Simultaneous Determination of 4 Kinds of Components in Yishen Yansi Pills by Double Wavelength HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of ferulic acid,catalpol,emodin and emodin methylether in Yishen yansi pills. METHODS:Double wavelength HPLC method was adopted. The separation was performed on Kromasil-C18 column with mobile phase methanol-0.1% phosphate solution(78:22,V/V)at flow rate of 1.0 mL/min. The detection wavelengths were set at 210 nm for catalpol,280 nm for ferulic acid,emodin and emodin methylether. The column temperature was 30 ℃,and sample size was 10 μL. RESULTS:The linear ranges were 0.0908-2.2700 μg/mL for ferulic acid(r=0.9999), 0.1990-4.9750 μg/mL for catalpol (r=0.9993),0.3540-8.8500 μg/mL for emodin (r=0.9999),0.3360~8.4000 μg/mL for emodin methylether(r=0.9997),respectively. The limits of quantitation were 14.66,8.75,10.26,13.68 ng,and the limits of de-tection were 5.66,2.67,3.78,4.25 ng,respectively. RSDs of precision,stability and reproducibility tests were lower than 2.0%. The recoveries were 101.15%-102.73%(RSD=0.58%,n=6),99.20%-101.69%(RSD=1.20%,n=6),100.90%-101.36%(RSD=0.16%,n=6),95.64%-98.96%(RSD=1.49%,n=6),respectively. CONCLUSIONS:The method is accurate,reliable,simple and suitable for simultaneous determination of 4 kinds of components in Yishen yansi pills.

Immunoregulatory effect of BuyangHuanwu Decoction on experimental autoimmune encephalomyelitis mice / 中国免疫学杂志

Objective:To explore the anti-inflammatory therapeutic effect and possible immunoregulatory mechanism of Buyang Huanwu Decoction (BYHWD) on the development of experimental autoimmune encephalomyelitis (EAE). Methods:Female C57BL/6 mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein peptides ( MOG35-55 ) ,and randomly divided into saline group,BYHWD group,with 13 mice in each group. At the 3th day,25 ml/kg of saline was orally given to each mouse of saline group,50 g/kg ig of crude BYHWD was orally given to each mouse in BYHWD group for 25 days. Clinical score and body mass were recorded every day. Inflammatory cell infiltrations of spinal cord were observed by HE staining Myelin staining observes the demyelination situa-tion. And the expression of ROCKⅡ in spleen was detected by immunofluorescence staining. The subtypes of CD4+ T cells were analyzed by flow cytometry. Western blot was used to detect the expression of TLR4,Myd88,NF-κB,COX-2,ROCKⅡ in spinal cord and ROCKⅡ in brain. Results: The neurologicalscore significantly decreased in EAE mouse of BYHWD group compared with the saline group (P<0. 001) . BYHWD inhibited the inflammatory cell infiltration and demyelination in the nervous centralis(P<0. 05). The treatment of BYHWD effectively reduced the increased the proportion of CD25+(P<0. 05),IL-10+(P<0. 05),TGF-β+(P<0. 01), IFN-γ+( P<0. 05 ) CD4+T cells , and inhibited the expression of peripheral and central ROCKⅡ( P<0. 05 ) ;BYHWD reduced the expression of TLR4,MyD88,NF-κB,COX-2 in spinal cord (P<0. 05). Conclusion:BYHWD can exert anti-inflammatory and immune regulation effect by the inhibition of ROCKⅡ/TLR4/ NF-κB signaling pathway and regulation of the proportion of peripheral T cell sub-sets.

Immunoregulatory effect of Buyang-Huanwu decoction on monocyte-mac-rophages in mice with experimental autoimmune encephalomyelitis / 中国病理生理杂志

AIM:To explore the therapeutic effect of Buyang-Huanwu decoction (BYHWD) on experimental au-toimmune encephalomyelitis ( EAE) and its immunoregulatory effect on monocyte-macrophages .METHODS: Chronic EAE was induced by myelin oligodendrocyte glycoprotein peptide fragment 35-55 ( MOG35-55 ) in the female C57BL/6 mice, which were randomly divided into saline group and BYHWD group .On day 3 after immunization , the mice in BYHWD group were orally administrated with BYHWD , while normal saline was given to the control mice .The clinical score and body mass were recorded every other day .At day 17 after immunization , the mice were sacrificed and spinal cords were obtained for HE staining and myelin staining .The M1 and M2 macrophage phenotypes of splenic cells were detected by flow cytometry and immunofluorescence staining .The protein expression of iNOS , TNF-α, arginase and IL-10 in the spinal cord macro-phages was determined by Western blotting .RESULTS:BYHWD delayed the onset of EAE , reduced the clinical scores of EAE, inhibited the inflammatory cell infiltration and demyelination in the spinal cord , and promoted the conversion of M 1 macrophages into M2 phenotype in the spinal cord and spleen .CONCLUSION:BYHWD intervention attenuates the be-havioral and pathological changes in the EAE mice , and its mechanism may be related to the macrophage conversion .

Immunoregulative effect of Fasudil on encephalomyelitic T cells in experimental autoimmune encephalomyelitis mice / 中南大学学报(医学版)

OBJECTIVE@#To explore the therapeutic effect of Fasudil-modified splenic mononuclear cells (MNCs) in experimental autoimmune encephalomyelitis (EAE) and the possible mechanisms.
@*METHODS@#C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to establish active immunity EAE model. Splenic MNCs were isolated on the 9th day after immunization and treated with or without Fasudil for 72 h in vitro. These cells were collected for analysis of the variance of T cell subtypes, the level of cytokines and the activity of Rho kinase (ROCK). MNCs (5×107 cells) were resuspended in 500 µL of phosphate buffer solution (PBS) and transferred into EAE model (intraperitoneal injection), which was divided into a PBS-MNCs group and a Fasudil-MNCs group. Changes of body weight and clinical symptom scores were observed.
@*RESULTS@#Splenic encephalitogenic MNCs from EAE mice on the 9th day after immunization could establish passive transfer EAE model. But Fasudil-treated MNCs did not trigger EAE development. Compared with the PBS-MNCs group, the loss of body weight was less in the Fasudil-MNCs group. The in vitro experiment indicated that Fasudil could suppress the activity of ROCK on MNCs (P<0.01), decrease the percentage of CD4+ T cells with the expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) (IFN-γ: P<0.01; IL-17: P<0.05), while increase the secretion of CD4+ T cells with the expression of transforming growth factor-β (TGF-β) and IL-10 (all P<0.001) . Furthermore, Fasudil could inhibit the release of IL-17 (P<0.001) and enhance the level of IL-10 (P<0.05).
@*CONCLUSION@#Fasudil-modified cell therapy affects the occurrence and development of EAE by inhibiting the inflammatory reaction of helper T cell 1 (Th1) and Th17 while enhancing the immunoregulative effect of Th2.

The immunoregulatory effect of Rho kinase inhibitor Fasudil on macrophages in a mouse model of ;experimental autoimmune encephalomyelitis / 中华微生物学和免疫学杂志

Objective To investigate the immunoregulatory effect of Fasudil-modified macrophages on cell transferred experimental autoimmune encephalomyelitis ( EAE) in a mouse model.Methods Fe-male C57BL/6 mice were immunized with MOG35-55 to establish the model of EAE.The encephalomyelitic mononuclear cells ( MNCs) were isolated from spleen of mice with EAE on day 9 after immunization and treated with or without Fasudil for 72 h in vitro.Several assays including the flow cytometry analysis, Griess reaction and ELISA were performed to analyze the M1 and M2 phenotypes of macrophages, the production of NO and the levels of cytokines, respectively.The cultured MNCs (5×107 cells) were resuspended in 500μl of PBS and transferred into na?ve C57BL/6 recipients via intraperitoneal injection.Two groups including the PBS-MNCs group and the Fasudil-MNCs group were set up.The body weights and clinical scores of the mice in each group were recorded in every other days after the induction of EAE in the recipients.Results The Fasudil treated MNCs affected the induction of EAE in adoptive cell transferred mice.The expression of CD16/32, iNOS and IL-12 on F4/80-macrophages were decreased, while the expression of CD206, CD23 and IL-10 on F4/80-macrophages were increased upon the treatment of Fasudil, indicating that Fasudil im-proved the differentiation of macrophages from M1 to M2 phenotypes.Moreover, Fasudil inhibited the pro-duction of NO and enhanced the expression of Arginase-1.Conclusion Fasudil ameliorated the clinical se-verity of EAE in mice by promoting the transformation of macrophages from M1 to M2 phenotype.

Lipoic acid protects dopaminergic neurons in LPS-induced model of Parkinson’s disease / 中国病理生理杂志

AIM:To evaluate the effect of lipoic acid ( LA) on LPS-induced Parkinson disease ( PD) model of mice.METHODS:Female C57BL/6 mice of 10-month-old were randomly divided into saline control group , PD group and LA group.The PD mouse model was induced by intranasal instillation of LPS .Assays of tyrosine hydroxylase , microglia and nuclear factor kappa B ( NF-κB) were performed by the methods of immunohistochemistry and Western blotting .RE-SULTS:Intranasal LPS instillation exhibited basic characteristics of PD model .However, LA administration significantly improved motor dysfunction , protected dopaminergic neurons from damage , and inhibited NF-κB activation in inflammatory microglia in the substantia nigra area of the brain .CONCLUSION:LA may exert a profound neuroprotective effect by an-ti-neuroinflammatory reaction to arrest the progression of PD .

Study on a novel Rho kinase inhibitor WAR5 for treating EAE / 中国病理生理杂志

AIM:To explore the therapeutic effect of a novel Rho kinase inhibitor WAR 5 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 MOG 35-55 peptide.WAR5 was in-jected 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 mye-lin staining .The splenocytes were isolated and the expression of CD 16/32 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 WAR 5 delayed the onset of EAE and attenuated the clini-cal symptoms .The results of the pathological examination revealed that WAR 5 inhibited the infiltration of inflammatory cells and improved myelination in spinal cords , accompanied with the poralization of M 1 macrophages to M2 phenotype in the spleen.WAR5 inhibited the expression of iNOS in the central nervous system , especially in the spinal cords .CON-CLUSION: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 .

Erythropoietin ameliorate experimental allergic encephalomyelitis / 中国免疫学杂志

Objective:To investigate the beneficial effect of erythropoietin(EPO)on experimental allergic encephalomyelitis(EAE).Methods: The severity of EAE mice with or without EPO therapy were evaluated through clinical symptoms score and histological observation.Mononuclear cells from spleens,lymph nodes, brains and lumber spinal cords were applied to flow cytometry to detect ratios of CD4~+T subgroup, scales of apoptotic and dead cells, levels of inflammatory cytokines.LDH release of peripheral mononuclear cells were examined after incubating with EPO at different concentrations.Results: EPO ameliorated clinical symptoms and infiltration of pathological inflammatory cells in EAE mice(P＜0.05); Ratio of CD4~+T subgroup, levels of IL-17 and IFN-γ in EAE mice with EPO therapy were significantly lower than those of EAE control group(P＜0.05).No statistical significant differences existed on scales of dead cells, levels of IL-10 and CD4~+ CD25~+ T cells (P＞0.05).EPO at the concentrstions of 1-1 000 U/ml had no effect on LDH release of peripheral mononuclear cells(P＞0.05).Conclusion: EPO play the role of neuroprotecion in EAE mice by the way of decreasing infiltrating inflammatory cells and lowering the levels of IL-17 and IFN-γ.

Comparison on the clinic features and neuromyelitis optica-IgG positive rate of neuromyelitis optica and multiple sclerosis / 中华神经科杂志

Objective To compare the clinic features of neuromyelitis optica (NMO) and multiple sclerosis(MS).To compare the positive rate of NMO-IgG in NMO,MS and other related diseases,and determine whether it can be considered as a biomarker for differential diagnosis.Methods Detected serum NMO-IgG in 34 NMO patients,22 MS patients,24 high risk syndrome,5 clinical isolated syndrome,and 35 patients with other neumlogical diseases.Compared the clinic features(onset age,severity,prognosis,MRI lesions,autoimmune antibodies,CSF)of 34 NMO patients and 22 MS patients.Results The onset age of NMO is older than that of MS.It is more severe and with worse prognosis than MS.Longitudinal spinal cord lesions are easily found in NMO.NMO-IgG positive rate in NMO and high risk syndrome patients are 58.8% (20/34)and 45.8%(11/24)respectively,which are higher than that in MS(1/22),clinical isolated syndrome(1/5)and other neurological diseases(1/35;x2=37.2,P<0.01).The positive rate may have a relationship with the length of spinal cord lesions.Condusions MS and NMO are probably different diseases.NMO-IgG positive rate in NMO is significandy higher than that in MS,it can be considered as a biomarker for differential diagnosis.

Cerebral ischemia induces astrocytes to express granulocyte colony stimulating factor and glial cell fine-derived neurotrophic factor / 中华神经科杂志

Objective To explore the neuroprotective mechanisms of granulocyte colony stimulating factor (G-CSF) in ischemic brain injury. Methods Brain tissues were taken out from MCAO and sham operated Sprague-Dawley rats 7 days after operation. The expression of G-CSFR, GDNF, MAP2 and GFAP was measured by using immunofluorescence co-staining. Results The expression of G-CSFR and GDNF were widely distributed in the neurons in normal brain tissues, not in the astrocytes. However, in ischemic peripheral zone, part of G-CSFR and GDNF positive cells merged with GFAP positive cells, in normal brain tissues, most G-CSFR positive cells were co-expressed with GDNF. Conclusion Cerebral ischemia induces astrocytes to express G-CSFR and GDNF, suggesting that endogenous neuroprotection by cerebral ischemia may be related with the expression and production of G-CSFR and GDNF in astrocytes in ischemic peripheral zone.

A comparative evaluation of enzyme linked immunospot assay and IS6110 polymerase chain reaction for early diagnosis of tuberculous meningitis / 中华神经科杂志

Objective To establish an early diagnostic test for tuberculous meningitis (TBM) with good sensitivity and specificity. Methods Twenty-five patients with a clinical diagnosis of TBM and 49 controls, including 27 patients with other infectious diseases of central nervous system and 22 patients with noninfectious neurological diseases, were enrolled in our research. We simultaneously detected antimycobacterium bovis BCG IgG secreting cells in both cerebral spinal fluid (CSF) and peripheral blood (PBL)by enzyme linked immunospot assay(ELISPOT),repeated insertion sequence IS6110 specific for mycobacterium tuberculosis in CSF by PCR and anti-BCG IgG titre in both CSF and PBL by enzyme linked immunosorbent assay(ELISA).Results The sensitivity of ELISPOT was 84.0%,much higher than that of PCR(75.0%)and ELISA(52.3%).The specificities of the three tests were 91.8%,93.7%and 91.8%respectively.The numbers of CSF cells secreting anti-BCG IgG tested by ELISPOT were even higher in the early phase of TBM, but declined along with the disease progressing(t=-3.183,P=0.008),which allowed an early diagnosis to be made. Conclusion ELISPOT technique is proved to be the most valuable test for the early diagnosis of TBM.

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens / 中国病理生理杂志

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-? release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.

An experimental study of dendritic cell-based vaccine therapy for intracranial gliomas / 中国免疫学杂志

Objective:To investigate the therapeutic efficacy of immunotherapy with dendritic cells-based vaccine against intracranial gliomas in rats.Methods:C6 glioma cells were injected into the brain of Wistar rat under stereotactic monitor to establish an animal model of glioma.The precursors of dendritic cells were isolated from bone marrow of rats,stimulated in vitro with recombinant rat granulocyte-macrophage colony-stimulating factor(rrGM-CSF)and interleukin-4(rrIL-4).Cultured cell populations were confirmed to be functional DCs.These DCs were then pulsed ex vivo with C6 tumor-lysates prepared by three cycles of freezing to -80℃ and thawing to 0℃ and subsequently injected subcutaneously into rats harboring intracranial C6 tumor.Rats from different group were treated with five weekly subcutaneous injections of either control media,unpulsed DCs,or DCs pulsed with tumor-lysates.The animals were followed for survival,the percentage of CD8 +T cells in peripheral blood and cytotoxicity assay in vitro were determined by FACS.The level of cytokine IFN-? and IL-10 were detected by ELISA.Results:The results indicated that C6 glioma model rats treated with tumor-lysate-pulsed DCs led to prolonged survival time,increased the percentage of CD8 + T lymphocyte in peripheral blood in comparing with control group.Cytotoxicity assays suggest that vaccination with these tumor-lysate-pulsed DCs can induce specific cytotoxic T lymphocytes against C6 tumor cells compared with control group.Furthermore,significantly enhanced IFN-? and reduced IL-10 (even undetectable)were observed in rats treated with pulsed-DCs.Conclusion:Data supported the therapeutic efficacy of systemic vaccination with DCs pulsed with tumor-lysates against intracranial glioma.