Oral administration of fasudil hydrochloride suppresses development of experimental autoimmune encephalomyelitis in mice

AIM:To explore the therapeutic effect of fasudil hydrochloride by the oral route on the development of experimental autoimmune encephalomyelitis (EAE) in mice and its possible mechanism. METHODS:The EAE model in female C57BL/6 mice was established by myelin oligodendrocyte glycoprotein 35-55 peptide(MOG35-55) immunization and the immunized mice were randomly divided into saline control group and fasudil intervention group, in which saline and fasudil were administered by the oral route once every day from post-immunization (PI) day 3 to day 27. Clinical score and body weight were recorded every other day. On PI day 28, the spinal cords were obtained for HE and myelin staining. The splenocytes were isolated and the expression of CD16/32, CD206 and interleukin (IL)-10 was analyzed by flow cytometry. The levels of IL-1β and tumor necrosis factor α (TNF-α) were detected by ELISA. RESULTS:Oral administration of fasudil delayed the onset of EAE, and attenuated the myelinoclasis of the model animals and the severity of EAE, accompanied by the phenotypic switch from M1 to M2 macrophages, the inhibition of the proinflammatory cytokine (IL-1β and TNF-α) production and the increase in IL-10 release. CONCLUSION: Oral administration of fasudil exhibits therapeutic effect on the development of EAE possibly through switching M1 macrophages to M2 phenotype and inhibiting inflammatory responses in mice.     

Study on a novel Rho kinase inhibitor WAR5 for treating EAE

AIM：To explore the therapeutic effect of a novel Rho kinase inhibitor WAR5 on the experimental autoimmune encephalomyelitis (EAE) and its possible mechanism. METHODS：Female C57BL/6 mice were randomly divided into EAE group and WAR5 group. EAE model was induced by the application of MOG35-55 peptide. WAR5 was injected intraperitoneally every other day from post-immunization (PI) day 3 to PI day 27. The clinical score and body weight were recorded every other day. On PI day 28, the animals were sacrificed and spinal cords were obtained for HE and myelin staining. The splenocytes were isolated and the expression of CD16/32 and CD206 were analyzed by flow cytometry. The protein extracts from the brains and spinal cords were collected for the measurement of inducible nitric oxide synthase (iNOS) by Western blotting. RESULTS：The administration of WAR5 delayed the onset of EAE and attenuated the clinical symptoms. The results of the pathological examination revealed that WAR5 inhibited the infiltration of inflammatory cells and improved myelination in spinal cords, accompanied with the poralization of M1 macrophages to M2 phenotype in the spleen. WAR5 inhibited the expression of iNOS in the central nervous system, especially in the spinal cords. CONCLUSION：The therapeutic effect of WAR5 on EAE may be related to the shift of M1 macrophages to M2 phenotype and inhibition of inflammation in the central nerve system.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells on mice with experimental autoimmune encephalomyelitis

AIM: To explore the neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells (BM-NSCs) on the mice with experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice (8~10 weeks old, n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) to establish chronic EAE model. The mice were randomly divided into control (ddH₂O) group, fasudil group, BM-NSCs group, and fasudil+BM-NSCs group. The clinical score and body weight were recorded every other day. The expression of neurotrophic factors was determined by immunofluorescence staining. RESULTS: In comparison with ddH₂O group, fasudil combined with BM-NSCs delayed onset and ameliorated severity of EAE. The numbers of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil+BM-NSCs group were all increased in various extents. In particularly, the expression of these neurotrophic factors in fasudil+BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01). CONCLUSION: Fasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system, thus playing a positive role in neural restoration and regeneration through a synergistic and superimposed effect.     

Study on the damage of auditory path of the experimental autoimmune encephalomyelitis

AIM: To explore if there is lesion in the auditory path in the brainstem of the Wistar rat with experimental autoimmune encephalomyelitis（EAE）.METHODS: EAE was induced by guinea pig spinal cord homogenate (GPSCH),then the evoked potential in both EAE and control groups was examined.The HE staining and myelin staining of the auditory nucleus were conducted in order to corroborate the damage of the auditory path in the brainstem.Through studying the pathological changes using light microscopy and behavior changes of rats,we defined that the model was successful.RESULTS: The latency period of auditory evoked potentials of EAE rats was much longer than that in control group.The pathological pictures manifested that auditory nucleus were invaded by a great number of lymphocytes,demyelinations were discovered in it.CONCLUSION: The auditory path in the brainstem of the Wistar rat with EAE is suffered from inflammation and demyelination.     

Fasudil ameliorates myocardial fibrosis by regulating polarization of macrophages in diabetic mice

AIM: To investigate the effect of hydroxyl fasudil (HF) on myocardial fibrosis and macrophage polarization in the diabetic (D) mice. METHODS: C57BL/6 mice (n=60) were randomly divided into normal saline group (NS group), normal+hydroxyl fasudil group (N+HF group), diabetes group (D+NS group), diabetes+low dose of HF group (D+LHF group), diabetes+middle dose of HF group (D+MHF group) and diabetes+high dose of HF group (D+HHF group). A mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ). The mice in treatment groups received different doses of fasudil through intraperitoneal injection for 8 weeks. At the end of the study, the effects of fasudil at different doses on the body weight and blood glucose were observed. The histopathological changes of the cardiac tissues were observed by HE staining. The myocardial collagen volume fraction (CVF) was calculated by Masson staining. Immumohistochemical staining was used to test the macrophage polarization and protein expression of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and IL-10 and Western blot was applied to determine the protein levels of p-MYPT1 Thr853, inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1).RESULTS: Compared with NS group, the body weight of the mice in D+NS group was decreased and the blood glucose was increased significantly(P<0.05). However, no statistically difference of blood glucose and body weight between the treatment groups and D+NS group was observed. Compared with NS group, CVF, the number of M1-type macrophages and the protein levels of IL-6, TNF-α, p-MYPT1 Thr853 and iNOS were increased markedly, while M2-type macrophages and the expression of IL-10 and Arg-1 were decreased in D+NS group (P<0.05). Compared with D+NS group, CVF, the number of M1-type macrophages, and the protein levels of IL-6 and TNF-α were relatively decreased, conversely the number of M2-type macrophages and the protein level of IL-10 was increased in treatment groups (P<0.05). Moreover, the protein levels of p-MYPT1 Thr853 and iNOS were reduced and the protein level of Arg-1 was increased in D+MHF and D+HHF group compared with D+NS group (P<0.05). No statistical difference in above mentioned indexes between NS group and N+HF group was observed. CONCLUSION: Fasudil significantly attenuates the myocardial fibrosis of diabetic cardiomyopathy in mice, which is possibly related to increased polarization of M2-type macrophages, decreased polarization of M1-type macrophages and inflammation.     

Establishment of experimental autoimmune encephalomyelitis model in mice induced by MOG and its effect on CD4+CD25+ T cells

AIM: To investigate the feasibility of inducing experimental autoimmune encephalomyelitis(EAE)model in C57BL/6 mice by TrxA-extracellular immunoglobulin domain of MOG(MOGIgd-TrxA)fusion protein produced by molecular cloning in our laboratory. Also to investigate the role of CD4+CD25+ T cells in the pathogenesis of EAE. METHODS: (1)The MOGIgd-TrxA fusion protein was induced and produced by molecular cloning and purified by metal chelate affinity chromatography and concentrated through ultrafiltration. The concentration of the protein was measured by Bradford method at last. (2)Animal experiment: C57BL/6 mice(12 mice in each group)were used. The mice in group MOG were immunized with MOGIgd -TrxA fusion protein. The mice in group GPSCH were received emulsion of spinal cord homogenate of guinea pigs(GPSCH), and mice in group TrxA or normal control group(group NC)were received the same volume emulsion of TrxA or saline/adjuvant, respectively. Clinical scores and histopathology were measured to value the models quality. (3)The percentages of CD4+CD25+ regulatory T cells in EAE mice were tested through flow cytometric analysis. RESULTS: (1)The purity of purified MOGIgd -TrxA fusion protein was about 98%, and its concentration was 2.3 g/L. (2)No significant difference between group MOG and group GPSCH in the clinical score was observed(P>0.05). Histologic sections of the brain and spinal cord taken from affected animals in both groups showed pathological change of different level throughout the central nervous system(CNS). (3)Percentages of CD4+CD25+ T cells in group MOG and group GPSCH were(4.71±1.61)%and(1.44±0.65)%,respectively, both of which were significantly lower than those in group NC(9.22±1.24)%and TrxA group(8.97±1.20)％(P<0.01). CONCLUSION: (1)The animal model of EAE in C57BL/6 mice induced by MOGIgd fusion protein produced through molecular cloning in our laboratory is stable and with high incidence. Thus, the author finds a good way to study the immune mechanisms of MS further and to search for the effective treatments as well. (2)The reduction of CD4+CD25+ T cells in EAE mice may have some relationship with the clinic.     

Dynamic changes and significance of axon damages in experimental allergic encephalomyelitis in Wistar rats

AIM: To study the dynamic changes and significance of axon damages in different stages of experimental allergic encephalomyelitis (EAE). METHODS: Tissue sections were processed with HE staining to observe inflammatory damages. The expression and distribution of β-amyloid precursor protein (β-APP) and tau protein were detected by the method of immunohistochemistry in acute and relapsing-remitting stages of EAE. RESULTS: Many inflammatory cells were infiltrated in the tissues of spinal cord and strong β-APP expression was observed in the area full of inflammatory cells in acute EAE rats. Meanwhile, a small amount of tau protein, the marker of neurodegeneration, was found. There were many perivascular cuffings in the tissues enclosed by the inflammatory cells, and there was also strong β-APP expression in the tissues where there was no inflammatory cell infiltration in paralyzed rats. Tau protein also largely precipitated in the spinal tissues of paralyzed rats with the repeated episodes, indicating that persistence of axon damages and evident axon degeneration existed. Positive cells of β-APP and tau protein in acute group and relapsing-remitting group were larger than those in normal group with statistical differences. A statistical difference between acute group and relapsing-remitting group was also observed. CONCLUSION: Axon damages are concerned with inflammation in the spinal cord. Axon damages accompany with neural degeneration during the process of chronic EAE. The permanent damages of axon are in the course of EAE. The degeneration of axon happens in the late time of relapsing-remitting EAE and results in irreversible neurological deficits.     

Regulatory effects of spirolactone on SiO2-induced lung macrophage subtype switching

AIM: To investigate the influence of spirolactone (SPI) on mouse pulmonary macrophage subtype switching induced by silicon dioxide (SiO₂). METHODS: C57BL/6 mice were divided into normal saline (NS) group, SiO₂ group, SiO₂+SPI group and SiO₂+NS group. A mouse model of silicosis was developed with crystalline SiO₂ particles (40 mg/kg, via oropharyngeal instillation). SPI (20 mg/kg) or (NS) was delivered daily by oral gavage after SiO₂ administration. The mice were sacrificed on days 3, 14 and 28. Alveolar washing, total cell counting, differential cell counting and flow cytometry analysis were performed. The right lower lobe of lavaged lungs was collected to prepare single-cell suspension for flow cytometry analysis. RESULTS: Compared with SiO₂ group, SPI treatment significantly alleviated SiO₂-induced inflammatory cell accumulation in brochoalveolar lavage fluid on day 3 and 14. Compared with NS group, the M1 alveolar macrophages and M1 pulmonary interstitial macrophages in SiO₂ group switched to M2 subtype dramatically, while SPI treatment reversed the switching effectively.CONCLUSION: SPI treatment alleviates SiO₂-induced inflammatory cell accumulation, which may be related to reversing macrophage subtype switching.     

Relationship between hippocampal structure and high incidence of depression after spinal cord injury in mice

AIM: To explore whether the high risk of depression caused by spinal cord injury is related to structural changes of hippocampus. METHODS: Mouse spinal cord injury model was established. The Basso Mouse Scale (BMS) was used to evaluate the postoperative motor function of mice at different periods after injury. The depression of mice was evaluated by behavior experiment. The morphological changes of cells in hippocampal tissue were observed by HE staining. The structural changes of hippocampal subcellular synapses were observed by electron microscopy. RT-qPCR was used to detect the changes of synaptic protein markers synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) at the mRNA level. RESULTS: The mice had significant depressive behaviors in model group, and the depression degree was the most serious on the 7th day (P<0.01). Compared with control group, the arrangement of hippocampal cells was disordered, the cell number was decreased, and the shape was irregular in model group. Compared with control group, the observation results of electron microscopy showed that the number of synapses in the hippocampal ultrastructure was decreased, the length of synaptic activity zone was shortened, the thickness of postsynaptic density was thinned, the width of synaptic gap was increased, and the curvature of synaptic interface was decreased in the model group (P<0.05). The mRNA expression levels of SYP and PSD-95 in model group were lower than those in control group (P<0.05). CONCLUSION: The change of hippocampal structure is one of the important reasons leading to high risk of depression caused by spinal cord injury.     

Effects of amifostine on formation of abdominal aortic aneurysm in mice induced by benzo[a] pyrene

AIM: To study the role of amifostine on the formation of benzo[a]pyrene (BaP)-induced abdominal aortic aneurysm (AAA) in C57BL/6J mice and the underlying mechanism. METHODS: RAW246.7 mononuclear macrophage in vitro were divided into control group, DMSO group, BaP group, low dose (1 μmol/L) amfostine treated group, middle dose (5 μmol/L) amfostine treated group and high dose (25μmol/L) amfostine treated group. The influence of BaP on the expression of matrix metalloproteinase (MMP)-9, MMP-12, TNF-α, NF-κB in the RAW246.7 mononuclear macrophages in vitro was determined by Western blot. Male C57BL/6J mice (8 months old) were divided into control group, model group (AngII+BaP group), low dose (50 mg/kg) amfostine treated group and high dose (100 mg/kg) amfostine treated group. After 6 weeks, the abdominal aorta were isolated. The aortic tissues were subjected to HE and Masson staining. The vascular wall structure, infiltration of macrophage, the expression of MMP-9, MMP-12, TNF-α, NF-κB were evaluated by Western blot and immunochemistry staining. RESULTS: Amifostine attenuated BaP-induced expression of TNF-α, MMP-9, MMP-12, NF-κB in the RAW246.7 mononuclear macrophages (P<0.05). The results of animal experiments showed that the incidence of AAA in high dose amifostine treated group were significantly lower than that in low dose amifostine treated group and model group (P<0.05). Immunohistochemistry staining observation showed that amifostine inhibited the aortic macrophage infiltration more obviously in high amifostine treated group compared with model group and low dose amifostine treated group (P<0.05). Compared with model group and low dose amifostine treated group, the MMP-9, MMP-12, TNF-α and NF-κB expression of abdominal aorta in high amifostine treated group was reduced significantly (P<0.05). CONCLUSION: Amifostine inhibits BaP-induced activation of macrophages, and also prevents the formation of abdominal aortic aneurysm in C57BL/6J mice induced by BaP by inhibition of the NF-κB pathway, macrophage infiltration and the expression of TNF-α and MMPs.     

Matrine attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in mice

AIM: To investigate the influence of matrine (MA) on the phenotype switching of mouse monocytes and alveolar macrophages induced by bleomycin (BLM).METHODS: All mice were randomly divided into normal saline (NS) group, BLM group, BLM+NS group and BLM+MA group. The mice were administered with BLM at 2.5 mg/kg via oropharyngeal instillation. The mice in BLM+MA group were treated with MA (15 mg·kg^-1·d^-1) by oral gavage following BLM administration. The mice were sacrificed on days 3, 7, 14, and 21. The lungs were removed for pathological analysis. The circulating monocyte subsets and polarization state of bronchoalveolar lavage fluid (BALF)-derived alveolar macrophages were analyzed by flow cytometry.RESULTS: The results of HE and Masson trichrome staining in BLM and BLM+NS groups exhibited classical pathological stages of lung fibrosis, including acute inflammation phase and later fibrosis phase. Compared with BLM+NS group, MA treatment alleviated the inflammatory response and the degree of fibrosis induced by BLM (P<0.05). There was a rapid change of circulating Ly6C^hi monocytes and its magnitude was positively associated with the pulmonary inflammatory response. An expansion of M2-like alveolar macrophages was positively correlated with the magnitude of lung fibrosis. Moreover, MA treatment partially normalized the phenotype switching of monocytes and alveolar macrophages.CONCLUSION: Matrine treatment attenuates BLM-induced pulmonary injury partially via modulating the phenotype switching of monocytes and alveolar mocrophages.     

Polydatin inhibits lipid peroxidation induced by ox-LDL and down-regulates CD36 expression in macrophages

AIM: To investigate the inhibitory effect of polydatin on the reaction of oxidation and regulation of CD 36 expression in mouse macrophages. METHODS: C57/BL6 mice were divided into two groups and injected with normal saline or 1.5% polydatin (0.2 mL/d) for 3 days. On the 5th d after the first injection, the mice were sacrificed and abdominal macrophages were collected. The activity of superoxide dismutase (SOD), the levels of oxidative burst and lipid peroxide (LPO), and the expression of CD36 in the cells were assayed. RESULTS: In polydatin group, SOD activity in the macrophages was higher, the oxidized low density lipoprotein(ox-LDL) induced macrophage respiratory burst level and the accumulation of lipid peroxide (LPO) were significantly lower than those in PBS control group. Polydatin also prevented ox-LDL-induced expression of scavenger receptor CD36 on the macrophages. CONCLUSION: Polydatin enhances the anti-oxidant ability in macrophages by inhibiting the mitochondrial oxidative burst and expression of scavenger receptor CD36, indicating that polydatin is an appropriate reagent for anti-aging and anti-atherosclerosis.     

Botulinum neurotoxin type A heavy chain intervenes in expression of general proteins in spinal cord after unilateral spinal cord injury in rats

AIM: To observe the effect of botulinum neurotoxin type A heavy chain (BoNT/A HC) on the pattern of spinal protein expression by intrathecal injection after spinal cord injury in rats, and to explore the role of BoNT/A HC intervention in spinal protein expression and some of its mechanisms in nerve regeneration after injury. METHODS: The model of unilateral lumbar spinal cord injury was established. The effects of BoNT/A HC intervention at different doses (2 μg, 4 μg, 6 μg and 8 μg) on the general pattern of protein expression in the spinal cord tissues at the injury site and the cranial part adjacent to the injury site was measured and evaluated by SDS-PAGE and Coomassie brilliant blue staining first, and then by two-dimensional SDS-PAGE. RESULTS: The histological structure of the ipsilateral side of lumbar spinal cord showed obvious destruction and degradation, mainly affecting both gray and white matter of the left side of the cord. The result of SDS-PAGE with Coomassie brilliant blue staining from injured spinal cord tissue displayed that the expression of some proteins after one-time BoNT/A HC treatment appeared obviously different from that without BoNT/A HC treatment. Moreover, the pattern of the protein expression affected by BoNT/A HC was similar to that of the normal spinal cord. The more detail information from two-dimensional SDS-PAGE indicated that more than 10 proteins with different molecular weight and isoelectronic points were differentially expressed at day 2 and day 20 after local injection of 6 μg BoNT/A HC. This altered expression actually appeared a tendency toward the pattern shown in normal group. CONCLUSION: The immediate application of BoNT/A HC at the injury site after unilateral lumbar spinal cord injury is able to affect the pattern of local protein expression. The altered protein expression by injury could be reversed back to normal or approximately normal by local BoNT/A HC administration.     

Effects of PPARγ agonist on calpain expression in model of acute experimental autoimmune encephalomyelitis

AIM: To explore the effects of peroxisome proliferator-activated receptor γ （PPARγ） agonist on calcium-activated neutral proteinase, calpain, expression in the brain of rats with acute experimental autoimmune encephalomyelitis (EAE). METHODS: EAE model was established in rats by inoculating the homogenate contained spinal cord of guinea pig and complete Freunds adjuvant. Respectively, the PPARγ agonist rosiglitazone and non-steroid anti-inflammatory drug (NSAID) ibuprofen were used to treat the EAE rats. Outcome measures (Kohs scale) were applied at baseline and after treatment to assess the improvement of clinical symptoms. Calpain expression levels were detected by RT-PCR and Western blotting. RESULTS: All the groups showed significant improvements of scales scores after treatment with rosiglitazone and ibuprofen. No significant difference of the expression of calpain mRNA was found among EAE group, rosiglitazone and ibuprofen groups (P>0.05), but the expression of calpain reduced markedly in rosiglitazone and ibuprofen groups compared with that in EAE group (P<0.05). CONCLUSION: Rosiglitazone and ibuprofen inhibit the expression of calpain and improve the clinical symptoms of EAE rats. PPARγ agonist plays a neuroprotective role in EAE rats.     

Protective effects of grape polyphenol on pancreatic tissues of mice with caerulein-induced acute pancreatitis

AIM:To investigate the effects of grape polyphenol (GP) on caerulein-induced acute pancreatitis (AP) in mice. METHODS:Two-month-old female mice of ICR strains (n=21) were randomly divided into 3 groups: normal control (NC) group, AP group, and GP-treated AP group. Before AP induction, the mice in GP-treated AP group were continuously administrated with 1.5 g/kg GP aqueous solution by gavage for 7 d, while those in NC group and AP group were treated with saline as a vehicle control. On the 7th day, the mice in AP group and GP-treated AP group were intraperitoneally injected with caerulein (50 μg/kg) in 1 h interval for 7 serial injections in total. The mice in NC group were treated with saline according to the same procedure in experimental group. All the mice were sacrificed 24 h after AP induction, and the pancreatic tissues and lung tissues were harvested for further investigation of the pathological changes, macrophages infiltration, myeloperoxidase (MPO) activity and expression of inflammatory and oxidative stress factors. RESULTS:Compared with AP group, the mice in GP-treated AP group showed milder morphological changes and lower pathological scores, including the scores of edema, inflammation and vacuolization (P<0.05), but the necrosis scores and total scores showed no statistical difference between these 2 groups. Besides, the mice in GP-treated AP group had fewer macrophage infiltration, lower lung MPO activity (P<0.01), and lower expression of inflammatory factors, tumor necrosis factor α (TNF-α)and monocyte chemotactic protein 1 (MCP-1) (P<0.05), and oxidative stress factors, superoxide dismutase (SOD)-1, SOD-2 and NADPH oxidase 2 (NOX-2) (P<0.01). CONCLUSION: Grape polyphenol has remarkable protective effect on pancreatic tissues of mice with caerulein-induced acute pancreatitis, and the mechanisms may be related to down-regulation of inflammatory and oxidative stress factors.     

Aberrant SYK expression in cholangiocarcinoma and its effect on M2 macrophage polarization in tumor microenvironment

AIM To investigate the expression of spleen tyrosine kinase (SYK) in 2 murine cholangiocarcinoma (CCA) progressive models and human CCA tissues, and to explore the effects of SYK expression on the polarization of M2 macrophages. METHODS SD rats were given drinking water containing thioacetamide (TAA) daily. BALB/c mice were treated with left median bile duct ligation combined with diethylnitrosamine (DEN) administration. The expression of SYK and M2 macrophage infiltration (CD163) in the animals and human CCA tissues were detected by immunohistochem?istry, and their correlation was analyzed. Ten SD rats, which were given drinking water containing TAA daily for 24 weeks, were randomly divided into control group (n=5) and SYK inhibitor group (n=5). After the rats received SYK inhibitor through gavage for 4 weeks, the effects of SYK inhibitor on tumor growth and macrophage polarization were analyzed. RESULTS The hepatic bile duct hyperplasia, dysplasia (or cholangioma), and CCA occurred at different time points in the rats and mice. During the development of CCA, SYK expression was gradually increased (P<0.01), accompanied by enhanced infiltration of M2 macrophages, while the M1 macrophages were decreased in the hepatic bile duct tissues (P<0.01). SYK and CD163 expression levels were significantly up-regulated (P<0.01), and a positive correlation (r=0.57, P<0.01) in human CCA tissues was observed. In the rat CCA model, the number of tumor nodules and infiltration of M2 macrophages in SYK inhibitor group were decreased compared with its control group (P<0.01). CONCLUSION In the murine CCA models, SYK expression was progressively increased during the evolution of CCA, which may promote tumor progression via the polarization of M2 macrophages, and SYK inhibitor effectively inhibits the tumor growth and M2 macrophage polarization in the rat CCA model.     

Role of CD163/TWEAK pathway in atherosclerosis

AIM:To investigate the effect of CD163/tumor necrosis factor-like weak inducer of apoptosis (TWEAK) pathway on atherosclerosis in mice. METHODS:APOE^-/- mice and wild-type (WT) C57BL/6 mice were divided into 4 groups (8~10 weeks, n=10):APOE^-/- +normal diet (ND) group, APOE^-/- +western diet (WD) group, WT+ND group, and WT+WD group. Detection of blood lipid levels and oil red O staining of aorta artery were performed to confirm whether the atherosclerotic model was well established in 16 weeks after feeding. The aortic tissues were harvested to measure CD163 and TWEAK protein levels by Western blot, and immunohistochemical staining was also performed to localize CD163 and TWEAK protein expression on atherosclerotic plaque in each group. The cell experiments were conducted to study whether CD163 regulated TWEAK expression in M1 macrophages and foam cells, and the possible downstream pathway was investigated. RESULTS:The blood lipid levels and aorta oil red O staining showed that the animal model of atherosclerosis was successfully established in APOE^-/- +ND group and APOE^-/- +WD group. The protein level of CD163 was significantly increased in the aortic tissue in APOE^-/- mice (P<0.05) as compared with C57BL/6 mice (P<0.05). Consistently, the protein level of TWEAK was also markedly higher in APOE^-/- +ND group and APOE^-/- +WD group than that in WT+ND group and WT+WD group (P<0.05). Immunohistochemical staining showed that CD163 was mainly expressed in the parts away from the lipid core, and TWEAK was found in all parts of the atherosclerotic plaque. CD163 significantly inhibited the protein expression of TEWAK in the M1 macrophages, and also significantly down-regulated the level of nuclear factor-κΒ (NF-κB) in the M1 macrophages and foam cells (P<0.05). CONCLUSION:The protein levels of CD163 and its ligand TWEAK are significantly increased in atherosclerotic mice. The CD163 positive macrophages are mainly located at the site far away from the lipid core, and CD163 may play an anti-atherosclerotic effect by inhibiting TWEAK/NF-κB pathway.