Immunomodulatory effect of astragaloside IV on T cells of experimental autoimmune encephalomyelitis mice / 中国组织工程研究

BACKGROUND:In the initial stage of multiple sclerosis,central immune cells activate and release a large number of inflammatory factors,causing white matter demyelination and even involving gray matter neurons.The equilibrium of differentiation between different subsets of CD4+ T cells plays an important role in the progression of experimental autoimmune encephalomyelitis.The previous results of the research group showed that the active ingredient astragalus glycoprotein in astragalus can regulate the immune response in experimental autoimmune encephalomyelitis mice,and whether it has a regulatory effect on the differentiation of T cell subsets has not been determined. OBJECTIVE:To explore the therapeutic effects and immune regulatory mechanisms of astragaloside IV on experimental autoimmune encephalomyelitis mice. METHODS:Female C57BL/6 mice were divided into the normal control group,experimental autoimmune encephalomyelitis disease model group,and astragaloside IV treatment group(n=8 per group).Myelin oligodendrocyte glycoprotein peptides 35-55 were used for experimental autoimmune encephalomyelitis model induction in the last two groups.On day 10 to 28 after immunization,the astragaloside IV treatment group was treated with 40 mg/kg per day astragaloside IV intragastrically.Body weight and clinical scores of mice in each group were recorded from the immunization day to the 28th day.On the 28th day after immunization,the mouse spinal cord was taken and made into frozen sections for hematoxylin-eosin staining and Lux fast blue staining to observe pathological changes in the spinal cord.Percentage of splenic T cell subsets was detected using flow cytometry.Western blot assay was used to determine the protein expression of interferon-γ,interleukin-17 and interleukin-6 in the spinal cord.Levels of interferon-γ,interleukin-17,interleukin-6 and interleukin-4 in supernatants of cultured splenocytes were determined by ELISA. RESULTS AND CONCLUSION:(1)Compared with the experimental autoimmune encephalomyelitis disease model group,astragaloside IV could reduce the degree of weight loss in experimental autoimmune encephalomyelitis mice(P<0.05),ameliorate clinical symptoms(P<0.05),inhibit the infiltration of inflammatory cells and alleviate myelin loss(P<0.01,P<0.05).(2)Compared with the experimental autoimmune encephalomyelitis disease model group,astragaloside IV could inhibit the proportion of CD4+T cell subsets expressing interferon-γ(P<0.001)and interleukin-17(P<0.001),but increase percentages of CD4+ interleukin-10+(P<0.001)and CD4+ transforming growth factor-β+(P<0.01)T cell subsets.(3)Astragaloside IV could inhibit the expression of interferon-γ(P<0.05,P<0.01),interleukin-17(P<0.05,P<0.05),and interleukin-6(P<0.05,P<0.05)in the spinal cord and spleen,and up-regulate the expression of interleukin-4(P<0.01)in spleen.(4)These findings confirm that astragaloside IV alleviates clinical symptoms in experimental autoimmune encephalomyelitis mice,which may be related to regulating the splenic T cell subsets,therefore,inhibiting the infiltration of inflammatory cells into the center and reducing the demyelination.

Astragaloside inhibits astrocyte activation and inflammatory response induced by inflammation / 中国组织工程研究

BACKGROUND:Astrocytes play an important role in the pathology of central nervous system diseases.The phenotypic and functional changes in astrocytes suggest that it may be an effective therapeutic target for central nervous system diseases.Our previous studies have confirmed that astragaloside can inhibit the lipopolysaccharide-induced astrocyte inflammatory response.Whether astragaloside can regulate the phenotype and function of astrocytes through Notch-1 and its downstream signaling pathway remains unclear. OBJECTIVE:To explore the effect of astragaloside on astrocyte activation and inflammatory response induced by inflammation and its possible mechanism. METHODS:Cerebral cortex astrocytes derived from neonatal C57BL/6 mouse were cultured in vitro.CCK-8 assay was used to determine the optimum concentration of astragaloside and Notch active inhibitor DAPT.The astrocytes were divided into five groups:PBS group,lipopolysaccharide group,lipopolysaccharide + astragaloside group,lipopolysaccharide + DAPT group and lipopolysaccharide + DAPT + astragaloside group.The secretion level of inflammatory factors was detected by ELISA,and the level of nitric oxide was detected by Griess method.The astrocytes and splenic mononuclear cells were co-cultured in Transwell chamber to observe the migration of CD4T cells.The expression of astrocyte activation marker GFAP,A1 marker C3 and A2 marker S100A10 as well as Notch 1 and Jag-1 was detected by immunofluorescence staining.The expressions of CFB,C3,S100A10,PTX3,Notch-1,Jag-1,and Hes were detected by western blot assay. RESULTS AND CONCLUSION:(1)According to the results of CCK8 assay,the final concentration of astragaloside was selected as 25 μmol/L and the final concentration of DAPT was 50 μmol/L for follow-up experiments.(2)Compared with PBS group,interleukin-6,interleukin-12 and nitric oxide secretion levels in the lipopolysaccharide group were significantly increased(P<0.05,P<0.05,P<0.01).Compared with the lipopolysaccharide group,interleukin-6(all P<0.05),interleukin-12(P>0.05,P<0.05,P<0.05)and nitric oxide(P<0.05,P<0.01,P<0.01)secretion significantly reduced in the lipopolysaccharide + astragaloside group,lipopolysaccharide +DAPT group,lipopolysaccharide + DAPT + astragaloside group.(3)Compared with the PBS group,the expression of GFAP that is the marker of activated astrocytes and the migration of CD4 T cells were significantly increased in the lipopolysaccharide group(P<0.01).Compared with the lipopolysaccharide group,astrocyte activation was significantly inhibited and CD4 T cell migration was significantly reduced in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group(P<0.05,P<0.05,P<0.01).(4)Compared with the PBS group,the expressions of A1 markers C3 and CFB in the lipopolysaccharide group were increased(P<0.01,P<0.05),and the expressions of A2 markers S100A10 and PTX3 were decreased(P<0.01,P<0.05).Compared with the lipopolysaccharide group,C3(all P<0.01)and CFB(both P<0.05)were significantly reduced and S100A10(all P<0.01)and PTX3(P<0.05,P<0.05 and P>0.05)were increased in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group.(5)Compared with the PBS group,the expressions of Jag-1,Notch-1 and Hes in the lipopolysaccharide group were significantly increased(all P<0.01).Compared with the lipopolysaccharide group,the expressions of Jag-1(all P<0.01),Notch-1(all P<0.01)and Hes(P<0.05,P<0.01 and P<0.01)were significantly reduced in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group.(6)The results indicate that astragaloside can promote the transformation of astrocytes from A1 to A2 by regulating Notch-1 signaling pathway,reduce the secretion of inflammatory factors and the migration of CD4 T cells,and thus inhibit astrocyte activation and inflammatory response.

Knock-down of ROCK2 gene improves cognitive function and reduces neuronal apoptosis in AD mice by promoting mitochondrial fusion and inhibiting its division / 细胞与分子免疫学杂志

Objective To explore the effect of knocking down Rho-associated coiled-coil kinase (ROCK2) gene on the cognitive function of amyloid precursor protein/presenilin-1 (APP/PS1) double transgenic mice and its mechanism. Methods APP/PS1 double transgenic mice were randomly divided into AD model group (AD group), ROCK2 gene knock-down group (shROCK2 group), ROCK2 gene knock-down control group (shNCgroup), and wild-type C57BL/6 mice of the same age served as the wild-type control (WT group). Morris water maze and Y maze were employed to test the cognitive function of mice. Neuron morphology was detected by Nissl staining. Immunofluorescence histochemical staining was used to detect the expression of phosphorylated dynamin-related protein 1 (p-Drp1) and mitochondrial fusion 1 (Mfn1). Western blot analysis was used to detect the expression ROCK2, cleaved-caspase-3 (c-caspase-3), B-cell lymphoma 2 (Bcl2), Bcl2-related protein X (BAX), p-Drp1, mitochondrial fission 1 (Fis1), optic atrophy 1 (OPA1), Mfn1 and Mfn2. Results Compared with AD group mice, the expression of ROCK2 in shROCK2 group mice was significantly reduced; the cognitive function was significantly improved with the number of neurons in the hippocampal CA3 and DG areas increasing, and nissl bodies were deeply stained; the expression of c-caspase-3 and BAX was decreased, while the expression of Bcl2 was increased; the expression of mitochondrial division related proteins p-Drp1 and Fis1 were decreased, while the expression of mitochondrial fusion-related proteins OPA1, Mfn1 and Mfn2 were increased. Conclusion Knock-down of ROCK2 gene can significantly improve the cognitive function and inhibit the apoptosis of nerve cells of APP/PS1 mice. The mechanism may be related to promoting mitochondrial fusion and inhibiting its division.

Preoperative MR T2WI signal characteristics of adenomyosis are closely related with the outcome of high-intensity focused ultrasound ablation: a propensity score-matched cohort study / 南方医科大学学报

OBJECTIVE@#To evaluate the correlation of magnetic resonance (MR) T2-weighted image (T2WI) signal characteristics of adenomyosis and the efficacy of high-intensity focused ultrasound (HIFU) ablation.@*METHODS@#Based on the presence or absence of patchy hyperintense foci on preoperative MR T2WI, the patients with adenomyosis undergoing HIFU treatment were divided into homogeneous signal group and heterogeneous signal group, and the heterogeneous group was further divided into heterogeneous hypointense group and heterogeneous isointense group according to signal intensity of the lesions. The patients in heterogeneous signal group were matched with the patients in the homogeneous group at a 1:1 ratio using the propensity score matching, and similarly, the patients in the heterogeneous hypointense group were matched with those in the heterogeneous isointense group at a 1:1 ratio. The non-perfused volume ratio (NPVR) and relief of dysmenorrhea were used to assess the therapeutic efficacy in the 4 groups.@*RESULTS@#A total of 299 patients were enrolled, who had a median preoperative dysmenorrhea score of 7.0 (6.0, 8.0) and a median NPVR of 53.5% (35.4, 70.1)%. After propensity score matching, the NPVR in homogeneous signal group was significantly higher than that in heterogeneous signal group [(60.3 ± 21.8)% vs (44.6±21.6)%, P < 0.05]. At 3, 6 and 12 months after HIFU, dysmenorrhea relief rates were higher in homogeneous signal group than in heterogeneous signal group, and the difference was statistically significant at 12 months (91.1% vs 76.8%, P < 0.05). The NPVR of heterogeneous hypointense group was higher than that of heterogeneous isointense group [(54.0±22.0) % vs (47.3± 22.9) %, P < 0.05]. At 6 months after HIFU, dysmenorrhea relief rate was significantly higher in heterogeneous hypointense group than in heterogeneous isointense group (91.5% vs 80.9%, P < 0.05).@*CONCLUSION@#The signal characteristics of adenomyosis on T2WI are closely related with the outcome of HIFU ablation, and its efficacy is better for homogeneous than for heterogeneous adenomyosis, and better for heterogeneous hypointense adenomyosis than for heterogeneous isointense adenomyosis.

Cellular senescence and type 2 diabetes / 中华内分泌代谢杂志

Cellular senescence is a state in which cells enter permanent cell cycle arrest, which is characterized by senescence-associated secretory phenotype secretion, macromolecular damage, metabolic dysregulation and so on. Recent studies have shown a close relationship between cellular senescence and type 2 diabetes. On the one hand, the glycolipotoxic microenvironment of type 2 diabetes can accelerate cell senescence and accumulation. On the other hand, cellular senescence can promote the development of type 2 diabetes. For example, senescence of pancreatic β-cells leads to β-cell dysfunction and adipocytes senescence results in the secretion of pro-inflammatory cytokines, causing disturbances in lipid metabolism and exacerbating insulin resistance. Moreover, senescence of endothelial cells, retinal endothelial cells, and other cell types contributes to the occurrence of chronic complications in diabetes. Cellular senescence is not only an important factor in the onset of type 2 diabetes but also a consequence of its progression. Targeting cellular senescence holds promise as a new strategy for the treatment of type 2 diabetes.

Expanded Applications of Traditional Chinese Medicine Four Examination Techniques in Asymptomatic Spleen-Stomach Diseases / 中医杂志

Asymptomatic spleen-stomach diseases refer to diseases without related symptoms and signs of abdo-minal pain， bloating， diarrhea an others in patients， but showing lesions or pathological changes discovered by modern medical techniques such as endoscopy， CT， MRI. The four examination techniques of traditional Chinese medicine （TCM） are based on symptoms and signs of patients， which are the advantage of TCM but also have certain limitations. In the context of the increasingly modernized diagnosis and treatment in TCM， it is proposed to expand the application of the four examination techniques from three aspects including microcosmic syndrome differentiation， data sharing， and artificial intelligence in asymptomatic spleen-stomach diseases， in order to achieve the goals of dynamically observing the disease process， collecting disease data in multiple dimensions， and intelligently processing disease data. This will strengthen the modern requirements of early diagnosis and treatment in TCM， and highlight the advantages of TCM in “treating disease before it arises and treating the symptoms beforehand”.

Biological Basis of Five-phase Evolution of Malignant Tumors from Perspective of "Immune Editing" / 中国实验方剂学杂志

Immune escape is one of the ten hallmarks of tumors， which plays an important role in the occurrence and development of tumors. Immune escape refers to a process where tumor cells remodel and edit the immune system through the model of immune clearance， immune balance， and immune escape to "transform" the immune cells into immunosuppressive cells in the tumor microenvironment， so as to support immune escape. The five-stage evolution is the summary of tumor pathogenesis by professor LI Jie. He believes that the gradual development of tumors follows the core pathogenesis of "deficiency-cold-toxin-obstruction-collapse"， in which "depression" runs through the whole process， and cancer toxin is the key. Based on immune editing， this paper combined phenotypic characteristics of tumor cells with the core pathogenesis of the five-stage evolution of professor LI to reveal the biological basis of malignant tumor five-stage evolution. The results indicate that the prominent change from deficiency to cold is the reduction of immune surveillance and the prominent change from toxin to obstruction is immune escape. The final stage of collapse is the outcome of immune failure. Depression is the booster of tumor immune editing. Therefore， the method of reinforcing the healthy Qi and removing toxins was proposed to regulate the immune editing and cut off the five-stage evolution of tumors. Supplementing Qi and warming Yang can reinforce the healthy Qi and restore immune surveillance. Removing toxins and dredging can reverse toxins and immune escape. The harmonizing method can maintain the dynamic balance of immune cells/immunosuppressive cells. Resolving depression can truncate tumor immune editing. Those methods can provide a certain reference for the treatment based on microscopic syndrome differentiation in traditional Chinese medicine （TCM）. In future studies， it is necessary to further explore the specific mechanism of the regulation of immune editing with the methods of supplementing Qi and warming Yang， removing toxins and dredging， their combination， and resolving depression， so as to find out specific Chinese medicines and targets and provide more sufficient evidence for the regulation of tumor immune editing by TCM.

Mutation analysis of SCN4A gene in a family with hypokalemic periodic paralysis / 中国医师杂志

Objective:To analyze the clinical features and SCN4A gentic background of a family with hypokalemic periodic paralysis.Methods:Peripheral blood samples and clinical data were collected from the proband, his brother and parents, and genomic DNA was extracted from these blood samples. Genome-wide exome sequencing was conducted to determine the mutation site in the proband and then allele-specific oligonucleotide primers were designed based on the mutation site. Polymerase chain reaction (PCR) was performed to detect the mutation site to further identify the causative gene in the family.Results:The patient was a 19-years-old male, Han nationality. The patient presented with periodic paralysis while hypokalemia at the same time. His father and grandpa have a similar medical history in the family. A hybrid missense variation (p.R672H) was identified in exon 12 of SCN4A gene in the proband. The same mutation was also detect in the proband's father.Conclusions:The heterozygous missense variation of SCN4A gene (p.R672H) found in this study resulted in familial hypokalemic periodic paralysis. Our research provided reference for the future genetic counseling of this patient and enriched the research data on the relationship between genotype and clinical manifestations.

Study on the Mechanism of Artemisia capillaries Based on Systems Pharmacology / 中国药房

OBJECTIVE:To investigate the possible mechanism of Artemisia capillaries,and to provide reference for further development and utilization of it. METHODS:The effective components and related target protein of A. capillaries were screened by Traditional Chinese Medicine Systems Pharmacology (TCMSP) analysis platform database. The effective compound-target protein visual network of A. capillaries was established by using Cytoscape 3.5.1 software,topology analysis was also performed. The protein-protein interaction (PPI) network was constructed and analyzed by STRING database. KEGG pathway enrichment of target protein coding gene was analyzed by DAVID bioinformatics resource database. RESULTS:A total of 13 kinds of effective compounds,189 target proteins and 34 enrichment pathways were selected. Quercetin,β-glutamol,isorhamnetin and artepillin C were main effective compounds. Prostaglandin G/H sythase 2(PTGS 2),heat shock protein 90(HSP 90),dipeptidyl peptidase Ⅳ, protein kinase A catalytic subunit Cα were main target proteins. Transcription factor AP-1 and cell tumor antigen p53 played a key role in PPI network. The target protein coding gene was rich in TNF-α signaling pathway,HIF-1 signaling pathway,Toll-like receptor signaling pathway,PI3K/Akt signaling pathway,T cell receptor signaling pathway,thyroid hormone signaling pathway, apoptotic signaling pathway,etc. CONCLUSIONS:Quercetin,β-glutamol and isorhamnetin of A. capillaries play an effect on PTGS2,HSP90,transcription factor AP-1 and other target proteins through TNF-α signaling pathway,HIF-1 signaling pathway and PI3K/Akt signaling pathway,so as to play anti-inflammatory and antitumor effect.

Effects of astragaloside Ⅳ on inflammatory responses in astrocytes induced by lipopolysaccharide in vitro and its mechanism / 中华微生物学和免疫学杂志

Objective To investigate whether astragaloside ( AST) Ⅳ could inhibit lipopolysac-charide (LPS)-induced activation of astrocytes and the possible mechanism. Methods Effects of different concentrations of AST Ⅳ on astrocyte viability were determined by MTT to select the optimum concentration for the following experiments. Primary astrocytes were induced by LPS to construct the inflammatory model. Astrocytes were divided into three groups: PBS, LPS and LPS+ASTⅣ(LPS+ASTⅣ) groups. The release of nitric oxide (NO) was detected by Griess method. Expression of glial fibrillary acidic protein ( GFAP), an astrocyte marker, and TLR4 were analyzed by immunohistochemistry and Western blot. Cytokines of IL-6, TNF-α, IL-4 and IL-10 in the supernatants of cell culture for 24 h collected after stimulation were meas-ured by ELISA. Results ASTⅣsignificantly inhibited the LPS-induced activation of astrocytes and NO re-lease (P<0. 01), suppressed the expression of TLR4 (P<0. 05) and reduced the secretion of IL-6 (P<0. 01) and TNF-α (P<0. 01), but increased the secretion of IL-4 and IL-10 (P<0. 05 and P<0. 01). Con-clusion AST Ⅳ could significantly inhibit the LPS-induced activation of astrocytes and suppress inflamma-tory responses, and the possible mechanism might be related to reduced secretion of inflammatory factors af-ter blocking the expression of TLR4.

Influence of Shenhong Tongluo Granules in apoptosis in atherosclerosis plaques of ApoE-/- mice and its mechanisms of anti-atherosclerosis / 吉林大学学报(医学版)

Objective:To explore the influence of Shenhong Tongluo Granules in the lipid levels,atherosclerosis plaques and apoptosis in the plaques of atherosclerotic apolipoprotein E (ApoE)-knokout mice,and to clarify the mechanism of Shenhong Tongluo Granules in anti-atherosclerosis.Methods:Forty SPF male mice aged six-week old were randomly divided into normal control group,model group,low dose of traditional chinese medicine (TCM) group and high dose of TCM group (n=10)after fed adaptively for 1 week.The mice in normal control group were fed with normal diet continuously, the mice in other three groups were fed with high fat diet to establish atherosclerosis model,and then the mice in low and high doses of TCM groups were respectively given Shenhong Tongluo Granules (5.06 and 10.12 g·kg-1 · d-1 )by gavage for 6 weeks. The levels of serum total cholesterol (TC),triglycerides (TG),low density lipoprotein cholesterol (LDL-C)and high density lipoprotein cholesterol (HDL-C)of the mice in various groups were measured after the eyeball blood was collected.The aortic arch tissue was separated,and the morphotogy and the areas of atherosclerotic plaques were observed and calculated by HE staining;the apoptosis in plaques of the mice was evaluated by TUNEL method, and the apoptosis index was calculated. The expressions of apoptosis-assoicated gene Bcl-2 and Bax protein were measured by immunohistochemical staining.Results:The serum level of TG in low dose of TCM group was lower than that in model group (P <0.05);the serum levels of HDL-C in low and high doses of TCM groups were lower than that in model group (P <0.05 or P <0.01);the serum levels of TG and HDL-C in high dose of TCM group was higher than that in low dose of TCM group (P <0.05).The areas of atherosclerotic plaques of the mice in low and high doses of TCM groups were significantly smaller than that in model group (P <0.01).The apoptosis index of mice in low and high doses of TCM groups were significantly lower than that in model group (P <0.01);the expression levels of Bcl-2 protein in atherosclerosis plaques of the mice in low and high doses of TCM groups were lower than that in model group,while the expression levels of Bax were significantly higher than that in model group (P <0.05 or P <0.01).Conclusion:Shenhong Tongluo Granules could effectively reduce the serum lipid level of ApoE-/-mice,meanwhile it could inhibit apoptosis by regulating the expressions of Bcl-2 and Bax protein,and then inhibit the progression of atherosclerotic plaques.