Effect of GRK5 on activation of rat astrocytes

AIM: To study the effect of G-protein-coupled receptor kinase 5 (GRK5) on the activation of astrocytes in the brain cortex of newborn Wistar rats. METHODS: GRK5 gene was silenced in the model of rat brain cortex astrocytes in vitro for 24 h. N-acetylcysteine (NAC), which is a known inhibitor of NF-κB, was added into the culture medium according to gene silencing for 24 h. The expression levels of GFAP and caspase-3 were detected by the method of immunofluorescence, and the mRNA levels of NF-κB, TNF-α, IL-1β and iNOS were determined by real-time PCR. Moreover, the activity of SOD and concentrations of TNF-α and NO were measured. RESULTS: GRK5 gene silencing increased the expression of NF-κB at mRNA and protein levels obviously (P<0.01), and the mRNA levels of IL-1β and iNOS increased synchronously (P<0.01). Furthermore, caspase-3-positive cells in GRK5 siRNA group were increased compared with control siRNA group (P<0.01). Treatment with NAC obviously reduced the activity of NF-κB and weakened the effects induced by GRK5 siRNA (P<0.05). CONCLUSION: GRK5 siRNA increases NF-κB activity and induces the activation of astrocytes.     

Study on COX activity and its mRNA expression,and PGE2 release from cerebral microvascular endothelial cells stimulated by IL-1β

AIM: To study the cyclooxygenase(COX) activity and its mRNA expression, and PGE₂ release from rats cerebral microvascular endothelial cells (rCEMC) stimulated by IL-1β(30 μg/L) at different times. METHODS: rCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor (Ⅷ factor, a marker for all endothelial cells) in cytoplasm of the cells. After rCEMC grew to confluency, they were stimulated with IL-1β for 0.5, 1, 2, 4, 8, 12 and 24 h, respectively. Activity of COX-1 and COX-2 in rCEMC and production of PGE₂ in the conditioned media were detected by ELISA. COX-1 and COX-2 mRNA expressions were measured by real-time quantity PCR. The amplification product was tested by melting curve and identified by electrophoretic gel. RESULTS: ① Positive immunostaining for Ⅷ factor was present diffusely in the cytoplasm in more than 90% rCMEC. ② Compared to the cells without IL-1β stimulation, the production of PGE₂ increased significantly (P<0.05) at 4 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ③ There was no significant difference on COX-1 activity between IL-1β group and non-IL-1β group. COX-2 activity increased significantly compared with those in non-IL-1β (P<0.05) at 8 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ④ There was no significant difference on COX-1 mRNA expression between IL-1β group and non-IL-1β group. COX-2 mRNA was induced and became detectable at 1 h, and reached the top level at 4 h, then declined thereafter at 8 h and became undetectable by 12 h and 24 h after incubation with IL-1β. The melting curve showed there was no nonspecific amplification and electrophoretic gel showed the lengths of amplification products accorded with the predicted lengths. CONCLUSION: While rCEMC are stimulated by IL-1β, the excretion of PGE₂ increases and reaches the top level at 12 h, which is related with its induction on COX-2 mRNA expression and COX-2 activity.     

Inhibitory effects of Am80 on proliferation in vascular endothelial cells and neointima hyperplasia of carotid arteries

AIM: To explore the inhibitory effects of Am80 on the proliferation in the vascular endothelial cells (VECs) and neointima hyperplasia of the carotid arteries after balloon injury in the rats. METHODS: The proliferation of EA-hy926 cells were detected by cell counting and MTS assay after the cells were treated with various doses of Am80 for 24 h. The cell cycle was analyzed by flow cytometry after the cells were stained with PI. The mRNA expression of cyclinB1, P21 and matrix metalloproteinase (MMP)-2 in the EA-Hy926 cells was detected by real-time PCR. The changes of neointima hyperplasia in the carotid arteries were observed under microscope with hemotoxylin and eosin (HE) staining, and the expression of cyclinB1 was examined by the method of immunohistochemistry. RESULTS: The proliferation of EA-Hy926 cells was obviously inhibited in a dose-dependent manner when the cells were treated with various doses of Am80 for 24 h. The cell cycle was arrested at G₂/S stage in response to Am80 treatment. The mRNA expression of P21 was increased, however, the mRNA expression of cyclinB1 and MMP-2 was decreased when the cells were treated with Am80 at 4 μmol/L for various times. In addition, the vivo experiment demonstrated that Am80 not only significantly reduced neointimal hyperplasia and the thickness ratio of intima to tunicae media compared with injured group, but also inhibited cyclinB1 expression in the carotid arteries. CONCLUSION: Am80 inhibits the proliferation of VECs and neointima hyperplasia in the carotid arteries after balloon injury by promoting P21 expression and decreasing cyclinB1 expression.     

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

AIM: To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A(PAPP-A)induced by TNF-α and IL-1β in endothelial cells. METHODS: The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method. The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups: blank control group: the cells were treated without any intervention; atorvastatin concentration groups: the cells were incubated with atorvastatin at the concentrations of 0.1, 1 and 10 μmol/L for 24 h; atorvastatin time groups: the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h; atorvastatin+inflammatory factors groups: the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h, then different concentrations of atorvastatin (0.1, 1.0, 10 μmol/L) were added for 6 h,12 h and 24 h. MTT reduction assay was used to observe the cell proliferation. The mRNA expression of PAPP-A was detected by RT-PCR. The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA. RESULTS: Compared with blank control group, no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h, 6 h, 12 h, 24 h and 48 h, indicating that the drugs had no toxic effects on the cells. No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found. Compared with TNF-α groups and IL-1β groups, PAPP-A expressions in atorvastatin intervention groups significantly decreased. The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration- and time-dependent manner. CONCLUSION: Atorvastatin doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration- and time-dependent manner in primary cultured rat aortic endothelial cells.     

Protective effect of deferoxamine on hypoxic-ischemic encephalopathy in neonatal rat

AIM:To investigate the possible mechanism of deferoxamine on angiogenesis in rat hypoxic-ischemic encephalopathy (HIE). METHODS:SD rats (7 days of age) were used to make HIE model. Model group and treatment group were injected with deferoxamine or normal saline alone 24 hours before hypoxic-ischemic insult. Rats were sacrificed at 1,3,7 or 14 days after hypoxic-ischemic insult. Brain capillary density index (BCDI),the number of proliferating capillary,brain water content and extent of brain atrophy were determined. The expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α(HIF-1α) mRNA was measured. RESULTS:Early water content and late atrophic ratio of the left brain were significantly improved in the treatment group compared to model group (P<0.01). The number of proliferating capillary in the treatment group was significantly higher than that in the model group ［(2.01±0.31)/HPF vs　(0.90±0.25)/HPF,P<0.01］. Deferoxamine markedly up-regulated the expression of VEGF and HIF-1α mRNA in the brain ［VEGF at 12 h: (1.41±0.07) vs (1.10±.15),P<0.05; HIF-1α at 12 h: (1.49±0.12) vs (1.11±0.16),P<0.05］.CONCLUSION:Deferoxamine may promote angiogenesis and attenuate hypoxic-ischemic induced brain injury via up-regulation of HIF-1α and VEGF expression.     

Effects of Scutellaria barbata flavonoids on abnormal expression of NOS,HSP70 and apoE induced by Aβ 25-35 in rat astrocytes

AIM:To study the effects of Scutellaria barbata flavonoids (SBF) on abnormal expression of nitric oxide synthase (NOS), heat-shock protein 70 (HSP70) and apolipoprotein E (apoE) induced by Aβ 25-35 in rat astrocytes. METHODS:The third generation of cultured rat astrocytes was divided into 5 groups. The cells in 3 drug treatment groups were given SBF at dose of 17.5 mg/L, 35 mg/L and 70 mg/L for 24 h, and then the cells in model group and 3 doses of SBF groups were exposed to Aβ 25-35 at concentration of 100 μmol/L for 24 h. The expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in the cultured cells was assayed by immunohistochemical method. The expression of HSP70 was estimated by Western blotting and the mRNA expression of apoE was assessed by RT-PCR. RESULTS:Compared with control group, the protein level of eNOS were significantly decreased and the protein level of iNOS increased (P<0.01) in model group. The protein expression of HSP70 and mRNA expression of apoE were notably increased (P<0.01) in model group. However, these disturbances were attenuated by SBF at dose of 17.5, 35 and 70 mg/L (P<0.01). CONCLUSION:SBF has an obvious protective effect on damaged astrocytes induced by Aβ 25-35, suggesting that SBF may be helpful for the treatment of Alzheimer disease.     

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.     

LPS and cerulein induce isolated rat pancreatic tissue injury and inhibit expression of HSP60 protein

AIM: To investigate the effects of lipopolysaccharides (LPS) or cerulein on the expression of HSP60 in isolated rat pancreatic tissues. METHODS: The tissue of rat pancreas was isolated by surgical operation and prepared into tissue snips. The isolated pancreatic tissue was cultured, and stimulated with low- and high-concentrations of cerulein (Cer, 10-11 mol/L, 10-5 mol/L) or lipopolysaccharides (LPS, 10 mg/L, 20 mg/L). Normal saline (NS) was used as control reagent. Before stimulation and 1 h or 4 h after stimulation, the following parameters for evaluating injury were detected: the viability and the level of trypsinogen activation peptide (TAP) in the pancreatic tissues, and the level of interleukin 6 (IL-6) in the culture supernatants. Meanwhile, real-time PCR and Western blotting were used to determine the HSP60 expression at mRNA and protein levels respectively. RESULTS: Under the stimulation with LPS or cerulein, the viability of the pancreatic tissues decreased slightly at 1 h and became much lower with prolonged treatment for 4 h (P<0.05). The TAP level in the pancreatic tissues increased obviously at both 1 h and 4 h (P<0.05) after treatment, except LPS at lower dose. The IL-6 level in the culture supernatant showed no significant change at 1 h stimulation, then increased remarkably at 4 h after stimulation, especially under the conditions of stimulating with LPS or cerulein at higher doses (P<0.05). The expression of HSP60 at mRNA and protein levels decreased sharply with the increase in the concentration of LPS and the prolonged stimulation time; In cerulein stimulation group, the expression of HSP60 mRNA showed an obvious increase (P<0.01), whereas HSP60 protein was reduced remarkably, especially stimulated with cerulein at higher dose and with longer stimulation time (P<0.05). CONCLUSION: LPS or cerulein induce injuries in isolated pancreatic tissues in dose and time dependent manners. Meanwhile, the protein expression of HSP60 is reduced significantly, indicating that the decrease in the cellular protection of HSP60 may involve in the injury of pancreatic tissues.     

Involvement of 26S proteasome LMP2 subunit in development of tolerance against oxidative stress in vascular endothelial cells

AIM: To observe the expression of 26S proteasome LMP2 subunit in vascular endothelial cells (VECs) under oxidative stress, and to evaluate its role in the development of tolerance against oxidative stress in VECs. METHODS: The cell model of H₂O₂ preconditioning-induced oxidative tolerance was established in VECs. The expression of LMP2 was detected by cellular immunofluorescent labeling and Western blotting. The LMP2 anti-sense and sense oligonucleotides were transfected into VECs by Lipofectamine^TM 2000. The damages of VECs were evaluated by detecting the activity of lactate dehydrogenase (LDH) and the concentration of malondialdehyde (MDA) in the culture medium. RESULTS: H₂O₂ (500 μmol/L for 3 h) induced oxidative stress in VECss in a dose- and the activity of time-dependent manner, characterized by the increase in the concentration of MDA and LDH in the culture medium. Pretreatment with H₂O₂ (10 μmol/L for 24 h) up-regulated the expression of LMP2. Meanwhile, the capacity of cellular tolerance against oxidative stress induced by H₂O₂ was increased as the concentration of MDA and the activity of LDH in the culture medium significantly decreased. Compared with H₂O₂ group, up-regulation of LMP2 by IFN-γ pretreatment (20 μg/L for 48 h) increased the tolerance of VECs against H₂O₂ injury, and the MDA conentration and the activity of LDH in the culture medium also significantly decreased. Transfection with LMP2 antisense oligonucleotide partly inhibited the increased expression of LMP2 induced by IFN-γ in VECs and abolished the tolerance against H₂O₂. CONCLUSION: The 26S proteasome LMP2 subunit is associated with the development of the tolerance against H₂O₂-induced oxidative stress in VECs.     

Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

Effect of recombinant rat CC16 protein on LPS-induced expression of TNF-α, IL-6 and IL-8 in rat tracheal epithelial cells

AIM: To explore the effect of recombinamt rat CC16 protein (rCC16) on LPS-induced expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-8 in the rat tracheal epithelial (RTE) cells.METHODS: The RTE cells were incubated with rCC16 at concentrations of 0.5, 1.0 and 2.0 mg/L in serum-free media for 2 h prior to LPS (0.1 mg/L) treatment for further 24 h. The cells were harvested for assessing the mRNA levels of TNF-α, IL-6 and IL-8 by RT-qPCR. The cell culture supernatants were collected for analyzing the protein levels of TNF-α, IL-6 and IL-8 by ELISA. In addition, the nuclear translocation of nuclear factor-κB (NF-κB) p65 was tested by Western blot.RESULTS: rCC16 inhibited LPS-induced IL-6 and IL-8 expression at both mRNA and protein levels in the RTE cells in a concentration-dependent (0~2 mg/L) manner, as demonstrated by RT-qPCR and ELISA. However, no concentration-dependent manner between the dose of rCC16 and TNF-α expression was observed, and rCC16 inhibited LPS-induced TNF-α expression at lower concentration (0.5 mg/L). rCC16 concentration-dependently inhibited the effects of LPS on the level of nuclear translocation of NF-κB p65.CONCLUSION: rCC16 suppresses LPS-mediated TNF-α, IL-6 and IL-8 production through inactivation of NF-κB activity in RTE cells.[KEY WORDS] CC16 protein; Airway inflammation; LPS; Inflammatory mediators; Nuclear factor-κB     

Effect of TRPC6 on PDGF-induced proliferation of rat airway smooth muscle cells

AIM:To investigate the role of canonical transient receptor potential channel 6 (TRPC6) in the proliferation of airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor (PDGF). METHODS:Rat ASMCs were cultured by enzyme digestion and tissue adhesion. The method of indirect immunofluorescence was applied to identify the ASMCs and to detect the expression of TRPC6 in ASMCs. The proliferation of ASMCs was determined by CCK-8 assay. The mRNA expression of TRPC6 was tested by real-time PCR. The protein expression of TRPC6 was analyzed by Western blotting. The influence of TRPC6 blocker at different concentrations on the proliferation of ASMCs was measured by CCK-8 assay. RESULTS:The results of immunofluorescence indicated that TRPC6 expression in ASMCs was positive. PDGF at concentration of 20 μg/L induced the proliferation of ASMCs compared with control group (P<0.05). When ASMCs were treated with both PDGF and different concentrations of TRPC6 blocker SKF96365, the proliferation of ASMCs was attenuated in dose-dependent and time-dependent manners as compared with the cells treated with PDGF alone (P<0.05). The mRNA expression of TRPC6 in PDGF group was significantly increased (P<0.05) after ASMCs were treated with PDGF for 12 h, 24 h and 48 h. The protein level of TRPC6 in PDGF group was significantly increased after ASMCs were treated with PDGF for 24 h and 48 h compared with control group (P<0.05). CONCLUSION: Up-regulation of TRPC6 at mRNA and protein levels is most possibly related to the proliferation of ASMCs induced by PDGF. Therefore, TRPC6 is involved in the proliferation of ASMCs.     

Effects of simvastatin on cigarette smoke extract-induced sEPCR and EPCR expression in human umbilical vein endothelial cells

AIM: To investigate the effects of simvastatin on cigarette smoke extract (CSE)-induced expression levels of soluble endothelial cell protein C receptor (sEPCR) and membrane-associated endothelial cell protein C receptor (mEPCR ) in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs at passage 4 to 6 were randomly divided into control group, 5% CSE group, simvastatin groups and simvastatin+CSE groups. In simvastatin groups, HUVECs were incubated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 24 h. In simvastatin+CSE groups, the cells were treated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 2 h, and then exposed to CSE for 24 h. The protein level of sEPCR in the culture supernatants was measured by ELISA. The cells were collected for determining the mRNA expression of mEPCR by real-time PCR. RESULTS: Compared with control group, the protein level of sEPCR was significantly increased, and the mRNA expression of mEPCR was significantly decreased in 5% CSE group (both P<0.05). The protein levels of sEPCR were significantly increased, and the mRNA expression of mEPCR was significantly decreased in 100 μmol/L and 200 μmol/L simvastatin groups. However, the protein levels of sEPCR were lower, and the mRNA expression of mEPCR was significantly higher in 100 μmol/L and 200 μmol/L simvastatin groups than those in 5% CSE group. Compared with 5% CSE group, the protein levels of sEPCR in simvastatin+CSE groups were significantly decreased, but higher than those in control group and simvastatin group with corresponding concentration. On the contrary, the mRNA expression of mEPCR in simvastatin+CSE groups was significantly increased, but lower than that in control group and simvastatin group with corresponding concentration (all P<0.05). CONCLUSION: Simvastatin obviously increases the mRNA expression of mEPCR, decreases the protein level of sEPCR, and attenuates the CSE-induced endothelial injury in vitro .     

Hydrogen sulfide inhibits inflammatory responses induced by acute myocardial ischemia in isolated rat hearts

AIM：To investigate whether hydrogen sulfide (H2S) protects the hearts against inflammatory responses induced by acute myocardial ischemia in isolated rat hearts. METHODS：Rat acute myocardial ischemia injury was induced by ligation of the left anterior descending coronary artery for 4 h, and the normal perfusate was replaced with NaHS (5 μmol/L, 10 μmol/L and 20 μmol/L) perfusate accordingly in NaHS groups 2 h after ischemia. The changes of cardiac function in the myocardial ischemic injury rats were observed. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and ICAM-1 was detected by real-time PCR. The protein level of nuclear factor-κB (NF-κB) in the myocardial tissues was detected by Western blotting. RESULTS：The cardiac function in ischemia group was lower than that in sham group (P<0.01). Compared with ischemia group, perfusion of NaHS resulted in the improvement of the cardiac function (P<0.05 or P<0.01). Compared with sham group, the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 in the cardiac tissues was significantly increased, and the mRNA expression of IL-10 in the cardiac tissues was significantly decreased in ischemia group (P<0.01). Compared with ischemia group, the perfusion of NaHS significantly decreased the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 (P<0.05 or P<0.01). The perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly increased the mRNA expression of IL-10 (P<0.01). The protein level of NF-κB in ischemia group was markedly higher than that in sham group (P<0.01). Compared with ischemia group, the perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly decreased the expression of NF-κB (P<0.05 or P<0.01). CONCLUSION：H2S protects the hearts against acute ischemia injury through inhibition of NF-κB activation and subsequent down-regulation of NF-κB-dependent inflammatory gene expression.     

Effects of resveratrol on TNF-α-induced injury and expression of MCP-1 in isolated rat pulmonary artery endothelial cells

AIM: To investigate the possible mechanism of resveratrol (Res) on tumor necrosis factor-α (TNF-α)-induced monocyte chemoattractant protein-1 (MCP-1) expression in primary rat pulmonary artery endothelial cells (RPAECs).METHODS: RPAECs were randomly divided into 4 groups:control group, solvent (1% DMSO) group, TNF-α group and Res group. Each group was divided into 1 h, 4 h and 8 h subgroups (n=6 per time point). The TNF-α+C1142 (a rodent chimeric mAb that neutralizes rat MCP-1) group was set up at the 8 h time point. At each time point, the protein and mRNA expression of MCP-1 was measured by Western blot and real-time PCR.RESULTS: Pretreatment of the RPAECs with C1142 significantly down-regulated the expression of MCP-1 (P<0.05). The protein and mRNA expression of MCP-1 was markedly increased in TNF-α group (P<0.05). Notably, incubation with Res down-re-gulated the protein and mRNA expression of MCP-1, which was significantly lower than that in TNF-α group (P<0.05).CONCLUSION: MCP-1 was involved in the process of TNF-α-induced injury of RPAECs. Res down-regulates the expression of MCP-1 in RPAECs, thus attenuating cell injury.     

Tristetraprolin attenuates subarachnoid hemorrhage-induced early brain injury in rats

AIM: To explore the expression level of tristetraprolin (TTP) in rats after subarachnoid hemorrhage (SAH) as well as the potential role of TTP in the early brain injury (EBI) after SAH in rats.METHODS: In the first experiment setting, total 56 adult male SD rats were randomly divided into sham group and SAH group. The SAH model was performed by endovascular perforation. The brain tissues were taken out after SAH at 5 different time points (0, 12, 24, 48, 72 h and 1 week). The expression of TTP in the brain tissues was detected by Western blot. In the second experiment, a total of 60 SD rats were divided into 4 groups:sham group, SAH group, SAH+vector group and SAH+TPP group. Neurological score, brain water content and blood-brain barrier were evaluated at 48 h after SAH. TUNEL staining was performed to detect cell apoptosis in the rat brain tissue. ELISA method was used for quantitative detection of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The protein levels of TTP, Bax, Bcl-2 and cleaved caspase-3 in the rat brain tissue were detected by Western blot.RESULTS: The protein expression of TTP in the brain was downregulated markedly from 12 h after SAH, reached the lowest level at 48 h, and then had an upward trend. After modeling for 48 h, Garcia neurological score was significantly reduced, and brain water content and Evans blue (EB) content of the brain tissue of the rats in SAH group were significantly higher than those in sham group (P<0.05). SAH induced significant increases in IL-6 and TNF-α levels in the brain tissue (P<0.05). The number of TUNEL-stained cells was increased in the subcortical brain region after SAH compared with sham group. In addition, a lower level of Bcl-2 and higher levels of Bax and cleaved caspase-3 in the rat brains were observed at 48 h after SAH. However, the neurological deficit score was significantly increased, and the brain water content and EB content in the rat brains were significantly reduced in SAH+TTP group in comparison with SAH+vector group (P<0.05). Over-expression of TTP dramatically suppressed the levels of IL-6 and TNF-α in the rat brains, and reduced the number of TUNEL positive cells. Furthermore, upregulation of TTP significantly decreased the levels of cleaved caspase-3 and Bax, and evidently enhanced the expression of Bcl-2 (P<0.01).CONCLUSION: The expression of TTP is significantly decreased in early period after SAH, and enhancing the level of TTP effectively inhibits EBI following SAH in rats.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.     

Effect of Chinese propolis on PC-PLC activity and TLR4 expression in LPS-treated vascular endothelial cells

AIM: To investigate the effect of Chinese propolis on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and the expression of Toll-like receptor 4 (TLR4) in LPS-treated vascular endothelial cells (VECs). METHODS: Confluent VECs were stimulated with LPS at the concentration of 100 μg/L in the presence of 0.5% fetal bovine serum. The cells were treated with Chinese propolis at the concentration of 12.5 mg/L for 12 h and 24 h. The viability of VECs and the level of nitric oxide (NO) were detected by sulforhodamine B (SRB) assay and chemical method, respectively. The activity of PC-PLC was measured using L-α-phosphatidylcholine as substrate. The protein levels of TLR4, nuclear factor-κB p65 (NF-κB p65) and p53 were determined by Western blotting. The level of intracellular reactive oxygen species (ROS) was examined using a fluorescent probe, 2,7-dichlorodihydrofluorescin (DCHF). For the measurement of mitochondrial membrane potential, the fluorescent dye JC-1 was used. RESULTS: Treatment with Chinese propolis for 24 h had no effect on the viability of VECs. However, the levels of NO and ROS were significantly decreased by Chinese propolis. PC-PLC activity and NF-κB p65 expression were significantly depressed by Chinese propolis treated for 12 h, and the expression of TLR4 and p53 were dramatically decreased by Chinese propolis treated for 12 and 24 h. No effect of Chinese propolis on mitochondrial membrane potential was observed. CONCLUSION: Chinese propolis depresses the activity of PC-PLC and the expression of TLR4, and then inhibits the downstream signal molecules such as NF-κB p65, p53, ROS and NO in VECs.