Indolepropionic acid inhibition of microglial cell M1 polarization for treatment of spinal cord injury / 中国组织工程研究

BACKGROUND:Indolepropionic acid has been shown to reduce diabetes-induced central nervous system inflammation.However,there is a lack of research on whether to inhibit microglia M1 polarization for the treatment of spinal cord injury. OBJECTIVE:To investigate the mechanism of indolepropionic acid inhibition of microglial cell M1 polarization for the treatment of spinal cord injury through cell and animal experiments. METHODS:(1)In vitro experiments:BV2 cell viability was assessed using the CCK-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,BV2 cells were categorized into control group,administration group(50 μmol/L indolepropionic acid),lipopolysaccharide group(100 ng/mL lipopolysaccharide),and treatment group(100 ng/mL lipopolysaccharide + 50 μmol/L indolepropionic acid).Nitric oxide content was quantified using the Griess method.Real-time quantitative PCR and western blot assay were employed to measure mRNA and protein levels of pro-inflammatory factors.Cell immunofluorescence staining was conducted to assess inducible nitric oxide synthase expression.The Seahorse assay was employed to assess glycolytic stress levels in BV2 cells.(2)In vivo experiments:30 SD rats were randomly divided into three groups:sham surgery group,spinal cord injury group,and indolepropionic acid group.Motor function recovery in rats after spinal cord injury was assessed using BBB scoring and the inclined plane test.Immunofluorescence staining of spinal cord tissue was conducted to evaluate the expression of inducible nitric oxide synthase in microglial cells.ELISA was employed to measure protein expression levels of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in spinal cord tissue. RESULTS AND CONCLUSION:(1)In vitro experiments:Indolepropionic acid exhibited significant suppression of BV2 cell viability when its concentration exceeded 50 μmol/L.Indolepropionic acid achieved this by inhibiting the activation of the nuclear factor κB signaling pathway,thereby suppressing the mRNA and protein expression levels of pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α),as well as the M1 polarization marker,inducible nitric oxide synthase,in BV2 cells.Additionally,indolepropionic acid notably reduced the glycolytic level in BV2 cells induced by lipopolysaccharides.(2)In vivo experiments:Following indolepropionic acid intervention in spinal cord injury rats,there was a noticeable increase in BBB scores and the inclined plane test angle.There was also a significant decrease in the number of M1-polarized microglial cells in spinal cord tissue,accompanied by a marked reduction in the protein expression levels of pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α).(3)These results conclude that indolepropionic acid promotes functional recovery after spinal cord injury by improving the inflammatory microenvironment through inhibition of microglia M1 polarization.

Application of 4M1E Refined Management Method in Reducing Dispensing Time of Long-term Prescription in Outpatient Pharmacy / 医药导报

Objective To investigate the effectiveness of 4M1E refined management method in reducing dispensing time of outpatient prescription in outpatient pharmacies under the implementation of a long-term prescription policy.Methods The hospital started to implement 4M1E refinement management in July 2022.Ten thousand prescriptions were randomly selected for each of the pre-implementation(from January to June 2022)and post-implementation(from July to December 2022)human-machine hybrid dispensing windows.The dispensing time of a single prescription,the volume of prescriptions dispensed and the number of drugs prescribed during peak periods,the use of intelligent equipment,and patient satisfaction before and after imple-mentation were compared.Results After implementation,the single prescription drug dispensing time at the mixed human-ma-chine dispensing window was reduced from(96.88±1 401.17)s to(55.84±526.24)s(P<0.01);The number of prescriptions dispensed in the window increased from(135.20±21.06)to(147.19±21.24)prescriptions per 2 h,and the number of drugs pre-scribed increased from(871.74±215.61)to(1 008.53±267.87)prescriptions per 2 h during peak hours.The rate of straight prescription and the rate of equipment automation have been greatly improved.Outpatient satisfaction was higher than before man-agement.The data showed statistical differences(P<0.05).Conclusion Under long-term prescription pressure,hospital outpa-tient pharmacies can greatly reduce prescription dispensing time and improve patient satisfaction after applying the 4M1E fine management method.

Effects of Siglec-E on parthenolide inhibiting microglia M1 polarization and targeting MAPK/NF-κB pathway / 陆军军医大学学报

Objective To explore the impact of the sialic acid binding lectin-E(Siglec-E)on the inhibitory properties of parthenolide(PTL)against lipopolysaccharide(LPS)-induced M1 polarization of microglia(BV2).Methods ①Single cell sequencing data of Siglece related mouse brain tissue was obtained from Gene Expression Omnibus(GEO)database and divided into the WT group(n=3)and the Siglece-/-group(n=4).The microglia cells were screened,and the enrichment analysis was performed to analyze related differential genes and pathways.BV2 cells were constructed by the shRNA interference technique and were divided into NC-shRNA and Siglece-shRNA to detect the expression level of Siglec-E(Siglece).② NC-shRNA and Siglece-shRNA cells were respectively divided into the Control group,LPS group,PTL group and PTL+LPS group(n=3).The mRNA levels of markers of M1 polarization in microglia,iNOS,IL-1 β and IL-6,were detected by RT-qPCR.Siglecefl/fl and Cx3cr1cre mice were mated to obtain microglia-specific Siglece deletion(Siglecefl/fl×Cx3cr1cre)mice,and LPS-induced neuroinflammation model was established.③ Nine WT and Siglecefl/fl×Cx3cr1cre male mice were assigned to the Control group,LPS group and PTL+LPS group(n=3).RT-qPCR,immunofluorescence assay and Western blotting were used to verify the knock-out effect and polarization-related pathways,and to investigate the mechanism of Siglec-E affecting PTL inhibition of M1 polarization of microglia.Results Compared with the NC-shRNA group,the expression of Siglec-E in the Siglece-shRNA group was significantly decreased(P<0.01),indicating that the Siglec-E knock-down cell model was successfully established.With the stimulation of LPS,mRNA levels ofiNOS,IL-1 β and IL-6 were significantly up-regulated compared with the Control group both in shRNA cells and Siglece-shRNA cells(P<0.01).With the influence of PTL and LPS,the markers of M1 polarization in NC-shRNA cells mentioned before were significantly decreased(P<0.05),while for Siglice-shRNA cells,there were no significant changes in the markers of M1 polarization.PTL inhibited the phosphorylation of JNK and IκB protein(P<0.01)and the nuclear translocation of NF-κB in BV2 cells,down-regulated Siglec-E,and weakened the inhibitory effect.Compared with mice in the WT group,the expression of Siglec-E in microglia of Siglecefl/fl×Cx3cr1cre mice was decreased significantly(P<0.01),and the inhibitory effect of PTL on the phosphorylation of NF-κB in microglia of Siglecefl/fl×Cx3cr1cre mice was also decreased.Conclusion The absence of Siglec-E in microglia attenuates the inhibition of M1 polarization by the MAPK/NF-κB pathway targeted by PTL.

AP2M1 inhibits proliferation and invasion of diffuse large B-cell lymphoma cells / 基础医学与临床

Objective To evaluate the regulatory effect of the adaptor related protein complex 2 subunit μ1(AP2M1)on proliferation and invasion of diffuse large B-cell lymphoma(DLBCL).Methods Human diffuse large B-cell lymphoma cell line OCI-LY8 was aliquoted into control group,NC-shRNA group,AP2M1-shRNA group,NC-LV group,and AP2M1-LV group.Lipofectamine 2000 was used for cell transfection.Cell proliferation was detected by tetramethylazolium salt(MTT)method,apoptosis was detected by flow cytometry and cell migration and invasion were detected by Transwell assay.The protein expression of AP2M1,epidermal growth factor receptor(EGFR),p-phosphatidylinositol 3 kinase(PI3K),PI3K,p-protein kinase B(Akt)and AKT was detec-ted by Western blot.Results Compared with control group,the relative expression of AP2M1 mRNA and protein in the AP2M1-shRNA group was decreased(P＜0.05).The relative cell viability was increased(P＜0.05).The cell apoptosis rate was decreased(P＜0.05).The counting number of migrating and invading cells was in-creased(P＜0.05).The relative expression level of EGFR protein and the phosphorylation level of PI3K and AKT were increased(P＜0.05).Compared with Control group,the expression of AP2M1 mRNA and protein relative ex-pression level in AP2M1-LV group was increased(P＜0.05).The relative cell viability was decreased(P＜0.05).The cell apoptosis rate was increased(P＜0.05).The number of migrating and invading cells was decreased(P＜0.05).The relative expression level of EGFR protein and the phosphorylation level of PI3K and AKT were all decreased(P＜0.05).Conclusions The over-expression of AP2M1 partially inhibits the proliferation and invasion of DLBCL cells by inhibiting the EGFR/PI3K/AKT signaling pathway.

Study on Serum FOXM1 and IGF2 Expression Levels and Prognostic Value in Elderly Patients with Heart Failure Complicated with Pneumonia / 现代检验医学杂志

Objective To explore the predictive value of the expression of serum forkhead box protein M1(FOXM1)and insulin-like growth factor 2(IGF2)on the prognosis of elderly patients with heart failure complicated with pneumonia.Methods A total of 126 elderly patients with heart failure complicated with pneumonia admitted to Handan Central Hospital from March 2021 to June 2022 were included in case group.According to the follow-up results,the 122 patients were grouped into poor prognosis group(n=33)and good prognosis group(n=89).Meanwhile,126 healthy people in the same period were included as the control group.The levels of serum FOXM1 and IGF2,forced vital capacity(FVC)and forced expiratory volume in the first second(FEV1)in the two groups(case group and control group)were measured.Spearman method was used to analyze the correlation between serum levels of FOXM1 and IGF2 and heart function classification in elderly patients with heart failure complicated with pneumonia.The predictive value of serum FOXM1 and IGF2 levels in elderly patients with heart failure complicated with pneumonia was analyzed by receiver operating characteristic(ROC)curve.Results Compared with the control group,the levels of FOXM1(2.39±0.55 vs 1.06±0.21)and IGF2(71.33±7.96pg/ml vs 47.82±5.14pg/ml)in the case group were significantly higher(t=25.358,27.581,all P<0.05).Compared with the good prognosis group,the levels of serum FOXM1(3.87±1.06 vs 1.95±0.51)and IGF2(85.88±9.54pg/ml vs 69.14±8.73pg/ml)in the poor prognosis group were significantly higher,and the differences were statistically significant(t=13.453,9.174,all P<0.05).There were significant differences in heart function classification between the good prognosis group and the poor prognosis group,and the differences were statistically significant(χ2=7.120,P<0.05).Compared with the poor prognosis group,FEV1(1.24±0.32 L vs 1.08±0.25 L)and FEV1/FVC(55.46%±5.77%vs 52.30%±5.38%)in good prognosis group were significantly higher,and the differences were statistically significant(t=2.592,2.735,all P<0.05).The levels of serum FOXM1 and IGF2(r=0.496,0.517,all P<0.05)in elderly patients with heart failure complicated with pneumonia were positively correlated with heart function classification.ROC curve results showed that the area under the curve(AUC)of serum FOXM1 alone in predicting the prognosis of elderly patients with heart failure complicated with pneumonia was 0.854(95CI%:0.779～0.912),with sensitivity and specificity of 75.76%and 86.52%,respectively,and the optimal cut-off value of 2.75.The AUC of IGF2 alone in predicting the prognosis of elderly patients with heart failure complicated with pneumonia was 0.874(95CI%:0.802～0.927),with sensitivity and specificity of 72.73%and 85.39%,respectively,and the optimal cut-off value of 78.30 pg/ml.The AUC of the combination of the two in predicting the prognosis of elderly patients with heart failure complicated with pneumonia was greater than the AUC diagnosed by serum FOXM1 alone and IGF2 alone(Z=2.737,2.413,P=0.006,0.016).Conclusion The serum levels of FOXM1 and IGF2 were increased in elderly patients with heart failure complicated with pneumonia,indicating the combined detection of the two may have a high predictive value for the prognosis of patients.

Role and application of early transient presence of M1 macrophages in bone tissue engineering / 中国组织工程研究

BACKGROUND:Early transient presence of M1 macrophages can play a beneficial role after the implantation of bone tissue engineering materials.Recently,strategies for manipulating M1 macrophages to produce an early moderate inflammatory response have been extensively studied and many research advances have been made in the design of bone tissue engineering materials. OBJECTIVE:To review the role of early transient presence of M1 macrophages in bone tissue engineering and recent research advances in the strategy for activating early transient presence of M1 macrophages in the field of bone tissue engineering. METHODS:Relevant literature included in PubMed,WanFang database,and CNKI Database from January 2012 to October 2022 was searched.Search terms were"M1,macrophage,bone immunoregulation,bone defect,osteogenesis,osteoimmunology,angiogenesis"in English and Chinese.After excluding articles irrelevant to the research purpose and repetitive articles,63 papers were finally included for review. RESULTS AND CONCLUSION:The early transient presence of M1 macrophages play a key role in bone tissue engineering by promoting angiogenesis,facilitating osteogenic differentiation of bone marrow mesenchymal stem cells and promoting an M2 macrophage phenotype.Strategies for inducing and activating early transient presence of M1 macrophages can modulate the local immune microenvironment for bone defect repair in a manner consistent with early natural bone healing,including modulation of the physicochemical properties of bone tissue engineering materials to promote appropriate M1 macrophage-mediated inflammatory responses,sequential delivery of cytokines,microRNAs or bioactive ions to facilitate the M1-to-M2 transition of macrophages,and controlled release of anti-inflammatory substances to achieve the maintenance of early inflammatory responses.

Th17/Treg balance and macrophage polarization ratio in lower extremity arteriosclerosis obliterans

@#Objective: To explore the balance of peripheral blood T helper 17 cells/regulatory T cell (Th17/Treg) ratio and the polarization ratio of M1 and M2 macrophages in lower extremity arteriosclerosis obliterans (ASO). Methods: A rat model of lower extremity ASO was established, and blood samples from patients with lower extremity ASO before and after surgery were obtained. ELISA was used to detect interleukin 6 (IL-6), IL-10, and IL-17. Real-time RCR and Western blot analyses were used to detect Foxp3, IL-6, IL-10, and IL-17 expression. Moreover, flow cytometry was applied to detect the Th17/Treg ratio and M1/M2 ratio. Results: Compared with the control group, the iliac artery wall of ASO rats showed significant hyperplasia, and the concentrations of cholesterol and triglyceride were significantly increased (P<0.01), indicating the successful establishment of ASO. Moreover, the levels of IL-6 and IL-17 in ASO rats were pronouncedly increased (P<0.05), while the IL-10 level was significantly decreased (P<0.05). In addition to increased IL-6 and IL-17 levels, the mRNA and protein levels of Foxp3 and IL-10 in ASO rats were significantly decreased compared with the control group. The Th17/Treg and M1/M2 ratios in the ASO group were markedly increased (P<0.05). These alternations were also observed in ASO patients. After endovascular surgery (such as percutaneous transluminal angioplasty and arterial stenting), all these changes were significantly improved (P<0.05). Conclusions: The Th17/Treg and M1/M2 ratios were significantly increased in ASO, and surgery can effectively improve the balance of Th17/Treg, and reduce the ratio of M1/M2, and the expression of inflammatory factors.

PI3K/AKT/mTOR in synovial fluid extends the proinflammatory response of macrophage polarization in knee osteoarthritis / 安徽医科大学学报

Objective @#Given that the PI3K/AKT/mTOR signaling pathway is associated with the progression of knee osteoarthritis (KOA) , this study aims to investigate whether the polarization induction of synovial macrophages mediated by the PI3K/AKT/mTOR signaling axis is the cause of KOA progression . @*Methods @#The synovial fluid of KOA KL-Ⅱ and KL-Ⅲ patients and normal individuals was collected , and the percentage of M1 macrophages (CD80 , CD86) and M2 macrophages (CD163 , CD206) in the synovial fluid (M1 /M2 ratio) was measured to e- valuate the polarization of macrophage cytokines such as IL-1 , IL-6 , IL-10 , and tumor necrosis factor (TNF) -α, transforming growth factor ( TGF)-βExpression in KOA synovial fluid , and detect and analyze of key molecules PI3K/AKT/mTOR signaling axis PI3K , AKT3 , mTORC1 , and inducible nitric oxide synthase ( iONS) in KOA synovial fluid . @*Results @#Compared with the synovial fluid of normal individuals , the percentage of M1 macrophages (CD80 , CD86) in KOA patients increased (P < 0. 01) , and the M1 /M2 ratio increased ( P < 0. 001) ; The ex- pression of IL-1 , IL-6 , and TNF-αin the synovial fluid of the KOA group was also higher than that of the control group (P < 0. 01) , while the expression of IL-10 and TGF-βin the KOA group was significantly reduced ( P < 0. 01) ; The key proteins PI3K , AKT3 , mTORC1 , and downstream inflammatory factor iONS in the PI3K/AKT/ mTOR signaling pathway in the synovial fluid of the KOA group were higher than those in the control group (P < 0. 01) . @*Conclusion @#In KOA synovial fluid , M1 macrophage polarization plays a dominant role , and the inflam- matory response mediated by M1 macrophage polarization may be the cause of synovitis . At the same time , the PI3K/AKT/mTOR signaling pathway may mediate the polarization of M1 macrophages involved in KOA inflammato- ry response .

Effect of LPS-induced M1 polarization of macrophages on tumor growth / 中国现代医生

Objective To study the effect of lipopolysaccharide(LPS)-induced M1 polarization of macrophages on tumor growth.Methods Female Balb/c mice were randomly divided into control group and experimental group.Renca cells were used to establish subcutaneous tumor model.NaCl(100μl/mice,once every two days)or LPS(100μg/mice,once every two days)was injected to the tumor side when the tumor grew to 50mm3.The M1 polarization level of tumor-associated macrophages was detected by flow cytometry.Mouse tumor cell lines(ML-1,MC38,Renca)were divided into three groups:blank group(complete medium with 50％DMEM basal medium),control group(complete medium with 50％medium supernatant of cultured macrophages)and experimental group(complete medium with 50％medium supernatant of LPS pretreated cultured macrophages).The proliferation of tumor cells was detected by cell counting kit-8(CCK-8).The cell cycle and apoptosis of tumor cells were detected by flow cytometry.Results The proportion of tumor-associated macrophages M1 increased after LPS treatment(P<0.001),thus enhancing its anti-tumor function.LPS-induced M1 polarization of macrophages can significantly inhibit the proliferation of tumor cells(Renca:P=0.023,ML-1:P=0.045).LPS-induced M1 polarization of macrophages blocked G0/G1 phase(MC38:P=0.011,ML-1:P=0.022)or S phase(Renca:P=0.022)of tumor cell cycle,and then cell division was inhibited.LPS-induced M1 polarization of macrophages significantly induced apoptosis of tumor cells(Renca:P=0.04,ML-1:P=0.007).Conclusion LPS can play an anti-tumor role by inducing M1 polarization of macrophages.

Shakuyakukanzoto Relieves Ulcerative Colitis in Mice by Regulating The Expression of NDUFS1 and Inhibiting The Polarization of Macrophages to M1 / 生物化学与生物物理进展

ObjectiveThis study aims to explore and elucidate the possible mechanism of action of Shakuyakukanzoto (SKT) in improving ulcerative colitis (UC) in mice through regulating energy metabolism and polarization of macrophages. MethodsThe mouse UC model was constructed by administering 3% dextran sulfate sodium salt (DSS), and the mice were treated with SKT intragastrically. In addition, single-cell sequencing and enrichment of metabolic pathways against two datasets, GSE21157 and GSE210415, were conducted first. Second, the extraction and metabolomics of peritoneal macrophages from UC mice were verified. Then, the pathway of differentially abundant metabolite enrichment and the correlation of UC risk were analyzed depending on univariate Mendelian randomization of two samples weighted by standard inverse variance. Finally, the results were verified by qRT-PCR, Western blot, and flow cytometry. ResultsAccording to the HE staining results, SKT can significantly alleviate colon damage caused by DSS. Macrophages, NK cells, T cells, and more than 10 different types of cells, based on single-cell sequencing analysis, are detected in the intestinal wall. In the disease group, we can conclude that the activity of 49 macrophage metabolic pathways, mainly involved in energy metabolism, is significantly upregulated through a comparison of the two datasets. In energy metabolomics, 10 and 18 types of metabolites accompanied by significantly upregulated and downregulated differential expression were identified in the treatment group and the model group, as well as the model group and the blank group, respectively. Meanwhile, these differentially expressed metabolites present an obvious correlation with glycolysis and oxidative phosphorylation. Moreover, it can be inferred that glycolysis and the oxidative phosphorylation-related gene NDUFS1 (OR: 0.56, 95% CI: 0.48-0.98, P=0.000 068) are associated with a reduced risk of UC based on the univariate Mendelian randomization of two samples weighted based on standard inverse variance. By analyzing the difference in transcription levels between the two datasets, the transcription level of NDUFS1 in UC was decreased compared with that in the normal group. The results of qRT-PCR, Western blot, and flow cytometry indicate that SKT can promote the expression of the oxidative phosphorylation protein NDUFS1 in macrophages and inhibit the M1-type polarization of macrophages. Furthermore, knockdown/overexpression of NDUFS1 can affect the effect of SKT on M1-type polarization of macrophages. ConclusionBased on the results of this study, SKT inhibits macrophage polarization toward the M1 phenotype by regulating the level of the oxidatively phosphorylated protein NDUFS1 in macrophages; hence, UC is also relieved in mice. These conclusions not only reveal the therapeutic mechanism of SKT for UC but also provide a new theoretical basis for clinical application.

Discussion on the cultivation of scientific research ability of academic postgraduate students majoring in clinical laboratory diagnostics from the perspective of 4M1E method / 中华医学教育探索杂志

In the current scientific research training of the academic postgraduate students majoring in clinical laboratory diagnostics, there are many problems including insufficient scientific research level, poor scientific research environment and so on. Based on many years of experience on cultivating postgraduate students and starting from the five factors of 4M1E (man, machine, material, method and environment) method, a scientific strategy is proposed, and then the scientific research ability and level of academic postgraduate students majoring in clinical laboratory diagnostics could be improved by exerting its own self-motivation, strengthening the guidance of tutors, improving training tools, heightening scientific thinking, and enhancing environmental friendliness of scientific research.

Progress in the regulatory effects of tumor-derived exosomes on macrophage polarization / 中华微生物学和免疫学杂志

Mononuclear macrophages are versatile cells that can have different responses to various microenvironmental signals. Under different stimuli of circumstances, macrophages can be fully polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2), which are the extremes of a continuum of functional states. Nuclear factor-κB, cyclooxygenase 2, anoxia status, proto-oncogene MYC, Toll-like receptor signaling pathway, Notch signaling pathway and cytokines are all closely involved in the transition of tumor-associated macrophages from M1 to M2 phenotype. Macrophages that infiltrate tumor tissues are driven by tumor-derived cytokines to acquire a polarized M2 phenotype. These functionally polarized cells play a key role in the subversion of adaptive immunity and in inflammatory circuits that promote tumor development and progression. Exosomes derived from tumors have the characteristics of tumor cells and could participate in multiple processes of tumorigenesis and development. This review focused on exosomes derived from various cancer cells and discussed the role of the payloads of tumor-derived exosomes in modulating macrophage polarization in the tumor immune microenvironment and the intracellular signal mechanisms involved.

Evaluation of biological activity of an eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody / 中国生物制品学杂志

@#Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/（M*s）]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.

Effect of Zhuangyao Tongluo Formula（壮腰通络方） Containing Serum on TNF-α-mediated Vicious Circle of Pyroptosis of Nucleus Pulposus Cells / 中医杂志

ObjectiveTo explore the mechanism of Zhuangyao Tongluo Formula（壮腰通络方，ZTF） in delaying intervertebral disc degeneration. MethodsM1 macrophages were induced from THP-1 cells using LPS， IFN-γ and PMA. The induced M1 macrophages were then co-cultured with nucleus pulposus cells in a transwell system. Fetal bovine serum was used as the control serum， and the effects of different concentrations （5%， 10%， 15%， 20%） of serum from rats treated with ZTF on the activity of M1 macrophages and nucleus pulposus cells were analyzed using MTT assay. Experiment 1 was established， including the nucleus pulposus cell control group， M1 macrophage control group， nucleus pulposus cell + ZTF group， nucleus pulposus cell + TNF control group， nucleus pulposus cell + TNF + ZTF group， co-culture group， and co-culture + ZTF group. The levels of IL-1β， and IL-18 in the culture supernatant were detected using ELISA. The mRNA expression of IL-1β and IL-18 in nucleus pulposus cells was detected using qPCR. Additionally， the expression of GSDMD protein in nucleus pulposus cells was detected using cell immunofluorescence. In experiment 2， co-culture groups were constructed using TNF-α overexpression （OE） or empty vector （EV） plasmids， including co-culture group， TNF-EV + co-culture group， TNF-EV co-culture group + ZTF， co-culture + ZTF group， TNF-OE co-culture group + ZTF， and TNF-OE + co-culture group. The mRNA and protein expression of TNF-α in M1 cells in each group were detected using qPCR and WB. ResultsThe ZTF with 10% serum was selected for subsequent experiments. The results of experiment 1 showed that compared to the control group of nucleus pulposus cells， there was no statistically significant difference in the levels of IL-1β， IL-18， mRNA， and GSDMD expression in the nucleus pulposus cells + ZTF group （P＞0.05）. However， the TNF-α + co-culture group showed a significant increase in IL-1β， IL-18 levels， mRNA， and GSDMD expression （P＜0.01）. When compared to the co-culture group， the ZTF+ co-culture group showed a significant decrease in IL-1β， IL-18 levels， mRNA， and GSDMD expression （P＜0.01）. The results of experiment 2 showed that there was no statistically significant difference in TNF-α mRNA and protein expression between the empty vector plasmids + co-culture group and the co-culture group （P＞0.05）. Compared to the empty vector + co-culture group， the expression of TNF-α mRNA and protein was significantly reduced in the empty vector co-culture + ZTF group （P＜0.01）. Compared to the co-culture group and the empty vector + co-culture group， the expression of TNF-α mRNA and protein was significantly reduced in the co-culture + ZTF group （P＜0.01）. Compared to the co-culture + ZTF group， the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture + ZTF group （P＜0.01）. Compared to the overexpression vector co-culture + ZTF group， the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture group （P＜0.01）. ConclusionZTF serum can inhibit the TNF-α-induced apoptosis of nucleus pulposus cells and delay lumbar disc degeneration by reducing the expression of TNF-α in M1 macrophages.

Advances on research of structure and function of puromycin sensitive aminopeptidase / 药学学报

Puromycin-sensitive aminopeptidase (PSAP) belongs to the M1 family of aminopeptidases, characterized by the N-terminal substrate binding sequence GAMEN, the enzyme activity center HEXXH(X)18E motif, and the C-terminal ERAP-1-like superfamily structural domain. Encoded by the gene NPEPPS located at 17q21.32, PSAP consists of 919 amino acids and is widely distributed throughout the human body, with the highest expression in the brain, followed by the heart and skeletal muscle. It is also found in the liver, renal tubular epithelium, small intestine, large intestine epithelium, and gastric epithelial cells. PSAP primarily relies on its aminopeptidase hydrolytic activity to remove toxic protein aggregates such as Tau, poly Q, and Cu, Zn-superoxide dismutase 1, making it an important factor in the development of diseases such as Alzheimer's disease, Huntington's chorea, and tumors. Existing PSAP inhibitors include bestatin, amastatin, leuhistin, actinonin, and purinomycin, some of which are already available or in clinical trials. This review provides an overview of the structural and biological functions of M1 family aminopeptidases, with a focus on PSAP, to facilitate further research and targeted drug development.

Identification of M1 macrophage-related genes in rejection after kidney transplantation based on weighted gene co-expression network analysis / 器官移植

Objective To identify M1 macrophage-related genes in rejection after kidney transplantation and construct a risk prediction model for renal allograft survival. Methods GSE36059 and GSE21374 datasets after kidney transplantation were downloaded from Gene Expression Omnibus (GEO) database. GSE36059 dataset included the samples from the recipients with rejection and stable allografts. Using this dataset, weighted gene co-expression network analysis (WGCNA) and differential analysis were conducted to screen the M1 macrophage-related differentially expressed gene (M1-DEG). Then, GSE21374 dataset (including the follow-up data of graft loss) was divided into the training set and validation set according to a ratio of 7∶3. In the training set, a multivariate Cox's model was constructed using the variables screened by least absolute shrinkage and selection operator (LASSO), and the ability of this model to predict allograft survival was evaluated. CIBERSORT was employed to analyze the differences of infiltrated immune cells between the high-risk group and low-risk group, and the distribution of human leukocyte antigen (HLA)-related genes was analyzed between two groups. Gene set enrichment analysis (GSEA) was used to further clarify the biological process and pathway enrichment in the high-risk group. Finally, the database was employed to predict the microRNA (miRNA) interacting with the prognostic genes. Results In the GSE36059 dataset, 14 M1-DEG were screened. In the GSE21374 dataset, Toll-like receptor 8 (TLR8), Fc gamma receptor 1B (FCGR1B), BCL2 related protein A1 (BCL2A1), cathepsin S (CTSS), guanylate binding protein 2(GBP2) and caspase recruitment domain family member 16 (CARD16) were screened by LASSO-Cox regression analysis, and a multivariate Cox's model was constructed based on these 6 M1-DEG. The area under curve (AUC) of receiver operating characteristic of this model for predicting the 1- and 3-year graft survival was 0.918 and 0.877 in the training set, and 0.765 and 0.736 in the validation set, respectively. Immune cell infiltration analysis showed that the infiltration of rest and activated CD4+ memory T cells, γδT cells and M1 macrophages were increased in the high-risk group (all P < 0.05). The expression level of HLA I gene was up-regulated in the high-risk group. GSEA analysis suggested that immune response and graft rejection were enriched in the high-risk group. CTSS interacted with 8 miRNA, BCL2A1 and GBP2 interacted with 3 miRNA, and FCGR1B interacted with 1 miRNA. Conclusions The prognostic risk model based on 6 M1-DEG has high performance in predicting graft survival, which may provide evidence for early interventions for high-risk recipients.

Effects of Total Saponins from Dioscorea Nipponica Makino on Monosodium Urate-Induced M1-Polarized Macrophages through Arachidonic Acid Signaling Pathway: An in vitro Study / 中国结合医学杂志

OBJECTIVE@#To investigate and reveal the underlying mechanism of the effect of total saponins from Dioscoreae nipponica Makino (TSDN) on the arachidonic acid pathway in monosodium urate (MSU) crystal-induced M1-polarized macrophages.@*METHODS@#M1 polarization of RAW264.7 cells were induced by 1 µ g/mL lipopolysaccharide (LPS). The methylthiazolyldiphenyl-tetrazolium bromide method was then used to screen the concentration of TSDN. MSU (500 µ g/mL) was used to induce the gouty arthritis model. Afterwards, 10 µ g/L TSDN and 8 µ mol/L celecoxib, which was used as a positive control, were added to the above LPS and MSU-induced cells for 24 h. The mRNA and protein expressions of cyclooxygenase (COX) 2, 5-lipoxygenase (5-LOX), microsomal prostaglandin E synthase derived eicosanoids (mPGES)-1, leukotriene B (LTB)4, cytochrome P450 (CYP) 4A, and prostaglandin E2 (PGE2) were tested by real-time polymerase chain reaction and Western blotting, respectively. The enzyme-linked immunosorbent assay was used to test the contents of M1 markers, including inducible nitric oxid synthase (NOS) 2, CD80, and CD86.@*RESULTS@#TSDN inhibited the proliferation of M1 macrophages and decreased both the mRNA and protein expressions of COX2, 5-LOX, CYP4A, LTB4, and PGE2 (P<0.01) while increased the mRNA and protein expression of mPGES-1 (P<0.05 or P<0.01). TSDN could also significantly decrease the contents of NOS2, CD80, and CD86 (P<0.01).@*CONCLUSION@#TSDN has an anti-inflammation effect on gouty arthritis in an in vitro model by regulating arachidonic acid signaling pathway.

Total Saponin Fraction of Dioscorea Nipponica Makino Improves Gouty Arthritis Symptoms in Rats via M1/M2 Polarization of Monocytes and Macrophages Mediated by Arachidonic Acid Signaling / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of effects of total saponin fraction from Dioscorea Nipponica Makino (TSDN) on M1/M2 polarization of monocytes/macrophages and arachidonic acid (AA) pathway in rats with gouty arthritis (GA).@*METHODS@#Seventy-two Sprague Dawley rats were randomly divided into 4 groups (n=18 in each): normal, model, TSDN at 160 mg/kg, and celecoxib at 43.3 mg/kg. Monosodium urate crystal (MSU) was injected into the rats' ankle joints to induce an experimental GA model. Blood and tissue samples were collected on the 3rd, 5th, and 8th days of drug administration. Histopathological changes in the synovium of joints were observed via hematoxylin and eosin (HE) staining. The expression levels of arachidonic acid (AA) signaling pathway were assessed via real-time polymerase chain reaction (qPCR) and Western blot. Flow cytometry was used to determine the proportion of M1 and M2 macrophages in the peripheral blood. An enzyme-linked immunosorbent assay (ELISA) was used to detect interleukine (IL)-1 β, tumor necrosis factor-alpha (TNF-α), IL-4, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4).@*RESULTS@#HE staining showed that TSDN improved the synovial tissue. qPCR and Western blot showed that on the 3rd, 5th and 8th days of drug administration, TSDN reduced the mRNA and protein expressions of cyclooxygenase (COX)2, microsomal prostaglandin E synthase-1 derived eicosanoids (mPGES-1), 5-lipoxygenase (5-LOX), recombinant human mothers against decapentaplegic homolog 3 (Smad3), nucleotide-binding oligomerization domain-like receptor protein 3 (NALP3), and inducible nitric oxide synthase (iNOS) in rats' ankle synovial tissues (P<0.01). TSDN decreased COX1 mRNA and protein expression on 3rd and 5th day of drug administration and raised it on the 8th day (both P<0.01). It lowered CD68 protein expression on days 3 (P<0.01), as well as mRNA and protein expression on days 5 and 8 (P<0.01). On the 3rd, 5th, and 8th days of drug administration, TSDN elevated the mRNA and protein expression of Arg1 and CD163 (P<0.01). Flow cytometry results showed that TSDN decreased the percentage of M1 macrophages while increasing the percentage of M2 in peripheral blood (P<0.05 or P<0.01). ELISA results showed that on the 3rd, 5th, and 8th days of drug administration, TSDN decreased serum levels of IL-1 β, TNF-α, and LTB4 (P<0.01), as well as PGE2 levels on days 3rd and 8th days (P<0.05 or P<0.01); on day 8 of administration, TSDN increased IL-4 serum levels and enhanced IL-10 contents on days 5 and 8 (P<0.05 or P<0.01).@*CONCLUSION@#The anti-inflammatory effect of TSDN on rats with GA may be achieved by influencing M1/M2 polarization through AA signaling pathway.

Advances in the role of microglia polarization in the pathogenesis of retinal degenerative diseases / 眼科新进展

Microglia is involved in the occurrence and development of retinal degenerative diseases,thus its activation is a marker for retinal degenerative diseases.Microglia can be activated by different external stimulating factors and polar-ized into two phenotypes with different functional states and surface markers.The classical activation pathway produces M1 phenotype microglia,which mainly exerts pro-inflammatory and neurotoxic effects;the selective activation pathway pro-duces M2 phenotype microglia,which mainly exerts anti-inflammatory and neuroprotective effects.Precise modulation of microglia polarization is a promising approach for treating retinal degenerative diseases.This paper reviews the role of mi-croglia polarization in the pathogenesis of retinal degenerative diseases,aiming to pioneer new avenues and research ideas for treating retinal degenerative diseases.

Homocysteine induces inflammatory polarization in mouse microglia by up-regulating Rap1a / 中国病理生理杂志

AIM:To investigate the impact of homocysteine(Hcy)on the inflammatory response mediated by BV2 mouse microglia,and to explore the mechanism of Ras-related protein 1a(Rap1a)in the Hcy-induced inflammatory response in BV2 cells.METHODS:Mouse microglial cell line BV2 was cultured in vitro,and Hcy intervention was used to establish a hyperhomocysteinemia cellular model.The cells were divided into 4 groups:blank control group,and 50 μmol/L,100 μmol/L and 150 μmol/L Hcy groups.The mRNA expression levels of M1 polarization markers,inflammatory factors IL-6,IL-1β and TNF-α,and Rap1a in BV2 cells were detected by RT-qPCR.Additionally,the levels of inflamma-tory factors IL-6,IL-1β and TNF-α in BV2 cells were measured using an ELISA kit.The protein expression level of Rap1a was detected by Western blot assay.To verify the function of Rap1a,viral transfection was employed for both over-expression and knockdown experiments.RESULTS:Under the intervention of Hcy concentration above 100 μmol/L,BV2 cells exhibited inflammatory polarization,as indicated by the increased mRNA expression of M1 polarization markers CD80 and CD86(P＜0.05).The mRNA and protein expression levels of inflammatory factors IL-6,IL-1β and TNF-α were significantly up-regulated(P＜0.05).Additionally,the mRNA and protein expression levels of Rap1a also showed a significant increase(P＜0.05).Moreover,Rap1a mRNA level was positively correlated with CD80 mRNA,IL-1β content and TNF-α content(P＜0.05).The multiplicities of infection of the viruses with Rap1a overexpression and Rap1a knock-down were both 80,and the effective transfection was observed through fluorescence microscopy.Overexpression of Rap1a exacerbated the inflammatory polarization of BV2 cells induced by Hcy(P＜0.05),while knockdown of Rap1a attenuated this polarization(P＜0.05).CONCLUSION:Hcy can promote M1 polarization of BV2 mouse microglia,leading to in-flammatory response,which indicates that Rap1a could potentially serves as a critical regulatory factor in the Hcy-induced inflammatory response of BV2 cells.