzer. The expression of oxidative stress-related indexes, nitric oxide (NO), malondialdehyde (MDA) and superoxide dismutase (SOD), were detected by colorimetry. HE staining was used to detect the degree of atherosclerosis, and Western blot was used to detect the expression of Rho-associated kinase (ROCK)/c-Jun N-terminal kinase (JNK) signaling pathway-related proteins, RhoA, ROCK1 and JNK. RESULTS After 4 weeks of treatment, compared with NC group, the blood pressure, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and MDA, and the protein expression of RhoA, ROCK1 and JNK in aortic tissues of the rats in model group were significantly increased (P<0.05), and the serum levels of high-density lipoprotein cholesterol, NO and SOD were significantly decreased (P<0.05). HE staining showed that the diameter of aortas in the rats was thickened, a large number of foam cells were formed under the endothelium, and the proliferation of smooth muscle cells was observed. Compared with model group, the blood pressure, the serum levels of TG, TC, LDL-C and MDA, and the protein expression of RhoA, ROCK1 and JNK in aortic tissues of the rats in CKL, CHSGD-L, CHSGD-M and CHSGD-H groups were significantly decreased (P<0.05), and the serum levels of NO and SOD were significantly increased (P<0.05). HE staining showed that the structure of each layer of rat aortas gradually returned to normal, the vascular cells were in good order, and the inflammatory cell infiltration was slight. Compared with CKL group, the blood pressure, the serum levels of TG, TC, LDL-C and MDA, and the protein expression of RhoA, ROCK1 and JNK in aortic tissues of the rats in CHSGD-L and CHSGD-M groups were significantly increased (P<0.05), and the serum levels of NO and SOD were significantly decreased (P<0.05). No significant difference of the above indexes between CHSGD-H group and CKL group was observed (P>0.05). CONCLUSION Chaihu-Shugan decoction may attenuate the oxidative stress response via inhibition of ROCK/JNK signaling pathway, thus alleviating the symptoms of atherosclerosis in SHR.     

Influence of metformin on LPIN1 expression and AMPK signaling in high-fat diet-induced insulin resistant rats

AIM: To explore the regulatory mechanism of LPIN1 in hepatic insulin resistance by investigating the influence of metformin on the expression of LPIN1 and AMP-activated protein kinase(AMPK) signaling in the rats with high-fat diet-induced insulin resistance. METHODS: Thirty-six 4-week-old male Wistar rats were randomly divided into 2 groups: control group and high-fat diet (HF) group. The rats in HF group were fed with high-fat diet for 8 weeks and then were randomly divided into 2 subgroups: HF group and metformin intervention group, and the animals were continuously raised for 8 months. The mRNA levels of α1 and α2 subunit of AMPK as well as LPIN1 were measured by real-time RT-PCR. Phospho-AMPKα (Thr-172) was detected by Western blotting to evaluate the activity of AMPK. RESULTS: After 4 months, the rats in HF group showed significant increase in the levels of body weight, fast plasma glucose and insulin, and the levels of triglyceride and total cholesterol significantly elevated.Significant decrease in LPIN1 and phospho-AMPKα (Thr-172) expression in the rat livers were also observed. After treated with metformin, the metabolic indexes of the HF rats were improved. The mRNA and protein expression of AMPKα1 and AMPKα2 had no significant difference among the 3 groups. Metformin treatment also increased the expression of LPIN1 in the liver tissues of HF rats. CONCLUSION: The decrease in LPIN1 expression and AMPK activity may contribute to hepatic insulin resistance in diet-induced obese rats. Metformin improves the LPIN1 expression and AMPK activity through the interaction between LPIN1 and AMPK signal pathways.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.     

Effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats

AIM:To investigate the effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats. METHODS:The male Sprague-Dawley rats were induced as the model of the type 2 diabetic neuropathic pain rats, they were randomly divided into 6 groups (n=27): type 2 diabetic neuropathic pain (DNP) group, type 2 diabetic neuropathic pain and intraperitoneal injection of curcumin (Cur) group, type 2 diabetic neuropathic pain and solvent control (DSC) group, type 2 diabetic neuropathic pain and JNK inhibitor (DJ) group, type 2 diabetic neuropathic pain and JNK inhibitor solvent control (DJS) group, type 2 diabetic neuropathic pain and monocyte chemoattractant protein 1 (MCP-1) agonist (DM) group. Another 27 normal SD rats were selected as control group. Mechanical withdrawal threshod and thermal withdrawal latency were measured at 3rd d, 7th d and 14th d after dosing, then the lumbar segment 4~6 of the spinal cord and L4~6 DRG were removed at the same time. ELISA was used to measure MCP-1 level. The expression of p-JNK was determined by Western blotting. RESULTS:Compared with DNP group, p-JNK was significantly decreased at 7th d and 14th d in Cur group, DJ group and DM group after treatment (P<0.05). Compared with C group, the MCP-1 was significantly declined in other 6 group after streptozotocin injection (P<0.05). Compared with DNP group, MCP-1 were significantly increased at 7th d and 14th d in Cur group and DJ group after treatment (P<0.05), and that in DM group was greatly decreased (P<0.05). CONCLUSION: The expression of p-JNK and MCP-1 was increased in DNP rats with spinal cord and dorsal root ganglion. The mechanism of curcumin reducing the neuropathic pain in type 2 diabetic rats might be through regulating the JNK/MCP-1 pathway.     

Effects of three kinds of calcium activator on the proliferation of cultured vascular smooth muscle cells in rats

AIM: To investigate the effects of intracellular free calcium ([Ca²⁺]i) from different resources on the proliferation mediated by mitogen activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs were used in all experiments. Calcium influx was stimulated by angiotension Ⅱ(Ang Ⅱ). The release of intracellular calcium stores was induced by inositol trisphosphate (IP₃) and ryanodine (RY). MAPK activity was measured by [γ-³²P]-ATP incorporation MAPK protein expression by western blot, VSMCs proliferation by [³H]-Leucine ([³H]-Leu) and [³H]-Thymidine ([³H]-TdR) incorporation. RESULTS: Compared to the control VSMCs, Ang Ⅱ, IP₃ and RY significantly increased [Ca²⁺]i concentration activity of MAPK and its protein content in VSMCs. The promotion of [³H]-Leu and [³H]-TdR incorporation in VSMCs was also observed (P<0.01). CONCLUSION: The study indicated that calcium activator-induced increase in the activity and protein content of MAPK was involved in the proliferation of VSMCs, which was closely related to the [Ca²⁺]i concentration but independent to its origin.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.     

Effect of BNP- mediated cAMP-PKA signaling pathway on bFGF expression in rats with spleen-qi deficiency syndrome

AIM: To explore the changes and the mechanism of heart functions in the rats with spleen-qi deficiency syndrome. METHODS: The rats were randomly divided into blank control group and spleen-qi deficiency model group. The changes of cardiac functions in the rats were determined by ultrasonic imaging with a high-resolution in vivo imaging system. HE staining was used to observe the pathological changes. The protein expression of brain natriuretic peptide (BNP) in the myocardium was assessed by Western blotting. The contents of BNP and cAMP in the serum and myocardium were measured by ELISA. The mRNA expression of basic fibroblast growth factor (bFGF) and protein kinase A (PKA) was detected by real-time PCR. RESULTS: Compared with blank control group, the myocardial cells in the model group had different degrees of necrosis and degeneration. Stroke volume and ejection fraction were decreased. The contents of cAMP and BNP in the serum and myocardium were increased in model group. The protein expression of BNP and the mRNA expression of bFGF and PKA were also increased.CONCLUSION: Spleen-qi deficiency syndrome causes heart function decline in rats. The expression of BNP, cAMP, PKA and bFGF is all increased.